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„_ Cornell University Library 
 
 QP 601.G79 1901 
 
 The soluble ferments and fermentation, 
 3 1924 003 185 513 . 
 
The original of tliis book is in 
 tlie Cornell University Library. 
 
 There are no known copyright restrictions in 
 the United States on the use of the text. 
 
 http://www.archive.org/details/cu31924003185513 
 
Cambrftfff Natural ^titmt iManuafe. 
 
 Biological Series. 
 
 General Editor :— Arthur E. Shipley, M.A. 
 tellow and tutor op chrisl's college, cambkidgb. 
 
 THE 
 
 SOLUBLE FEEMENTS 
 
 AND 
 
 FEEMENTATION. 
 
aoniJon: C. J. CLAY and SONS, 
 
 CAMBEIDGB UNIVEESITT PEESS WAEEHOUSE, 
 
 AVE MAEIA LANE, 
 
 AND 
 
 H. K. LEWIS, 
 136, GOWEE STEEET, W.C. 
 
 ©laSBoto: 50, WELLINGTON STEEET. 
 
 ILeipjifl: V. A. BROCKHAUS. 
 
 i^eto Botii: THE MACMILLAN COMPANY. 
 
 aSombaa: E. SEYMOUR HALE. 
 
 [411 Eights reserved.'] 
 
THE 
 
 SOLUBLE FEEMENTS 
 
 AND 
 
 FEEMENTATION 
 
 BY 
 
 Jf !rEYNOLDS GEEEN, ScD., rrs. 
 
 TKINITY COLLEGE, CAMBBIDGE ; 
 
 PROFESSOR OF BOTANY TO THE PHARMACEUTICAL SOCIETY OF GREAT BRITAIN 
 
 FORMERLY SENIOR DEMONSTKATOR IN PHYSIOLOGY IN 
 
 THE T3NIVEBBITY OF CAMBRIDGE. 
 
 SECOND EDITION. 
 
 CAMBRIDGE : 
 
 AT THE UNIVERSITY PRESS. 
 
 190X 
 
First Edition, 1899. 
 Second Edition, 1901. 
 
PEEFACE. 
 
 THE various problems connected with the phenomena of 
 fermentation have received attention during the past ten 
 years from so many investigators in different countries, and are 
 occupying the minds of so many people to-day, that it has 
 seemed desirable to put together, as far as possible, the results 
 reached up to the present time, and to put forward a view of 
 our present position with regard to the whole subject. 
 
 The recent discovery by Biichner of zymase, the enzyme 
 which is capable of setting up alcoholic fermentation, has 
 attracted renewed attention to the whole question, and has for 
 the first time clearly shown what has long been suspected, that 
 the production of alcohol must fall into line with the other 
 processes of decomposition which have for some years now been 
 included under the one name of fermentation. 
 
 It has appeared to the writer desirable for many reasons to 
 lay stress on the relations existing between fermentation in the 
 broad sense and the general metabolic phenomena of living 
 organisms. Recent discoveries have shown more and more 
 plainly what a prominent part is played by enzymes in intra- 
 cellular metabolism, till it has become clear that the distinction 
 drawn between organised and unorganised ferments is based 
 upon an incomplete acquaintance with the metabolic processes 
 in both higher and lower organisms, and must now be 
 abandoned entirely in the light of fuller knowledge. The dis- 
 covery of zymase, already alluded to, causes the disappearance 
 of almost the last resting-place of the distinction. 
 
VI PREFACE. 
 
 As soon as we cease to associate fermentation solely with 
 the lowly forms of living organisms, the vitalistic theory of the 
 writers of the school of de Latour and Pasteur, which makes 
 alcoholic fermentation especially the expression of that life 
 under different conditions of nutrition or aeration, is seen to 
 be no longer sufficient to embrace all the facts. The more 
 recent work takes the subject beyond the stage at which it 
 was left by Pasteur, showing us that precisely similar opera- 
 tions are incidental to the life of the higher organisms. It 
 thus becomes necessary to enquire into the relationship of 
 protoplasm to metabolism, and to the association of ferments 
 or enzymes with the living substance, and so to establish the 
 intimate relationship of fermentation to the ordinary metabolic 
 processes. It becomes possible to go further than this, and 
 to consider by what chemical or physical processes the observed 
 changes or decompositions are effected by protoplasm or by 
 its secretions. 
 
 The comparatively recent speculations of Emil Fischer upon 
 the configuration of the enzymes, and the various bodies 
 they attack, based as they are upon careful and painstaking 
 investigations, direct us towards a new chemical hypothesis of 
 their action, which, while not contravening the views of the 
 vitalistic school of the earlier observers, certainly extends, if it 
 does not complete them. That the work of enzymes will be 
 ultimately shown to be chemical rather than physical in nature 
 is rendered probable by the able researches of Croft Hill, to 
 which attention is called at the conclusion of this work. 
 
 J. REYNOLDS GREEN. 
 
 CAUBRIDaE, 
 
 April, 1899. 
 
PREFACE TO THE SECOND EDITION. 
 
 TN the two years which have elapsed since the publication of 
 this work there has been a large increase in the number of 
 memoirs bearing upon its subject. I have endeavoured, without 
 unduly extending the size of the volume, to incorporate into it 
 the bulk of the new matter and to include the principal 
 memoirs, numbering more than a hundred, into the bibliography 
 at the end. Several new enzymes have been discovered, hadro- 
 mase, tannase, lotase, galactase and vesiculase, besides several 
 oxidases, and some which effect reduction instead of oxidation. 
 These will be found treated of in their appropriate connexions. 
 
 In issuing this new edition I should like to express my 
 appreciation of the kindness of my scientific friends in wel- 
 coming the work as they have done. 
 
 J. EEYNOLDS GREEN. 
 
 Cambridge, 
 April, 1901. 
 
X CONTENTS. 
 
 CHAPTER IV. 
 
 PREPARATION OF DIASTASE AND ITS COURSE OF ACTION. 
 
 Methods of preparation ; work of Cohnheim, Krawkow, von Wittich 
 Mialhe, Lintner, Loew, and Musculus. Action of diastase on starch ; 
 products of the action ; theories of starch transformation ; nature of 
 the sugar produced ; maltose ; isomaltose. Transformations of starch in 
 plants. Is diastase a mixture of enzymes ? Action of diastase on glycogen. 
 
 pp. 45—63. 
 
 CHAPTER V. 
 
 CONDITIONS OF THE ACTION OF DIASTASE. 
 
 Liquefying and hydrolysing powers of diastase ; diastases from dif- 
 ferent sources and their relative activities ; effect of various foreign 
 substances on diastasic action. Conditions of the secretion of diastase ; 
 stimulus of nutrition ; stimulus; of starvation ; nervous action ; object of 
 the secretion. pp. 64 — 76. 
 
 CHAPTER VI. 
 
 IXULASE. 
 
 Occurrence, properties and distribution of inulin ; discovery of inulase ; 
 distribution of the enzyme ; products of its action ; conditions necessary 
 for its activity. pp. 77 — 85. 
 
 CHAPTER VII. 
 
 CYTASE, AND OTHER CELLULOSE-DISSOLVING ENZYMES. 
 
 Composition of the cell-wall ; celluloses and pectoses. Cytase in 
 Peziza, and in Botrytis ; mode of preparation from these fungi. Cytase 
 in the cereal grasses ; the scutellum and the aleurone-layer ; cytase in 
 the endosperm of Tamus and in the seeds of Palms ; in Bacteria. Pro- 
 ducts of the action of cytase. Caroubinase. Hadromase, the wood- 
 dissolving enzyme. Wiesner's Gum-ferment. pp. 86 109. 
 
CONTENTS. XI 
 
 CHAPTEK VIII. 
 
 SUGAR-SPLITTING ENZYMES. 
 
 Characters of the sugars. Inversion of cane-sugar by acids. Discovery 
 of invertase ; its distribution in the vegetable and in the animal organism ; 
 modes of preparation of invertase ; its course of action and the efiFect 
 of various conditions upon it; its importance in metabolism. 
 
 pp. 110—127. 
 
 CHAPTER IX. 
 
 SUGAR-SPLITTING ENZYMES (continued). 
 
 Glucase and its action on malt-sugar. Digestion of maltose in the 
 small intestine ; glucase in the secretions of the pancreas and the small 
 intestine; in various fungi, in malt and maize; in serum and lymph; 
 in other animal secretions ; in the liver and other organs ; in yeast. 
 Action of glucase on glucosides. Trehalase, its distribution and action ; 
 preparation of trehalase ; diiFerences between glucase and trehalase. 
 Eaffinase. Melizitase. Lactase. pp. 128 — 145. 
 
 CHAPTER X. 
 
 GLUCOSIDE-SPLITTING ENZYMES. 
 
 Nature of glucosides. Emulsin and its action. Distribution of emul- 
 sin ; researches of Guignard, G&ard, and Bourquelot ; conditions of the 
 action of emulsin. Myrosin and its action ; localisation of myrosin ; 
 peculiarities of its action. Erythrozyme. Rhamnase. Gaultherase. 
 Tannase. Lotase. Other enzymes of this class. pp. 146 — 171. 
 
 CHAPTER XL 
 
 PROTEOLYTIC ENZYMES. PROTEOLYSIS. 
 
 Products of the decomposition of proteids by acids. Eesearches of 
 Eiibne. Decomposition of proteids by pepsin and trypsin. Researches 
 of Meissner and Kuhne. Later work of Neumeister and of Chittenden. 
 Hydrolysis. Objections to Kuhne's views. pp. 172 — 185. 
 
Xll CONTENTS. 
 
 CHAPTER XII. 
 
 PROTEOLYTIC ENZYMES {continued). 
 
 Pepsin. Methoiis of preparation. Work of Scheff'er, Briicke, Maly, 
 Loew, and Pekelharing. Distribution of pepsin ; peculiarities of its 
 action ; eflfect of various conditions on pepsin ; decomposition of vegetable 
 proteids and gelatin. Action of pepsin on other substances. Trypsin ; 
 its occurrence in pancreatic juice ; separation from other enzymes ; 
 KUhne's method of preparation ; distribution of trypsin in the Inverte- 
 brata. Conditions of the action of trypsin ; products of its activity. 
 Intracellular digestion by trypsin. Differences between pepsin and 
 trypsin. Galactase. pp. 186 — 208. 
 
 CHAPTER XIII. 
 
 PROTEOLYTIC ENZYMES {continued). VEGETABLE TRYPSINS. 
 
 Bromelin ; its occurrence in the Pine-apple ; modes of preparation ; 
 course of action ; conditions affecting it. Papain and its preparation ; 
 action on animal and vegetable proteids ; conditions affecting the action 
 of papain. Trypsin in seeds ; action on fibrin and on the proteids of the 
 seed. Zymogen of trypsin. Proteolysis in cereal grasses. Trypsin of 
 Agave, Fieus, Cucwmis. Proteolytic enzymes of the insectivorous plants. 
 Trypsin in Yeast and in Bacteria. Casease. Identity of animal and 
 vegetable trypsins. pp. 209-^35. 
 
 CHAPTER XIV. 
 
 FAT-SPLITTING ENZYMES. LIPASE. {PIALYN, STEAPSIJV.) 
 
 Digestion of fat in the alimentarj' canal. Discovery of lipase ; its 
 distribution in the animal kingdom ; methods of identification ; differences 
 between serum lipase and pancreatic lipase ; lipase in the vegetable 
 kingdom. pp. 236—249. 
 
 CHAPTER XV. 
 
 THE CLOTTING ENZYMES. RENNET. 
 
 Characteristics of the clotting enzymes. Rennet, and its action on 
 milk ; casein and tyrein. Sources of rennet, modes of preparation. 
 Relation of rennet to calcium salts ; theories of the action of rennet. 
 Peculiarities of pancreatic rennet. Vegetable rennet, and its distribution. 
 
 pp. 250—265. 
 
CONTIfNTS. xiii 
 
 CHAPTER XVI. 
 
 THE CLOTTING ENZYMES (continued). 
 THROMBASE, {THROMBIN), THE FIBRIN-FERMENT. 
 
 Phenomena of the coagulation of the blood ; early researches ; the work 
 of Schmidt and Hammarsten. Discovery of thrombase ; its preparation 
 from serum and from fibrin ; nature of thrombase. Conditions affecting 
 coagulation; influence of calcium salts; zymogen of thrombase ; inhibition 
 of coagulation, and conditions of action of thrombase ; its possible nucleo- 
 proteid nature ; mtravascular clotting. Crustacean fibrin-ferment. Myosin- 
 ferment ; comparison of myosin and fibrin ; preparation of the enzyme and 
 peculiarities of its action. Vesiculase. pp. 266 — 286. 
 
 CHAPTER XVII. 
 
 THE CLOTTING ENZYMES {continued). PECTASE. 
 
 The formation of vegetable jellies ; early work of Fremy ; discovery 
 of pectase. Eesearches of Mangin on the pectic bodies in plants. Work 
 of Bertrand and Mall^vre on pectase. Preparation of the enzyme and its 
 relation to calcium salts ; conditions of the action of pectase ; its distri- 
 bution in the vegetable kingdom ; difierences in the composition of the 
 cell- wall ; formation of the middle lamella. pp. 287—300. 
 
 CHAPTER XVIII. 
 
 AMMONIACAL FERMENTATION. UREASE. 
 
 Changes which take place in urine on standing. Organisms causing 
 ammoniacal fermentation. Decomposition of urea and of hippuric acid. 
 Urease ; its detection ; experiments of Musculus ; of Lea ; of Miguel. 
 Conditions of activity of urease. Histozyme. pp. 301—307. 
 
xiv CONTENTS. 
 
 CHAPTER XIX. 
 
 OXIDASES, OR OXIDISING ENZYMES. 
 
 Changes set up by oxidases. Laccase : discovery by Yoshida ; pre- 
 paration ; action ou urushic acid ; work of Bertrand ; action of laccase on 
 various phenols ; , distribution of laccase ; association with manganese ; 
 laccase in fungi. Tyrosinase j occurrence in fungi ; action on tyrosin ; 
 separation of tyrosinase from laccase ; its action on various aromatic 
 compoimds. CEnoxydase : the breaking of wines due to it ; presence of 
 oenoxydase in fruits ; condition of the action of oenoxydase. Oxidase 
 of cider-apple. Other vegetable oxidases ; presence in yeasts, plants 
 yielding indigo, olives, aconite and belladonna, bran, &c. Animal oxidases ; 
 glycolysis in blood ; oxidase of the pancreas ; action on salicyl-aldehyde ; 
 relation to nucleo-proteids ; oxidase in molluscs. Keducing enzymes. 
 
 pp. 308—332. 
 
 CHAPTER XX. 
 
 ALCOHOLIC FERMENTATION. 
 
 Early researches ; work of Lavoisier, Gay-Lussac, Th^nard, and 
 de Saussure. Inversion of cane-sugar before fermentation. Nature of 
 the decomposition ; formation of alcohol and carbon dioxide ; formation 
 of succinic acid and glycerine ; sugars capable of undergoing fermentation. 
 Eelation of yeast to fermentation ; researches of Hansen ; conditions of 
 the action of yeast. Fermentation of other carbohydrates -than sugars. 
 Koumiss. Kephir. The ginger-beer plant and other similar organisms ; 
 Koji ; Arrack. Fermentation caused by other organisms. Production of 
 alcohol by fruits and other parts of the higher plants ; conditions of such 
 production. Significance of fermentation. Discovery of zymase ; method 
 of preparation, properties and distribution of zymase. pp. 333 — 362. 
 
 CHAPTER XXI. 
 
 THE FERMENTATIVE POWER OF PROTOPLASM. 
 
 Behaviour of protoplasm in metabolism ; intracellular decompositions 
 without enzymes ; modifications caused by absence of oxygen. Lactic 
 fermentation and the microbes causing it. Butyric, propionic, citric and 
 oxalic fermentations. The formation of acetic acid from alcohol : dis- 
 cussion as to its cause ; views of Liebig, Pasteur, and later writers. The 
 organisms causing acetic fermentation. Nitric fermentation ; organisms 
 producing nitrous and nitric acids ; researches of Winogradsky. Viscous 
 fermentation. Putrefaction ; its complex character ; nature of the de- 
 compositions taking place ; organisms concerned : ptomaines. Relation- 
 ship between organised and unorganised ferments. Views of Naegeli and 
 Sachs. pp. 363—395. 
 
CONTENTS. XV 
 
 CHAPTER XXII. 
 
 THE SECRETION OF ENZYMES. 
 
 Conditions antecedent to secretion ; nature of stimuli causing its 
 occurrence ; process of secretion in animal cells ; sequence of changes 
 which may be observed ; process in vegetable tissues. Zymogens. Part 
 played by the nucleus in the act of secretion ; work of Macallum ; 
 formation of enzyme a gradual process; relation between zymogens and 
 enzymes. Zymogens of pepsin, trypsin, rennet, diastase. Vegetable zymo- 
 gens. The zymogen of thrombase. Differences in behaviour between 
 zymogens and enzymes. pp. 396 — 417. 
 
 CHAPTER XXIII. 
 
 THE CONSTITUTION OF ENZYMES. 
 
 Eeactions of enzymes ; fallacious character of colour tests ; association 
 of enzymes with various proteids; relation existing between the proteid 
 and the enzyme. Are enzymes proteids? Difl&culties in the way of 
 accepting this view. Study of invertase hy O'Sullivan and Tompson. 
 Theory of the nucleo-proteid constitution of enzymes ; work of Pekelharing, 
 Macallum, Spitzer. pp. 418—435. 
 
 CHAPTER XXIV. 
 
 THE MODE OF ACTION OF ENZYMES. 
 THEORIES OF FERMENTATION. 
 
 Early theories of fermentation. Views of Valentinus, Libavius, Lemery, 
 Willis and Stahl, Gay-Lussac. Discoveries of de Latour and his contem- 
 poraries ; the vitaUstic theory. Controversy between the supporters of 
 the physical and the vitalistio theories. Views of Liebig, Pasteur, and 
 NaegeU. Discovery of enzymes. Differences supposed to exist between 
 their action and that of organised ferments. Views of Naegeli and Sachs. 
 Fallacies underlying such supposed differences. Relation of fermentation 
 to metabolism. Modern theories of fermentation. Eesearches of Fischer 
 and the configuration hypothesis ; theory of electric hydrolysis ; theory of 
 de Jager and Maurice Arthus ; chemical hypothesis of Bunsen and 
 Hiiiher ; researches of Croft Hill and their support of this theory. 
 Destruction of enzymes by heat. pp. 436—462. 
 
 Bibliography. pp. 463 — 504. 
 
 Index. pp. 505 — 512. 
 

 
 ERRATA. 
 
 
 
 On p. 149, 
 
 line 21 /oi- 4 cm. 
 
 
 read excess 
 
 p. 212, 
 
 )j 
 
 I „ amides 
 
 
 „ amido-acids 
 
 p. 302, 
 
 ») 
 
 25 J 
 
 
 
 p. 233, 
 
 11 
 
 35) 
 
 
 
 p. 341, 
 
 }i 
 
 23 > „ casein 
 
 
 „ caseinogen 
 
 p. 342, 
 
 11 
 
 16J 
 
 
 
 p. 245, 
 
 1, 
 
 33 „ two years 
 
 later 
 
 „ in 1891 
 
 p. 268, 
 
 M 
 
 30 „ tyrein 
 
 
 „ casein 
 
 p. 427, 
 
 „ 
 
 12 „ page 177 
 
 
 „ page 187 
 
CHAPTER I. 
 
 THE NATURE OF FERMENTATION AND ITS RELATION 
 TO ENZYMES. 
 
 i Like so many of the phenomena which are associated with 
 the presence of life, fermentation has been the subject of careful 
 study extending over centuries of time. It has been the object 
 of experiment at the hands of many investigators, and its 
 nature has been the theme of vigorous controversy. Hypo- 
 thesis after hypothesis has been advanced to explain the 
 various phenomena which it presents, each in turn marking 
 an advance in knowledge, but none of them sufficing to account 
 satisfactorily for the whole of the facts observed. Though 
 within the past two centuries very substantial advances have 
 been made, much still remains undiscovered. 
 
 The term fermentation was first applied to the process 
 which leads to the formation of alcohol, the knowledge of which 
 goes back to very remote antiquity. The name probably arose 
 from the copious evolution of gas which accompanies the pro- 
 duction of the spirit, and which gives the liquid in which it is 
 taking place the appearance of a gentle ebullition. 
 
 It is not surprising that in early times very mistaken ideas 
 arose as to the nature of the process, based upon this ap- 
 pearance. Wherever a disengagement or evolution of a gas was 
 noticed it was thought to be due to similar causes. Hence 
 we find some of the older writers classing with fermentation 
 the effervescence which ensues when an acid is brought into 
 contact with chalk. The evolution of gas in the animal 
 intestine was held more correctly to be a kindred phenomenon. 
 
 G. F. 1 
 
2 THE NATURE OF FERMENTATION [CH. 
 
 Though many of the reactions thus grouped together were 
 possessed of very little in common, one process which was of 
 very early date was associated correctly with fermentation. 
 This was the action of leaven in the preparation of bread. The 
 evolution of gas was observed in connection with the raising 
 of the dough, though no further resemblance to the alcoholic 
 fermentation was recognised. Indeed the speculations con- 
 cerning leaven and its action were of the wildest order, some 
 writers comparing it to the hypothetical philosopher's stone, 
 and attributing to it the power of transforming the dough into 
 something resembling itself. 
 
 One fact of importance came out amidst all the mass of 
 confusion, though its interpretation left much to be desired. 
 This was the discovery that a very small quantity of leaven was 
 capable of fermenting or transforming an almost indefinite 
 amount of dough. The latter was however considered to be 
 converted into leaven. 
 
 Another point of resemblance between these two fermen- 
 tative processes was known at a comparatively early date. 
 Just as the aeration of dough was associated with the 
 presence of leaven, alcoholic fermentation was found to be 
 accompanied by the formation of a deposit in the fermenting 
 liquid, which took the form sometimes of a sediment, sometimes 
 of a scum floating on the surface. By many writers con- 
 siderable importance was attached to this deposit, and, as in 
 the case of the leaven, some special occult force was attributed 
 to it, by virtue of which it set up the changes which could be 
 observed in the liquid. It was consequently called & ferment. 
 
 The true nature of this deposit, like that of leaven, for a 
 very long period was not investigated, nor the part it plays 
 at all understood. 
 
 About the end of the sixteenth century the process of 
 putrefaction was associated with the two fermentations so far 
 mentioned, and the three shown to have much in common. 
 The confusion however which led to the association of efferves- 
 cence with them was maintained for some considerable time. 
 It was not till 1659 that de la Boe pointed out that they 
 differ considerably from effervescence, the chief reaction of 
 
l] AND ITS RELATION TO ENZYMES. 3 
 
 the latter being one of combination, while in the cases of 
 fermentation it is one of decomposition. 
 
 The first clear pronouncement upon alcoholic fermentation 
 was made by Becher in 1682, and it marks an epoch in the 
 development of our knowledge of the subject. This author 
 ascertained that only saccharine liquids are capable of under- 
 going alcoholic fermentation, and he showed that the alcohol 
 does not exist as such in the original " must " of wine, as had 
 been supposed, but is formed during the operation of fermenta- 
 tion. Becher considered its formation to be due to a kind of 
 combustion of the sugar, as he ascertained that air is needed to 
 set up the phenomenon. 
 
 About the same time the deposit or scum which had been 
 observed as an invariable accompaniment of fermentation was 
 examined microscopically by Leuwenhoek, who showed it to 
 be composed of little ovoid or spherical globules, but he was 
 unable to determine their nature. 
 
 Though the subject was somewhat vehemently discussed 
 and various theories were advanced to explain the observed 
 phenomena, but little real advance was made during the next 
 century. The acetic fermentation was discovered and the 
 similarity of putrefaction and fermentation in general insisted 
 upon. But the nature of Leuwenhoek's globules remained 
 undetermined, many observers, especially Fabroni, holding 
 them to be of animal origin. The work of Lavoisier towards 
 the close of the eighteenth century threw a flood of light upon 
 the process. The great chemist studied quantitatively the 
 relations of the sugar to the derivatives formed from it during 
 the fermentation, and came to the conclusion that the operation 
 consisted of a splitting up of the sugar into two parts, one of 
 which became oxygenated to form carbonic dioxide, while the 
 other was converted into alcohol. He said that if it were 
 possible to recombine these two bodies, sugar would again be 
 formed. 
 
 About the year 1815 analyses by Gay-Lussac, Th^nard and 
 de Saussure fixed definitely the composition of sugar and 
 alcohol. During the early years of the present century indeed 
 the views of Lavoisier were the basis of much research from the 
 
4 THE NATURE OF FERMENTATION [CH. 
 
 chemical side, and the work resulted in determining the changes 
 which take place in the sugar. 
 
 During the same period inquiries were made by many 
 observers into the nature of the globules and their relation to 
 the fermentation. Astier in 1813 and Desmazi^res in 1826 
 adhered to Fabroni's view that they were of animal origin, 
 Astier holding that they could only live at the expense of the 
 sugar which they decomposed. 
 
 Shortly afterwards Cagniard de Latour, repeating Leu- 
 wenhoek's experiments, saw that the globules consisted of a 
 definite organism, capable of reproduction by budding, and 
 belonging apparently to the vegetable kingdom. He concluded 
 that probably they disengaged the carbonic dioxide and fer- 
 mented the liquid by some effect of their vegetation. 
 
 This discovery, which is really the basis of the present 
 views of the subject, was also made almost simultaneously by 
 Schwann at Jena, and by Kiitzing at Berlin, who were con- 
 firmed by Quevenne, Turpin and Mitscherlich. The organism 
 was referred by some to the Fungi and by others to the Algae, 
 but its true systematic position was first ascertained by Meyen, 
 who pronounced it a fungus and placed it in a new genus, 
 to which he gave the name Saccharomyces. 
 
 These researches laid the foundation for the more complete 
 and satisfactory views of Pasteur, whose investigations have 
 thrown so much light upon the whole process of fermentation. 
 The association of a definite organism with the decomposition 
 of the sugar and the idea that the latter was in some manner 
 connected with the manifestation of its vital processes, removed 
 the question from the realm of chemistry in the narrower sense 
 and gave it a place among the problems of physiology, 
 Pasteur in studying the subject from the latter point of view 
 made the very important discovery that the production of 
 alcohol is accompanied by the coincident formation of glycerine 
 and succinic acid, and he determined by quantitative methods 
 that about 4 per cent, of the sugar which disappears during 
 the progress of a fermentation gives rise to these two new 
 derivatives. 
 
 Pasteur came to the conclusion that the exercise of the 
 
l] AND ITS RELATION TO ENZYMES. O 
 
 fermentative power by the Saccharomycete, or yeast, was 
 connected with nutrition in the absence of free oxygen, and 
 that it was really the expression of the effort of the organism 
 to obtain oxygen in the absence of a free supply. In this 
 connection some investigations of Lechartier and Bellamy may 
 be recalled. These observers made a number of experiments 
 with ripe succulent fruits, which they kept for several months 
 in an atmosphere devoid of oxygen. Under these conditions 
 the fruits gave off continuously a certain quantity of carbon 
 dioxide, and at the end of the time of observation the pulp 
 contained a measurable amount of alcohol. Microscopic ob- 
 servation showed that this fermentation took place in the 
 absence of yeast cells, and was in fact carried out by the living 
 substance of the cells of the pulp. Pasteur confirmed these ob- 
 servations and regarded them as strengthening his hypothesis. 
 
 Schutzenberger has argued with some force against the 
 view that the decomposition of the sugar is related to the 
 respiratory process of the yeast, preferring to regard it as 
 ministering to its nutrition. A discussion of this point must 
 however be left to a subsequent chapter. 
 
 Though the views of the Pasteur school did not at once 
 meet with universal acceptance, being opposed strongly by 
 Liebig and by Naegeli, both of whom supported a theory of 
 molecular vibration as explaining the decomposition of the 
 sugar, no doubt is now entertained that the living protoplasm 
 of the vegetable cell is the ultimate cause of the fermentative 
 process, and that the latter is the expression of some activity 
 connected with the maintenance of its life. That it is not a 
 specific peculiarity of the protoplasm of the yeast cell, but is 
 shared by that of much higher plants, is equally clearly shown 
 by the work of Lechartier and Bellamy already alluded to. 
 
 The influence of living cells of yeast in the fermentation or 
 leavening of dough, and that of other vegetable organisms in the 
 processes of putrefaction established during the present century 
 show how essentially similar these processes are to the alco- 
 holic fermentation. Micro-organisms have been found capable of 
 setting up also many other decompositions comparable with all 
 these. 
 
6 THE NATURE OF FERMENTATION [CH. 
 
 Whilst Pasteur's views were gradually making themselves 
 felt, work in another direction was progressing which was destined 
 to materially enlarge our views of the whole subject. Quite in 
 the early part of the present century (1814) Kirchhoff observed 
 that germinating barley contained something which was capable 
 of liquefying starch-paste and that in the process it formed a 
 kind of sugar. Kirchhoff associated this power with the albumi- 
 noid material or gluten of the grain. In 1823 the observation 
 was repeated by Dubrunfaut, and in 1833 Payen and Persoz 
 extracted from germinating barley the substance which effected 
 the decomposition. They steeped the grain in water for some 
 time and filtered off the extract. On adding a large excess of 
 alcohol to the filtrate they obtained a white flocculent precipi- 
 - tate, which when dried and dissolved in water was found capable 
 of converting starch-paste into sugar. The change induced by 
 this substance, which they called diastase, was held to be a 
 kind of fermentation. It appeared to act in the same way as 
 the globules of Leuwenhoek, the nature of which was at that 
 time undetermined, as already explained. At the same time it 
 was clearly an unorganised substance, though very little was 
 definitely known about it. On this account it was distinguished 
 as an unorganised ferment. 
 
 Two years earlier Leuchs had noticed that saliva possessed 
 the same property as the germinating barley in that it was 
 capable of converting starch-paste into sugar. In 1845 Mialhe 
 showed that a preparation of diastase could be prepared from 
 this animal secretion by the same process as Payen and Persoz 
 had adopted for their barley extract. 
 
 This discovery was followed by others. In 1836 Schwann 
 demonstrated the existence of pepsin in gastric juice, and 
 showed that it decomposed indiffusible albuminous bodies 
 into others that were capable of passing through membranes. 
 Berthelot found that a watery extract of yeast, quite free from 
 the cells of the plant, was capable of converting cajie-sugar into 
 two other sugars of simpler composition. Liebig and Woehler 
 discovered a similar body in the seeds of the almond, which 
 decomposed amygdalin with the formation of sugar and other 
 products, and they were again struck by the similarity of the 
 
l] AND ITS REI.ATION TO ENZYMES. 7 
 
 action to that of yeast in alcoholic fermentation. About 1860 
 Bi'ucke prepared from the mucous membrane of the stomach 
 the ferment first observed by Schwann which split up insoluble 
 " proteids, causing the formation of soluble peptone. 
 
 All these various ferments were recognized as associated 
 in some way with the living substance of either animal or 
 vegetable cells, and they were soon held to play an important 
 part in the life of the organism from which they were extracted. 
 
 Two classes of ferments appeared to be thus indicated, the- 
 one a living organism, working only during its own processes of 
 growth and multiplication ; the other consisting of substances 
 which could be extracted by solvents from the cells in which 
 they were formed, and capable of setting up decompositions 
 apart from the life of such cells. The two categories were 
 consequently called organised and unorganised or soluble 
 ferments. The term enzyme is now generally used to indicate 
 the latter class. 
 
 In studying these various bodies and the ultimate relations 
 of the one class to the other it is evident that attention must 
 be given to the vital phenomena of protoplasm, and to the 
 changes which take place in its substance during the manifesta- 
 tion of its life. The work of recent years has thrown much 
 light upon the various operations that take place in cells, and 
 we now know that these are for the most part, if not entirely, 
 regulated by the behaviour of the living substance. 
 
 Various views have been put forward as to the arrangement 
 of the living protoplasm. Though this is a matter which can 
 by no means be regarded as finally established, there is some 
 probability that it is disposed in the form of a network, the 
 meshes of which are occupied by a material which is similar in 
 composition but which is not living. The great characteristic 
 of protoplasm is its instability; it is continually undergoing 
 decomposition and reconstruction. Some of the residues of its 
 breaking down are capable of being built up again into its 
 substance ; others are thrown off from it. Of these some are 
 eliminated entirely from the organism, others are retained 
 within it to carry on some of the more subordinate processes 
 of its life or its nutrition. 
 
8 THE NATURE OF FERMENTATION [CH. 
 
 An aaimal or vegetable cell is hence the centre of very 
 vigorous activity ; work is going on within it in the direction of 
 incorporating material for the growth of the living substance, 
 or of preparing material brought to it, so that it may be capable 
 of such incorporation. Again some of its substance may be 
 undergoing decomposition with a view to supplying the energy 
 which it needs for the maintenance of its vital processes. 
 
 The chemical changes involved may be of three kinds. The 
 decompositions may involve the incorporation of material into 
 the actual substance of the protoplasm and the subsequent 
 splitting off of various residues from the latter. Such appear 
 to attend the formation of the various enzymes such as diastase ; 
 also the formation of fat, starch, and other compounds which can 
 be seen in various cells. Other changes may take place without 
 the establishment of such an intimate relationship with the 
 protoplasm. They may be carried out by the protoplasm 
 outside its own substance, the materials affected not being 
 incorporated in it while the change is taking place. Such 
 decompositions have been alluded to by various writers as 
 caused by the fermentative action of protoplasm itself 
 
 A third class of reaction may take place in the cell with- 
 out the actual intervention of the protoplasm at all. It is 
 probable that processes of oxidation and reduction are taking 
 place among the substances which occupy the meshes of the 
 protoplasmic network, and that quiescent as the cell appears it 
 is the seat of many chemical reactions of this kind. Thus the 
 formation of sugar in the cells of leaves under the influence of 
 chlorophyll, which probably involves the polymerisation of some 
 form of aldehyde, need not necessarily involve the action of the 
 living substance, as such polymerisation is very frequent among 
 aldehyde bodies. 
 
 Some of the decompositions of the latter class may be distin- 
 guished from the others by the fact that though protoplasm is 
 not immediately concerned in bringing them about, it prepares 
 from its own substance an enzyme by which the transforma- 
 tion is effected. The secretion or formation of this new 
 factor belongs to the first class of reactions mentioned, but 
 the material once secreted is endowed with the power of 
 
l] AND ITS RELATION TO ENZYMES. 9 
 
 setting up and maintaining the decompositions in question. 
 The ultimate purpose of the secretion is usually the digestion 
 of some form of food material to prepare it for incorporation 
 into the living substance itself But recently reason has been 
 found to believe that the processes of oxidation and reduction 
 axe carried out by similar agencies. We can consequently 
 recognise in many organisms both digestive and respiratory 
 enzymes. So many of these bodies are now known that it 
 does not seem very unreasonable to put forward the view 
 that all decompositions of this kind will ultimately be found 
 to be carried out by such a mechanism. The number of cases 
 in which the direct rather than the indirect intervention of 
 protoplasm appears to be involved is continually growing smaller 
 as further work upon metabolism proceeds. A very important 
 addition to our knowledge in this direction has been made 
 during the past two years by Buchner, who has ascertained 
 that even the earliest known fermentative process, the forma- 
 tion of alcohol from sugar, is carried out by a soluble ferment 
 which can be extracted from the yeast cell. 
 
 The constructive processes which take place in the cell 
 have not been so clearly shown to be carried out by such 
 secretions. In most cases in which a building up of complex 
 from relatively simple substances takes place in the cell, it is 
 carried out by the direct intervention of the protoplasm. Hence 
 the study of fermentation is mainly directed to processes of 
 degradation whereby complex substances are broken down into 
 more simple ones. To this point we shall return later. 
 
 We may consequently for the present define fermentation 
 to be the decomposition of complex organic material into 
 substances of simpler composition by the agency either of pifoto- 
 plasm itself or of a secretion prepared by it. 
 
 We find instances of both these methods in unicellular 
 and in multicellular organisms, as well in the animal as in the 
 vegetable , kingdom. By far the greater number of instances 
 of the fermentative activity of the protoplasm as apart from 
 secreted enzymes may be found in the so-called organised fer- 
 ments, the yeasts and the great group of micro-organisms or 
 Schizophyta. So prominent indeed are these plants in their 
 power of exciting various decompositions that they were origin- 
 
10 THE NATURE OF FERMENTATION [CH. 
 
 ally regarded as the only "ferments," the fact that the "fermen- 
 tation" was merely incident to their own biological process 
 passing almost unnoticed. The theory that this power or 
 property puts them into a class, distinct biologically from other 
 and higher plants is however quite disproved by the discovery 
 that many of them provoke the decomposition associated with 
 them by means of enzymes, which can with a little care be 
 prepared and separated from them with almost as much ease as 
 from the higher plants themselves. Moreover the enzymes they 
 secrete are identical with those which are prepared by the latter. 
 
 It follows from this that those processes of fermentation in 
 which the protoplasm is directly involved are intracellular. It is 
 only comparatively lately that this has been realised in the case 
 of many vegetable organisms. The presence of a cell-wall 
 clothing the living substance would render impossible the con- 
 tact necessary to produce externally the decompositions observed. 
 This point still needs emphasising in the case of many of the 
 bacterial fermentations. Not only in unicellular but in multi- 
 cellular organisms also this can be observed. Instances are 
 afforded by the alcoholic fermentation of ripe fruits noticed by 
 Lechartier and Bellamy, and more recently studied by Gerber ; 
 by the transformation of glycogen into sugar in the muscles, 
 and possibly the liver of the higher animals, though it is not 
 certain that the latter is not the seat of a fermentation by 
 a variety of diastase. The power of forming acids possessed 
 by various bacteria is shown also by the cells of the succulent 
 parenchyma of the higher plants. Though acetic acid is formed 
 by Mycoderma aceti from alcohol, and such parenchyma appears 
 to form the acids it contains from sugar, the protoplasm in 
 both eases is the active agent. The acids are more complex 
 in the latter case, but this is probably due to the character of 
 the metabolism of the two classes of cells respectively. 
 
 Turning to the processes of fermentation which are carried 
 out by means of enzymes we find more complexity. In the 
 simplest cases of unicellular plants and animals intracellular 
 fermentation is most general. The complex substance, which 
 is usually a food-material, is absorbed into the cell ; an enzyme 
 is secreted there, and the work of transformation follows. 
 From various unicellular plants enzymes have been extracted 
 
l] AND ITS RELATION TO ENZYMES. 11 
 
 which do not escape during life, but which are responsible for 
 various chemical reactions in the cells. Evidence of the exist- 
 ence of others in other cells has been obtained, though the 
 enzymes have never been extracted. Thus in the digestive 
 processes of certain unicellular animals, notably Amoeba, Car- 
 chesium, and Actinosphserium, ingested food has been found, 
 soon after its absorption, to be surrounded by a vacuole con- 
 taining an acid liquid, and has been seen to dissolve gradually 
 under the influence of the contents of the vacuole, in just the 
 same way as similar food-material can be digested in vitro by 
 artificial gastric juice. 
 
 But the process of intracellular digestion is not the only 
 one which is carried on. Many cells after forming the enzyme 
 excrete it from their substance into the surrounding medium, 
 where it brings about decompositions the products of which 
 are subsequently absorbed by the organism. Many bacteria 
 have been found to behave in this way, but it is a phenomenon 
 which is associated more generally with multicellular structures, 
 both animal and vegetable. So long as a single cell or mass of 
 protoplasm constitutes the whole organism, its metabolic and 
 nutritive processes go on throughout its whole substance. But 
 with the multiplication of cells in the body and the attendant 
 differentiation of members and organs, special cells are found 
 devoted to the preparation of enzymes. Others in turn are 
 specially set apart for the absorption of nutritive materials. 
 It is evident that in such cases the utilisation of the enzymes 
 is only possible when they are excreted from the body and 
 allowed to do their work in such positions that the products 
 which they form can be brought easily into the absorptive 
 region. Hence we get gradual differentiation of the complex 
 glands, found for instance in the alimentary tract of the human 
 body, which excrete their enzymes into a region which is 
 external to the cells ; indeed for many reasons the alimentary 
 tract itself may be regarded as merely an invagination of the 
 external surface of the body. 
 
 The distinction which was formerly drawn, as already 
 mentioned, between organised and unorganised ferments is 
 thus seen to be misleading. The fundamental difference which 
 
12 THE NATURE OF FERMENTATION [CH. 
 
 ajjpears when the two modes of activity are compared is found 
 to be a difference of differentiation of the organism. The 
 unicellular being appears in most cases to carry out its work 
 throughout its whole substance, and the magnitude of the sphere 
 of the changes it sets up being far greater than its own dimen- 
 sions it appears only as something provoking large disturbances 
 in some organic compound, the fact that these are only 
 incidental to its own biological needs being thereby obscured. 
 The multicellular organism devoting only a definite part of its 
 substance, and that generally a small one, to the transforma- 
 tions, these are easily recognised as biological in their end and 
 aim. Both unicellular and multicellular structures as we have 
 seen carry out the work by identical means, either the fer- 
 mentative activity of their protoplasm or the secretion and 
 sometimes subsequent excretion of enzymes. 
 
 The study of fermentation thus resolves itself very largely 
 into an investigation of the destructive or decomposing power 
 of protoplasm, exerted either directly or by means of secreted 
 enzymes. This may perhaps be narrowed still more, for as 
 already noted many of the cases in which protoplasm has been 
 supposed to act directly have been found to be additional 
 instances of enzymic powers. The improved methods of pre- 
 paration which have been discovered have in many cases led 
 to the isolation of enzymes where they have not been suspected. 
 The search for the enzyme which produces alcohol from sugar 
 has been carried on by many investigators, and as mentioned 
 above has now been crowned with success. The importance 
 of this discovery can hardly be overestimated, as the yeast 
 cell has so long been held to occupy the most prominent 
 position as an organised ferment. There still remain however 
 many cases in which the existence of an enzyme is only 
 hypothetical. 
 
 Turning to the consideration of the enzymes as a particular 
 group of substances we find that certain general features can 
 be ascribed to them in common, though they present great 
 differences among themselves. A discussion of their possible 
 or probable composition must be deferred to a later chapter, 
 but for the present we may note that it is very difficult to 
 
I] AND ITS RELATION TO ENZYMES. 13 
 
 hazard more than a conjecture on the subject, as we have no 
 criterion of their purity, nor indeed any satisfactory test for 
 their existence except the demonstration of their powers. 
 
 Their activity is very largely dependant upon temperature, 
 not being manifested at low temperatures approaching the freez- 
 ing point of water ; it increases gradually as the temperature 
 rises, reaching its maximum at a point which varies somewhat 
 for each enzyme, but which ranges between 30° and 50° C. ; 
 beyond this so-called optimum point it gradually diminishes, 
 and finally disappears. By the action of a temperature 
 approaching 100° C. they permanently lose their characteristic 
 properties and are destroyed or decomposed. The deleterious 
 effect of this high temperature is apparently dependant upon 
 their being in contact with water, for if perfectly dry they will 
 survive heating to a much higher point. 
 
 It is generally held that they do not themselves enter into 
 the reactions which they set up, but provoke those changes 
 without undergoing any alteration. Further, they are not 
 destroyed by their own activity, the energy with which they 
 work not apparently proceeding from any decomposition of 
 their substance. 
 
 Certain facts which have been observed recently throw a 
 certain amount of doubt on the accuracy of these generally 
 accepted statements, but a discussion of these points must be 
 deferred to a subsequent chapter. 
 
 The enzymes show a peculiar sensitiveness to the environ- 
 ment in which they find themselves. They are peculiarly 
 influenced by the reaction of the solutions in which they are 
 working, some being active only in acid, others in neutral, and 
 others again in alkaline media. Nor is their activity alone 
 affected, for slight alterations in the composition of a medium 
 favourable to them will frequently result not only in the 
 stoppage of their action but even in their own destruction. 
 Their activity again is in all cases lessened and finally paralysed 
 by the presence of an excess of the products which they form. 
 
 For convenience of discussion the enzymes have been 
 classified according to the materials on which they work. The 
 following groups are fairly well established. 
 
14 THE NATURE OF FERMENTATION, ETC. [CH. I 
 
 (1) Those which transform insoluble carbohydrates of 
 various kinds, ultimately producing soluble sugars. Of these 
 we recognise the various forms of diastase, which attack starch 
 and its allies; inulase, which decomposes inulin; cytase which 
 hydrolyses cellulose. 
 
 (2) Those which transform sugars of the biose type into 
 simpler sugars, usually hexoses. Of these we have instances in 
 invertase which attacks cane-sugar; glucose which splits up 
 maltose, and others decomposing other bioses. 
 
 (3) Those which decompose glucosides, giving rise to a 
 form of sugar together with various aromatic bodies. These 
 include emulsin, myrosin, and several others. 
 
 (4) The proteolytic group, whose members decompose 
 various forms of insoluble proteids. Among them are pepsin 
 and trypsin, which play so prominent a part in animal digestion. 
 
 (5) The clotting enzymes, which produce from various 
 soluble bodies jelly-like substances which involve a coagulation 
 of the lic[uid in which they are dissolved. Included in this group 
 are rennet, which coagulates milk, thrombose, which plays a part 
 in the coagulation of the blood, and pectase which is the leading 
 factor in the formation of vegetable jellies. 
 
 (6) The enzyme which decomposes oils or fats, which has 
 recently been named lipase. 
 
 (7) Oxidases, or oxidising enzymes, which, as their name 
 implies, assist in the oxidation of various substances. Among 
 them are laccase and tyrosinase. 
 
 This list is not a complete one, for a few other enzymes 
 are known, though they appear to occupy isolated positions. 
 Among them may be mentioned urease, which forms ammonium 
 carbonate from urea, and the new zymase of Buchner, which is 
 the alcohol-producing enzyme. 
 
15 
 
 CHAPTER II. 
 
 DIASTASE. {AMYLASM!, PTYALIN.) 
 
 Of the soluble ferments or enzymes diastase has been 
 longest known. It was first observed in 1814 by Kirchhoff that 
 germinating barley grains contain a principle which when 
 extracted from them by water is capable of converting starch 
 into sugar. The power at the time was considered to be due 
 to some property of the gluten of the grain, and was not put 
 down to a separate constituent of the barley. But little 
 notice was taken of the discovery for some years, but in 1823 
 Dubrunfaut repeated the observation and slightly extended it, 
 ascertaining that the transformation was most easily effected 
 at a temperature of 65° C, and that the sugar produced was 
 capable of alcoholic fermentation. In 1831 it was discovered by 
 Leuchs that saliva also possessed the power of transforming 
 starch into sugar. 
 
 These observations led the way to the work of Payen and 
 Persoz, who were the first to prepare diastase from the extracts 
 of germinated grain. In 1833 these observers steeped ground 
 germinating barley for some time in water, and after filtration 
 of the extract, precipitated firom it, by the addition of strong 
 alcohol, a white flocculent material which when dried and 
 redissolved was found to have the power possessed by the 
 original extract. Payen and Persoz extended their observations, 
 subsequently discovering the same body in oats, wheat, maize, 
 and rice during the process of germination and in the tubers 
 of the potato while they were in course of growth. 
 
 The name diastase was given by its discoverers to the 
 material precipitated by alcohol. 
 
16 DIASTASE (amylase, PTYALIN). [<-H. 
 
 The observation of Leuchs in 1831 was followed up in 1845 
 by Mialhe, who applied to saliva the same treatment as that 
 adopted by Payen and Persoz in the case of extracts of germin- 
 ated grain. He added to saliva absolute alcohol in excess, 
 and thereby produced, a precipitate which like the vegetable 
 diastase was found to possess a starch-transforming power. 
 
 He named this body salivary diastase, indicating its simi- 
 larity to, if not identity with, the body prepared by Payen and 
 Persoz. The name has fallen into disuse in works on physio- 
 logy, the term ptyalin having been generally adopted m its 
 place. 
 
 The power to saccharify starch was in the same year dis- 
 covered to be present in another animal secretion, that of the 
 mammalian pancreas. The discoverers were Bouchardat and 
 Sandras, and their work extended so far as to enable them to 
 claim that a variety of diastase can be extracted from that 
 organ. 
 
 Thus at comparatively early dates there were established 
 three sources of diastase, two of animal and one of vegetable 
 origin. For a long time these were regarded as the special 
 situations of the enzyme. Within the past three decades 
 however a far more widespread distribution has been established 
 both in the animal and the vegetable organism. 
 
 Dealing in the first place with investigations carried on in 
 the region of plant life, we find the subject taken up again in 
 1874 by von Gorup-Besanez, who found the enzyme in several 
 other varieties of germinating seeds. He was followed by 
 Kosmann in 1877, Baranetzky in 1878, and Krauch in 1879. 
 Owing to the investigations of this gi-oup of workers the presence 
 of diastase was established in the ordinary vegetative members 
 of the plant and in other resting bodies besides seeds. Thus 
 Kosinann and Krauch recognized its presence in the leaves and 
 shoots of the higher plants, and also in certain algae, lichens, 
 mosses and fungi, while Baranetzky found it both in buds and 
 potato-tubers. These extended observations led the latter 
 writer to suggest that it is universally present in vegetable 
 cells, so long as the latter are living. 
 
 In 1884 a very important research upon this subject was 
 
Il] DIASTASE (amylase, PTYALIN). 17 
 
 carried out by Brasse. He used the leaves of the potato, dahlia, 
 Jerusalem artichoke, maize, beet, tobacco, and castor-oil plant, 
 of all of which he made watery extracts. On the addition of 
 alcohol to these extracts a flocculent precipitate fell, which he 
 collected and dried. On adding some of this powder to starch- 
 paste the latter became liquefied and converted into sugar. 
 The amount of sugar formed was then ascertained by ob- 
 serving the weight of cuprous oxide which it formed when it 
 was boiled with Fehling's solution. 
 
 Brasse guarded his experiments from eiTor due to the 
 possible presence of micro-organisms by carrying out his diges- 
 tions in the presence of traces of chloroform, which prevents 
 the growth of such organisms. 
 
 The investigations of many writers during more recent 
 years have gone far to establish the truth of Baranetzky's 
 suggestion, while they have extended our knowledge of diastase 
 so far as to show that at least two varieties of it exist in the 
 vegetable organism. 
 
 The first of these is the more widespread; it apparently plays 
 the principal part in the transport of starch from place to place 
 about the plant and is most commonly met with in the vegeta- 
 tive organs. The other is almost if not entirely confined to 
 germinating seeds, particularly those of the Graminese, and is 
 probably the body first prepared by Payen and Persoz. These 
 two varieties have been called for purposes of distinction, 
 diastase of translocation and of secretion respectively. 
 
 Diastase of translocation. 
 
 This enzyme is the more widely distributed of the two, 
 not only occurring in the vegetative organs but being present 
 in the seed during the development of the embryo. Its 
 action can be studied upon grains of starch in the vegetable 
 tissues themselves, or it can be extracted from the tissues by 
 water or by glycerine, and its behaviour tested by mixing such 
 an extract with either a thin starch-paste or a solution of soluble 
 starch. In the former case the diastasic solution gradually 
 dissolves the starch grain, sometimes firom the outside only, 
 
 G. F. 2 
 
18 DIASTASE {AMYLASE, PTTALIN). [CH. 
 
 sometimes from the interior, but it never gives rise to any pitting 
 or corrosion, so that the shape and transparency of the grain 
 remain unaffected almost to the time of its disappearance. When 
 the diastase is allowed to work upon a thin starch-paste, made 
 by mixing about 1 gramme of starch with 100 c.c. of boiling 
 water, the opalescent liquid slowly becomes more and more 
 transparent till it is quite limpid ; it then gradually undergoes 
 almost complete conversion into sugar. If a true solution of 
 starch is substituted for the starch-paste the transformation 
 into sugar is much more rapid. The details of the action will 
 be discussed in a subsequent chapter. 
 
 The action of this form of diastase takes place most 
 energetically at a temperature of 45° — 50° C. 
 
 Diastase of translocation has been described as oc- 
 curring in leaves, shoots, and certain reserve-organs. In 1889 
 it was found by Kjeldahl to be present in the ungerminated 
 grains of barley, a discovery confirmed by Brown and Morris 
 the next year. Kjeldahl showed that while the extract of 
 ungerminated barley was able to convert solutions of soluble 
 starch rapidly into sugar its activity on thin starch-paste was 
 but slight. Brown and Morris studied the formation and 
 distribution of the enzyme during the process of ripening or 
 maturation of the barley grain. The chief place of its occur- 
 rence is the bulk of the endosperm, in which it may be found 
 during its development. It is always most plentiful in the part 
 of the endosperm which is nearest the young embryo, and it ap- 
 pears to prepare the material for the nutrition of the latter as it 
 increases in size. It thus appears in the barley grain at a much 
 earlier period than the other variety of diastase, to be described 
 later, which is only found after maturation and indeed at the 
 commencement of germination. The translocation-diastase 
 makes its appearance at a very early period in the development 
 of the endosperm, and gradually increases until that develop- 
 ment is completed but the grain not ripened. Brown and 
 Morris estimated carefully the amounts of diastase present in 
 barley grains at three stages of their formation ; — (i) when the 
 endosperm was half developed ; (ii) when the development had 
 reached two- thirds of its total amount; (iii) when it was 
 
II] DIASTASE (amylase, PTYALIN). 19 
 
 complete. The amount of the diastase was ascertained by 
 finding what amount of soluble starch could be converted into 
 sugar by the ground-up substance of the same number of barley 
 grains acting for the same time and under the same conditions. 
 The relative quantities were found to be in the proportion of 
 4'4 : 7'8 : 9'7, showing that as the embryo grows the quantity 
 of diastase found in its vicinity increases pari passu with the 
 growth, confirming the idea that the enzyme makes its appear- 
 ance with a view to the formation and nutrition of the embryo. 
 
 Not only does this variety of diastase appear in the seed 
 outside the embryo, but when the latter begins to grow during 
 germination the same diastase is found to be developed in its 
 cells, not so much in the cotyledon as in the plumule and 
 radicle, the parts namely in which growth is most vigorous. 
 Even at this early stage Baranetzky's idea of diastase as a 
 necessary constituent of every vegetable cell seems to be 
 confirmed. The function of this amount of diastase is not 
 connected with the absorption of sugar from the endosperm of 
 the parent but with translocations in the body of the embryo 
 itself. 
 
 The diastase of leaves and shoots has much in common with 
 that of ungerminated grain and appears to be the same variety. 
 The disappearance of starch fi?om the leaves of plants during 
 the hours of darkness, after its formation and deposition in the 
 chloroplastids during sunlight, has always been associated with 
 the presence of the enzyme, though no detailed investigations 
 into the conditions of the disappearance were made till recently. 
 In 1890 the existence of diastase in foliage leaves was contro- 
 verted by Wortmann, who, in consequence of failure to extract 
 it in many cases, asserted that the conversion of this starch 
 into sugar was effected by the immediate action of the proto- 
 plasm of the living cell itself, and not by the agency of an 
 enzyme. This statement led to the reinvestigation of the 
 subject of the starch-transformations in leaves by Vines and by 
 Brown and Morris, with the result of fully establishing the 
 accuracy of the observations of Brasse and other earlier writers. 
 At about the same time an elaborate paper by Krabbe was 
 published which showed that the visible mode of dissolution of 
 
 2—2 
 
20 DIASTASE {AMYLASE, PTYALIN). [CH. 
 
 the starch-granule within the cell corresponds to that observed 
 when it is treated with diastase outside the plant. 
 
 Vines carried on his work largely with the leaves of grasses, 
 which he extracted with water. Some of his extracts were 
 filtered, and others used while still turbid. The latter were 
 found to be more active than the former, a fact which suggests 
 that Wortmann lost a good deal of the diastase contained in 
 the leaf by the operation of filtration, the enzyme for some 
 reason not having fully passed into solution. Vines was able 
 to prepare a solution of diastase from the grass leaves which 
 exhibited a fair degree of activity. He suggests that though 
 the amount of enzyme at any given moment may be small, 
 there is a constant secretion or formation of it, so that the total 
 amount produced is sufficient to cause the conversion of all the 
 starch translocated during the night. 
 
 The work of Brown and Morris appeared in 1893 and is the 
 fullest and most detailed that has at present been carried out 
 on the diastase of foliage leaves. 
 
 At the outset of their investigations they were able to 
 explain the apparent absence of diastase from many leaves which 
 was supposed to be indicated by the very small diastasic power 
 of filtered extracts. They dried leaves of various plants in air 
 at a temperature of 40° to 50° C. and reduced them to a fine 
 powder. An extract of 10 grammes of such powder, the leaf of 
 Helianthus tuberosus being used in the experiment quoted, was 
 made by steeping it for some time in water to which a trace of 
 chloroform was added. Finally the extract was filtered. Two 
 digestions of solutions of soluble starch were then carried out, 
 the extract described being used in one of them, while 10 
 grammes of unextracted powdered leaves were employed in the 
 other. _ The relative diastasic activity in the two cases was 
 found to be in the proportion of 7 : 1 in favour of the powdered 
 leaves, the estimation being made by observation of the cupric- 
 reducing power of the products of the digestions. 
 
 The authors advance two reasons to explain the difference, 
 the first being the great resistance which protoplasm often 
 offers to the separation of the enzyme, and the second the action 
 of the tannin so frequently present in leaves, which renders 
 
n] DFASTASE (amylasi:, ptyalin). 21 
 
 it impossible in many cases to extract the enzyme from the 
 cells. 
 
 Brown and Morris investigated the leaves of 34 species of 
 plants belonging to a great many different Natural Orders and 
 found that all contained a measurable amount, though the 
 quantities differed very considerably. The Leguminosse were 
 especially rich, the common Pea (Pisum sativum) being ex- 
 tremely conspicuous among them; its diastasic activity was 
 nearly one-half that of malt. 
 
 Though exact quantitative determinations of the enzyme were 
 hardly if at all possible, comparative estimations were found 
 easy to make. The method of measuring the amount of starch 
 conversion which an extract is capable of carrying out under 
 certain standard conditions and in unit time has been shown 
 by Kjeldahl to yield accurate results, and from it he formulated 
 a "law of proportionality" which may be expressed thus'; 
 " providing a starch-transformation is not allowed to fall below 
 a cupric-reducing power of k 25 to 30 for the mixed products of 
 hydrolysis, then under identical conditions of time, temperature, 
 and concentration, the cupric-reducing power is proportional to 
 the amount of diastase originally present."^ 
 
 Brown and Morris further showed not only that diastase is 
 present in foliage leaves, but that it is possible to prove that its 
 amount varies considerably from time to time, under differences 
 in their environment. This point will be referred to more 
 fully in a subsequent chapter, but for the present it will 
 suffice to say that there is a considerable formation during 
 darkness and a conspicuous diminution during bright sunshine. 
 
 The work of Brown and Morris receives material support 
 from some investigations published' in the same year by 
 St Jentys. He has found that the diastase is formed only in 
 small quantities as it is required. When an aqueous extract of 
 leaves is prepared, it usually contains tannin, which causes the 
 precipitation of the starch. The latter is only acted on with 
 
 1 Brown and Morris. " On the Chemistry and Physiology of Foliage 
 Leaves," Joum. Chem. Soc. Trans. 1893, p. 637. 
 
 ^ K represents the apparent percentage amount of dei^trose which a substance 
 contains as determined by its ouprio reduction. Thus k 25 means that the 
 reducing power of the substance is one-quarter that of dextrose. 
 
22 DIASTASE {amylase, PTYALIN). [cH. 
 
 difficulty under such conditions. Moreover the tannin pre- 
 cipitates the diastase itself. 
 
 Besides occurring in the actively vegetative parts of the 
 plant this form of diastase is found also in the reservoirs of 
 starchy material, and is particularly conspicuous there at the 
 time when a call is made upon the reserve supply, as happens 
 for instance when growth recommences. 
 
 An investigation of the potato by Prunet has brought to 
 light several very interesting facts connected with the formation 
 and distribution of this variety of the enzyme in the tissue of 
 the tuber. On the onset of the germination of that organ it is 
 possible to notice that the buds near its apex develop more 
 rapidly than those situated lower down. Prunet separated the 
 different regions of the sprouting potato, and made watery 
 extracts of each. He ascertained the diastasic powers of the 
 several extracts thus prepared by precipitating the enzyme with 
 excess of alcohol, filtering, and dissolving the precipitate, and 
 then allowing it to act upon thin starch-paste. His results are 
 comparative merely and perhaps only approximately accurate, 
 as he used for an indication of the progress of the digestion 
 simply the colour obtained by the addition of iodine. As the 
 starch in such a digestion disappears, this colour, which is at 
 first blue, changes in the direction of purple, then becomes red, 
 and finally is not developed. The method is not so accurate as 
 the measurement of the sugar produced, but it yields reliable 
 comparative results. He found that while diastase is present 
 in all parts of the tuber during the progress of its germination 
 it is in greatest amount near the points where active growth is 
 proceeding. The commencement of the growth of the young 
 shoots is accompanied by a production of the enzyme in their 
 immediate neighbourhood, and as they become longer the 
 formation of the diastase can be traced farther and further 
 back. Prunet estimated at the same time the sugar existino- 
 in the same regions of the potato and found a correspondence 
 between the amount of the two bodies. 
 
 In 1893 the writer carried out a series of investigations on 
 the processes of germination of the pollen grains of various 
 plants, in the course of which the presence of this form of 
 
Il] DIASTASE (AMYLASH, PTYALIN). 23 
 
 diastase was recognized in those of several species. Starch was 
 found to be present in many of the pollens examined and to 
 occur also in the tissues of the styles down which the tubes 
 penetrate in the process of the germination of the pollen grain. 
 The diastase was looked for by similar methods to those already 
 described and was found in most cases. 
 
 The same enzyme was discovered in the tissue of the style 
 in some of the flowers examined. 
 
 The quantity of diastase which can be extracted from pollen 
 varies considerably from time to time. At the onset of germi- 
 nation of the grain it is usually considerably increased, and it 
 appears to accompany the tube as the latter elongates, which 
 suggests a formation not only in the grain but in its tube also. 
 As the pollen loses its power of germination with the increase 
 of age, it loses at the same time the diastase which it contains. 
 Thus, as in the potato, it is possible to trace clearly the in- 
 fluence which the enzyme exerts on the process of germination. 
 
 The statement of Kosmann that diastase can be extracted 
 from fungi was for a long time unsupported. It seems some- 
 what unlikely at first sight that it should occur there, as these 
 plants do not contain starch. It has however been shown that 
 glycogen is very frequently to be met with in them, and as 
 glycogen resembles starch so very closely this a priori difficulty 
 disapjDears. 
 
 In 1883 some investigations were made by Duclaux into the 
 behaviour of Aspergillus niger under different conditions, and 
 he ascertained that this fungus has the power of saccharifying 
 starch solutions in the same way as germinated barley. More 
 extended observations were carried out by Bourquelot ten years 
 later, and the latter botanist was successful in proving that 
 diastase can be extracted from a culture of the fungus at the 
 time when it is in full fructification. The estimation of the 
 amount is difficult if not impossible, for as we shall see later 
 Aspergillus niger contains other enzymes, one of which acts 
 upon the sugar to which the starch gives rise, decomposing it 
 during the progress of the experiment. 
 
 In 1895 Bourquelot and Ht^rissey demonstrated the presence 
 of the same diastase in the tissue of Polyporus sulphureus It 
 
24 DIASTASE (amylase, PTTALIN). ICH. 
 
 exists in this fungus, as it does in Aspergillus, side by side with 
 other enzymes, but can readily be extracted by expressing the 
 iuice of the large thallus and precipitating it with twice its 
 volume of 95 "/o alcohol. The diastasic power can be demon- 
 strated by using the juice without this treatment. 
 
 According to De Bary, diastase can be extracted from the 
 Plasmodium of Aethalium, a Myxomycete, and Stone states that 
 it is present in Taka, a preparation of Eurotium oryzae. 
 
 Morris and Wells have shown that diastase can also be 
 extracted from yeast cells, and many observers, especially 
 Lauder Brunton and MacFadyen, and Wortmann, have found 
 it to be excreted by bacteria. Its presence in these organisms 
 seems not to be constant, but to depend upon the medium in 
 which they are cultivated. 
 
 Hansen has detected diastase in the latex of Ficu-s elastica^ 
 Garica papaya, and Papaver somniferum. It can be separated 
 from the latex by precipitation with alcohol. 
 
 The existence of diastase in tissues containing starch or 
 glycogen is not surprising when we regard its importance in 
 the digestion of these carbohydrates. It occurs, however, in 
 other tissues in which they are very rarely present, if at aU. 
 In the case of the sugar beet, in which the reserve materials 
 take chiefly the form of cane-sugar, Gonnermann states that he 
 prepared from crushed germinating roots an enzyme which 
 digested starch-paste and amylodextrin. Baranetzky detected 
 diastase also in the carrot and the turnip. 
 
 Diastase of secretion. 
 
 This variety of diastase is especially connected with the 
 process of germination and can be most favourably studied in 
 the grains of grasses. If the progress of the development of the 
 embryo in the germinating seed is studied with the aid of 
 the microscope, changes in the starch-containing cells of the 
 endosperm can be observed at a time when the primary radicle 
 has attained a length of about 2 mm. The walls of the cells 
 become broken down, and the starch grains at this period begin 
 to undergo a process of dissolution which is entirely different 
 from that which can be observed in other regions of the plant. 
 
n] DIASTASE (amylase, PTYALIN). 25 
 
 Instead of gradually dissolving without alteration of their 
 shape, they show small indentations or pittings on their 
 surfaces. These gradually become deeper and wider, and the 
 grain is soon broken up into irregular pieces, while the 
 separate laminae tend to split off from each other. This process 
 of erosion is followed later by the gradual disappearance of the 
 several portions of the grain. 
 
 The formation and behaviour of the diastase which is the 
 instrument in effecting this change has been studied especially 
 by Brown and Morris and by Haberlandt, who carried out their 
 work chiefly on different cereals, principally barley and rye. 
 They agree in attributing its origin to a true process of secretion 
 carried out by the embryo, which thus prepares the way for its 
 own nutrition, pouring enzymes, of which diastase is one, iiito 
 the tissue of the endosperm by the side of whibh the embryo 
 rests. 
 
 The embryo in the grasses lies at one end of the grain and 
 the portion of it which is in contact with the tissue of the 
 endosperm is the so-called scutellum, a mass of parenchymatous 
 tissue whose morphological nature has been much disputed, 
 but which is probably a specially differentiated portion of the 
 cotyledon. Over the surface of the scutellum, abutting on the 
 cells of the endosperm, there is a well-marked external layer or 
 epithelium. This is composed of closely set columnar cells, 
 about three times as long as they are broad, which are arranged 
 with their long axes at right angles to the surface. The 
 cell- walls are delicate and thin, and made of unchanged cellulose. 
 Each cell contains a finely granular protoplasm and a fairly 
 large nucleus. Prior to the researches of Brown and Morris 
 the functions of this epithelium were thought to be confined to 
 the absorption of digested reserve-materials, passing from the 
 endosperm to the embryo. These observers have shown that 
 they are also the seat of the formation of one, if not two 
 enzymes, the first of which is the diastase under consideration, 
 and the other is an agent in the preliminary destruction of the 
 cell-walls. A few hours after placing the grains of barley under 
 conditions favourable for germination a marked change takes 
 place in the contents of the cells. The protoplasm becomes 
 
26 DIASTASE {AMYLASJi, PTTALIN). [CH. 
 
 visibly coarser in stiacture and the granules increase in size 
 and number, becoming so prominent in the cell that the nucleus 
 is almost hidden. These changes are complete in about 1 — 2 
 days, and the condition of the cells remains thenceforward 
 unaltered as long as germination proceeds. The tissue of the 
 endosperm above the scutellum becomes depleted of its starch, 
 while the coarse granularity of the epithelium persists, and the 
 tissues of the embryo itself, particularly the cells of the scutellum, 
 become more and more charged with newly formed starch 
 grains. When the reserve store of starch in the endosperm 
 has been thus absorbed, the epithelial cells again change in 
 appearance, losing their granularity and becoming transparent 
 as at first. There is, however, an important difference between 
 their first and their final condition, as in the latter the nucleus 
 is no longer to be found, indicating that the cells have finished 
 their work and are on the way to disintegration. The process 
 of germination thus shows that as it commences, active 
 metabolism goes on in the epithelium, synchronous with the 
 dissolution of the cell-walls and the transformation of starch ; it 
 continues as long as starch is being digested, and ceases when 
 that process is over. The obvious conclusion to be drawn from 
 the observations is that these metabolic changes are concerned 
 in the elaboration of the secretion, which causes the disap- 
 pearance of the reserves of cellulose and starch; that is, that 
 ' the epithelium is primarily a glandular structure. 
 
 That this conclusion is not merely hypothetical has been 
 demonstrated by careful experiments, carried out by Brown and 
 Morris, upon the artificial nutrition of barley embryos removed 
 from germinating grains. Such embryos were carefully dis- 
 sected out and their scutellar surfaces freed from adhering 
 matter by careful stroking by means of a fine camel-hair pencil 
 moistened with dilute sugar solution. They were then placed 
 upon a thin layer of moist barley starch, so that their scutella 
 rested in contact with the latter. Within a very short time 
 transitory starch grains could be detected in the parenchyma of 
 the scutella, and microscopic examination of the starch grains 
 in contact with the surface of the epithelium showed them 
 to be corroded and in process of disintegration. In further 
 
II] DIASTASE {aMYLASM, PTYALIN). 27 
 
 experiments the excised embryos were partially embedded in 
 a '5 per cent, gelatin solution in which small quantities of 
 barley starch had been suspended. The embedding was carried 
 out just as the gelatin solution was on the point of solidifying, so 
 that the curved surface of each scutellum was in close contact 
 with the cultivation medium. After a short time small pieces 
 of the gelatin cut out from the mass just below the growing 
 embryo showed the starch grains affected as before. Besides 
 barley starch, the diastase that passed out into the gelatin was 
 found to be capable of corroding and dissolving the starches of 
 wheat, rice, and maize, but to have no evident action on those 
 of the potato and kidney -bean. 
 
 Further proof of the connection between the secretion of 
 the diastase and the epithelium cells was afforded by paralysing 
 the latter by exposing them to chloroform vapour. After such 
 treatment they had no action on the starch. 
 
 Freshly dissected embryos of resting barley were sub- 
 sequently rubbed down in a mortar with a little chloroform 
 water and allowed to stand for 24 hours. The extract was 
 then filtered and added to an equal volume of a 2"5 °/o solution 
 of soluble starch and digested for an hour at a temperature of 
 30° 0. It was found to have no diastasic action. At the same 
 time some more embryos excised from grains of the same 
 sample of barley were cultivated for four days upon appropriate 
 culture media. They were then treated exactly as the first ones 
 and the extract was found to have under similar conditions 
 considerable power of converting the starch into sugar. By 
 both microscopical and chemical methods Brown and Morris 
 therefore showed t]\& formation of diastase to take place during 
 germination, and further, that when formed it slowly diffused 
 out of the epithelium. 
 
 These observers advanced further proof of the formation of 
 the diastase by the epithelium by carefully removing the latter 
 from the rest of the scutellum by gentle scraping. They found 
 that scutella so denuded of their covering were capable of 
 absorbing soluble carbohydrates if placed upon them, but they 
 had lost their power of corroding starch grains. Extremely 
 thin sections of the epithelium, taken in a plane tangential to 
 
28 DIASTASE {amylase, PTYALIN). [CH. 
 
 the surface of the scutellum, and placed upon the mixture of 
 gelatin and starch, exercised a corrosive and solvent action on 
 the starch grains. 
 
 This secretory action of the epithelium is only manifested 
 by grains in the process of germination. The power of forming 
 diastase seems only to be acquired when the barley is quite 
 ripe. Barley taken fresh from immature ears shows the gradual 
 acquirement of the structures of the grain as already described, 
 but the scutella of such unripe grains have no action on starch. 
 The diastase which we have already seen to be present in 
 ungerminated grain is located in the body of the grain itself 
 and not in the scutellum. Moreover by its general behaviour 
 it is proved to be of the translocation variety. 
 
 The increase in the amount of secretion diastase during the 
 progress of germination is very marked. Comparing embryos 
 after four days' germination with others taken three days later, 
 the diastasic activities of the two were found by Brown and 
 Morris to be as 16 : 66. Petit says that no diastase is found 
 before the fourth day of germination. 
 
 Haberlandt has put forward the view that the diastase of 
 secretion is not elaboi-ated only by the scutellum but that the 
 so-called aleurone-layer of the endosperm is also concerned in 
 its production. This layer is three or four rows of cells in 
 depth, and the constituent cells have thick and somewhat 
 cuticularised walls. It is situated in the peripheral portion of 
 the grain, just underlying the wall, which is composed of the 
 fused pericarp and seed-coat. The contents of the cells are 
 chiefly aleurone grains with a little oil, embedded in the 
 protoplasm. Each cell contains a well-defined nucleus. The 
 aleurone-layer covers the whole of the endosperm but becomes 
 much less conspicuous in the neighbourhood of the embryo, 
 where it is reduced to a single layer of cells. 
 
 Haberlandt's experiments were carried out upon rye, from 
 which he thinks histological and chemical evidence is forth- 
 coming to prove that in gerniination secretion of diastase takes 
 place in these cells as well as in those of the scutellum. He 
 states that during germination corrosion of the starch grains 
 first appears between the scutellum and the aleurone-layer on 
 
Il] DIASTASE (aMYLASF., PTYALIN). 29 
 
 the sides of the rye grain, and that as the process goes on 
 diastasic action upon the starch takes place earlier in the cells 
 underlying the aleurone-layer than in those of the central 
 portion of the endosperm. The cells of the aleurone-layer 
 assume the peculiar granular appearance of secreting cells, and 
 project in papilla fashion into the interior of the endosperm. 
 He removed pieces of the integument, which included the 
 aleurone-layer, from rye grains which had germinated to such a 
 point that the connecting tissue between the latter and the 
 endosperm had been broken down, leaving the aleurone-layer in 
 close connection with the seed-cat and pericarp. He then 
 washed them with a brush wetted with a weak sugar solution, 
 and placed them on moist filter-paper with the aleurone-layer 
 upwards. Upon such preparations a small quantity of starch 
 suspended in water was then carefully placed, and the whole 
 kept at a temperature of 18 — 20° C. Corrosion of the starch 
 gfrains took place within 24 hours. 
 
 Like the cells of the epithelium of the scutellum, those of 
 the aleurone-layer exhibited no power of corroding starch until 
 the onset of germination. 
 
 In their paper published in 1890 Brown and Morris opposed 
 the views of Haberlandt, and maintained the opinion that the 
 aleurone-layer does not play the part he suggested. In a more 
 recent investigation carried on during 1897 Brown and Escombe 
 made a very exhaustive series of experiments upon this point,, 
 and after carefully eliminating the influence of the action 
 of micro-organisms and of the possible presence of translocation 
 diastase in the starch-containing cells, they came to the con- 
 clusion that this layer does possess a secretory function, and 
 that it forms a certain amount of diastase, but from the mode, 
 of its action on starch grains they considered the latter to be 
 the translocation variety of this enzyme. The pitting and 
 erosion of the grains of starch, so characteristic of the action of 
 scutellar diastase, were not seen, but the process was one of 
 more regular solution, accompanied in some cases by the 
 splitting of the granule without previous pitting. 
 
 The aleurone-layer according to these observers is the chief 
 seat of the formation of another enzyme, cytase, which dissolves 
 
30 DIASTASE {amylase, PTTALIN). [CH. 
 
 the cell-walls, and which will be treated of in a subsequent 
 chapter. Like the scutellar epithelium therefore it secretes two 
 enzymes, cytase and diastase. Comparing the two regions it 
 appears that cjrtase is most prominent in the secretion of the 
 aleurone-layer, diastase in that of the scutellum. 
 
 The results of Haberlandt, Brown and Morris, and Brown 
 and Escombe, have been confirmed by the investigations of 
 other writers, especially Grliss, Hansteen, and Puriewitsch. 
 Grtiss holds further that the depletion of the starch-containing 
 cells below the aleurone-layer is partly due to residual diastase 
 existing in them. 
 
 The differences between the two varieties of vegetable 
 diastase have been studied by Lintner and Eckhardt, who have 
 compared them as they exist in raw and in germinated barley. 
 In their experiments they prepared the diastase of secretion 
 from extract of malt, of which it is the characteristic enzyme. 
 They made extracts of malt and of barley of such strengths 
 that both solutions had equal power of converting starch into 
 sugar and wore therefore comparable in the other peculiarities 
 of their action. Causing such extracts to act upon solutions of 
 soluble starch at several different temperatures, they were able 
 to construct a curve for each which showed how different 
 temperatures modified their behaviour. The curves were drawn 
 so that their abscissae represented the temperatures, and they 
 erected on the abscissas ordinates proportional to the cupric- 
 oxide-reducing power which was found to be possessed by the 
 solution at the end of the experiment. The two sets of curves 
 differed materially. Malt diastase showed itself to possess an 
 optimum point, or point of greatest activity, at 50° — 55° C. ; 
 barley diastase, which is the translocation variety, was most 
 powerful at 45° — 50° C, an average of about 5° C. lower. At 
 a temperature of 35° C. they were equally active ; the barley 
 diastase at 4° C. had as much power of starch conversion as the 
 malt diastase had at 14"5° C. 
 
 Kjeldahl also has published some comparative experiments 
 upon the two varieties. He confirms Lintner's results as to 
 the optimum point of the activity of secretion-diastase, but 
 makes the point at which it is destroyed by heat a little higher. 
 
[l] DIASTASE (amylase, PTYALIN). 31 
 
 The two vegetable diastases may be thus compared : — 
 
 (1) Translocation diastase. This dissolves stai-ch grains 
 without corrosion ; has a very slow action on starch-paste, 
 though it readily converts soluble starch into sugar; works 
 best at a temperature of 45° — 50° C. ; is much more active at 
 a low temperature than secretion diastase. 
 
 (2) Diastase of secretion. This corrodes starch grains and 
 disintegrates them before solution; rapidly liquefies starch-paste; 
 works most advantageously at a temperature of 50° — 55° C. 
 It will withstand heating to 70° C. without destruction. 
 
 Diastase is secreted by some of the lowliest plants as well 
 as those of higher organisation. In the latter the presence of 
 the first variety is connected with the transformation of starch 
 with a view to the transport of the latter from one part of the 
 plant to another, and the enzyme always remains within the 
 cells. Secretion diastase on the other hand is formed by a 
 special glandular tissue, and is excreted from this tissue into 
 a region where it may prepare nutritive substances to be 
 subsequently absorbed by the cells which in the first place 
 prepared the enzyme. The barley embryo in fact digests and 
 absorbs the endosperm on which it grows parasitically. 
 
 When we pass from the higher to the lower forms we can 
 recognize similar behaviour on their part. Diastase has been 
 observed to occur in several bacteria, from some of which it is 
 possible to extract it. These organisms do not uniformly 
 contain it, nor is it present at all times in those which do 
 secrete it. 
 
 Lauder Brunton and MacFadyen isolated it from Klein's 
 scurf-bacillus and the Welford bacillus, but these microbes only 
 secreted it when cultivated upon starch-paste. If the cultiva- 
 tion-medium was a meat extract, no diastase was produced. 
 Wortmann showed in 1882 that certain bacteria exert diastasic 
 powers on starch through excreting an enzyme when starch 
 grains are their only available food. 
 
 In Bacillus inesentericus vulgatus diastase has been found 
 to exist side by side with four other enzymes. The cholera 
 bacillus of Koch can liquefy starch-paste when cultivated upon 
 it, and some of the starch is transformed into sugar. It 
 
32 DIASTASE (AMYLASJE, PTYALIN). [CH. II 
 
 apparently does this by the action of its own protoplasm, as 
 Wood, who observed the phenomenon, says no enzyme is secreted, 
 or can at any rate be extracted from the cells. On com- 
 paring these results with the others already quoted it seems 
 probable that his methods of extraction were defective, as it is 
 difficult to see how the protoplasm of the bacillus and the 
 starch-paste could come into contact, on account of the cell- 
 wall clothing the organism. Protoplasm no doubt possesses a 
 starch-transforming power, but it must come into contact with 
 the starch in order to exercise it. This property of protoplasm 
 will be more fully considered in a subsequent chapter. 
 
 Pfeffer was able to determine the secretion of diastase by 
 Bacterium megatherium, and found that the amount this organism 
 prepares depends to a large extent upon the amount of cane- 
 sugar present in the culture medium. He extended his re- 
 searches in this direction to several of the mould fungi, and 
 found the same influence was exerted by the environment. 
 Penicilium glaiicum ceased to form diastase at all in the 
 presence of 10 per cent, of cane-sugar, and even when the latter 
 only amounted to 1"5 per cent, the starch was only slightly 
 attacked. Aspergillus niger behaved differently, producing 
 diastase even in the presence of 30 per cent, of cane-sugar. 
 Different sugars have different powers of checking the secretion 
 of the enzyme, cane-sugar and dextrose being more effective in 
 this direction than maltose. As the former are the sugars 
 which have the greatest nutritive value for the organisms this 
 result appears to indicate that the formation of diastase in 
 these plants only takes place under the stimulus of a semi- 
 starvation. This is rendered the more' probable in that if the 
 plants are excited to more vigorous growth by being supplied 
 with peptone, a greater proportion of sugar is necessary to 
 inhibit the production of diastase. 
 
33 
 
 CHAPTER III. 
 
 ANIMAL DIASTASE. 
 
 The existence of a diastasic property in animal cells was 
 first indicated in 1831 by Leuchs, who discovered saliva to 
 have the power of converting starch into sugar. He did not 
 take the matter further, and it was not till 1845 that the 
 enzyme was prepared from the secretion of the salivary gland. 
 This was effected by Mialhe, who obtained it by precipitation of 
 saliva with excess of absolute alcohol. It was prepared in a 
 purer form by Cohnheim in 1863 by a method that will be 
 described in a subsequent chapter (page 45). 
 
 In 1845 Bouchardat and Sandras prepared the same 
 enzyme from the secretion of the pancreas. 
 
 Further observations, made chiefly within the present decade, 
 show that diastase is as widely distributed in the animal as in 
 the vegetable body. Indeed though two varieties have not been 
 indicated so clearly as in the latter case, we find two modes of 
 action much resembling those of the translocation and secretion 
 varieties found in plants. Thus the secretions of the salivary 
 gland and of the pancreas are poured out into special regions 
 of the alimentary canal to convert into a diffusible form the 
 starch and glycogen of the animal's food, while in the cases of 
 tissues containing the latter of these carbohydrates, an enzyme 
 is produced which acts only on the glycogen in the interior 
 of the cell, and from the latter the diffusible products are 
 removed by a species of translocation. 
 
 Diastase is stated by Rohmann to exist in small amount in 
 the succus entericus or secretion of the small intestine, though 
 
 6. F. 3 
 
34 ANIMAL DIASTASE. [CH. 
 
 in much smaller amount than in either saliva or pancreatic 
 juice. Hamburger has made a similar observation. Comparing 
 the relative diastasic powers of saliva, pancreatic juice, and the 
 secretion of the small intestine, by observing their power of 
 forming cupric-oxide-reducing sugar from solutions of starch, 
 he finds that taking the reducing power of glucose as unity for 
 purposes of a standard, the products of the action of the three 
 secretions gave reducing powers of -31, '36, and -26 respectively. 
 This amount of reducing-sugar was produced by saliva and by 
 pancreatic juice in an hour or less, while it was only arrived at 
 under the influence of intestinal juice in rather longer than a 
 day. The last-named secretion therefore contains but a very 
 small amount of diastase. 
 
 The existence of the enzyme in the intestinal juice of the 
 sheep was proved by Pregl in 1895. 
 
 The other variety which in function at least corresponds to 
 translocation diastase is found in almost all tissues and fluids 
 of the body, though it is often present in very small quantities 
 and is difficult to extract. Different animals however show 
 considerable differences in these respects. 
 
 The liver and the muscles are the chief storehouses of 
 carbohydrate reserve-material in the body, where it takes 
 the form of large quantities of glycogen, which vary con- 
 siderably from time to time. Carbohydrates leaving the 
 liver are found to be in the form of sugar, most probably 
 glucose, and the conversion of glycogen into the latter must 
 therefore take place within the organ. Very conflicting state- 
 ments have been made as to the possibility of preparation of 
 a soluble enzyme from the liver cells, but there seems to be 
 a gradually accumulating body of evidence in favour of its 
 existence. 
 
 Leaving that question for the moment and turning to other 
 body fluids in which the existence of diastasic enzymes would 
 seem less probable than in saliva and the pancreatic and 
 intestinal juices, we find a certain amount of evidence of their 
 existence in blood and lymph. Bial in 1893 stated that diastase 
 could be extracted from the serum of both these fluids but not 
 from their corpuscles. Rohmann about the same time made the 
 
Ill] ANIMAL DIASTASE. 35 
 
 observation that if glycogen is injected into the lymphatics of 
 an animal and the lymph from the thoracic duct then allowed to 
 run into alcohol, the percentage of sugar in the Ijrmph is found 
 to have risen. The glycogen in his experiments was injected 
 in suspension in a small quantity of solution of common salt, 
 about "G'/o in strength (normal saline solution). The salt 
 solution produced no effect when injected alone. Rohmann 
 also found diastase in blood, existing side by side with other 
 enzymes, but being present only in relatively small amount. 
 Dastre also found diastase in lymph, which he says contains 
 •097 parts of glycogen per 1000. If the lymph is examined after 
 standing 24 hours, the glycogen has disappeared. In the paper 
 already quoted Hamburger also states that an enzyme capable 
 of producing sugar exists in the blood. He speaks of it as giving 
 rise very slowly to a sugar having a reducing power greater 
 than that formed by saliva. It is probable, as he points out, 
 that this sugar is not the product of the action of diastase 
 alone, but that the body formed by the latter is further changed 
 by another enzyme also present in the blood. He shows clearly 
 however that blood has the power of transforming starch into 
 sugar, which can only be due to diastase. 
 
 Foster has found that pericardial, pleural, and peritoneal 
 fluids have the power of converting starch into sugar, and 
 Grohe says that chyle behaves similarly. According to Cohn- 
 heim and Bechamp fresh filtered urine has the same power, 
 containing enough diastase to allow the enzyme to be obtained 
 from it by precipitation. Its presence in the urine probably 
 indicates that it is in process of being excreted from the blood, 
 as the amount is increased after meals. Foster says it can be 
 prepared from the natural deposits of urates, with or without 
 previous washing with alcohol. 
 
 The diastase which is present in saliva, pancreatic juice, and 
 succus entericus, is like that of the germinating barley grain, 
 a secretion by a definite tissue. Saliva in mammals is formed 
 in certain structures, known as salivary glands. There are three 
 pairs of these in the body, the parotids situated on the sides of 
 the face, in front of the ears, the submaxillary, just below- the 
 base of each inferior maxilla, and the sublingual, placed along 
 
 3—2 
 
36 ANIMAL DIASTASE. [CH. 
 
 the floor of the mouth between the tongue and the gums of 
 the lower jaw. Each is composed of a number of tubes opening 
 into ducts which ultimately coalesce to form a single large 
 outlet through which the whole secretion of the gland flows. 
 The tubes are much convoluted and bound together into lobes 
 by connective tissue, so that the whole gland forms a solid mass 
 of small size. The act of secretion is carried out in the 
 ultimate tubes or alveoli of the gland. Each of these consists 
 of a transparent basement membrane, on the inner face of which 
 the secreting cells are placed. There is a very small lumen or 
 canal in the centre, formed by the free margins of the cells. 
 Each alveolus is bathed by the lymph which exudes from a 
 close plexus of blood capillai-ies, situated in the connective 
 tissue which is outside the basement membranes and which 
 helps to bind the whole gland together. The cells thus receive 
 supplies of lymph at their bases, while the secreted saliva is 
 poured out from them into the delicate canal which is the 
 centre of the alveolus. Each gland is supplied with a double 
 set of nerve fibres, one from the cerebro-spinal, the other from 
 the sympathetic system. 
 
 The structure of the secreting cells differs in one important 
 respect from that of the cells of the scutellar epithelium of the 
 barley. There is no cellulose membrane investing them and 
 their protoplasm is therefore constantly bathed by the lymph 
 which reaches them freely. They are composed of protoplasm 
 which can be seen to be arranged in a network ; this is fairly 
 regular and close in some cells, while in others the size of 
 its meshes varies in different parts of the cell. The outer 
 part or limiting layer of the cell has much closer meshes than 
 any other, and forms an almost continuous layer. Between the 
 meshes of the protoplasm there is a more transparent, possibly 
 fluid material, often known as paraplasm. This may contain 
 various substances and may be crowded with granular matter. 
 There is always a conspicuous nucleus present but its position 
 varies in cells of different glands. 
 
 The process of formation of the diastase, or ptyalin, as it is 
 frequently called, can be traced by a microscopic examination 
 of the cells under different conditions of the animal. If 
 
Ill] ANIMAL DIASTASE. S7 
 
 examined after a period of fasting the meshes of the proto- 
 plasm are found to be filled with granules to such an extent 
 that the outlines of the cells are hardly if at all visible and the 
 nucleus is altogether obscured. If the gland is taken from the 
 body immediately after a copious outflow of saliva has taken 
 place, the granules are smaller and sparser, and are accumulated 
 on the sides of the cells next to the lumen, the outer border 
 appearing clear. The whole cell is somewhat smaller, as if 
 shrunken, and the nucleus is plainly visible. The examination 
 can be made upon the fresh tissue, or more easily after treat- 
 ment of it with osmic acid, which stains the granules black. 
 
 The process of secretion appears to consist of three stages, 
 absorption of nutritive matter Irom the lymph by the cell, the 
 manufacture and deposition of the granules in its interior, and 
 the subsequent solution of these and their coincident extrusion 
 from the cell into the duct. All these processes are under the 
 control of the nerves which are supplied to the glands, each of 
 which has a definite part to play in the sequence of events. 
 In the case of the sub-maxillary gland, the formation and 
 solution of the granules appear to be regulated by a branch of 
 the cervical sympathetic nerve, and their extrusion from the 
 cells by the chorda tympani, a branch of the 7th cranial nerve. 
 The whole nervous action is controlled by the brain. 
 
 The exact nature of the granules is not completely known. 
 They are probably not diastase itself, but an immediate ante- 
 cedent which becomes diastase in the process of dissolving and 
 leaving the cell. This view is however based rather upon 
 analogy with what happens in certain other glands than upon 
 direct observation of the salivary ones. 
 
 In some of the salivary cells the granules are certainly not 
 all diastase, as the secretion of the gland contains other bodies 
 as well, notably mucin. Still the granularity and the amount 
 of diastase obtainable from a gland appear to vary together. 
 
 The granular appearance thus noticed in the animal cell 
 recalls the fact that a similar granularity may be observed in 
 the scutellar epithelium of the barley grain while diastase is 
 being formed there. 
 
 Similar appearances to those described for the salivary 
 
38 ANIMAL DIASTASE. [CH. 
 
 gland may be observed in the cells of the pancreas, which has 
 a very similar structure. The cells of this organ however 
 secrete, in addition to diastase, other enzymes which will be 
 discussed later. The granules of these cells have been ascer- 
 tained not to be composed of the enzymes themselves, but of 
 antecedents of them known as zymogens. Possibly the granules 
 diifer from each other, some being those of one zymogen, others 
 those of another. 
 
 The amount of diastase in saliva and pancreatic juice varies 
 according to the animal from which those fluids are taken. 
 It is least in the juices of the herbivora, the saliva of the 
 horse being practically devoid of it. There is very little 
 again in the secretions of young animals so long as they 
 are suckled. 
 
 The diastase of blood and lymph may be compared with the 
 translocation diastase of plants. It acts upon substances in 
 those fluids and is not brought into contact with carbohydrates 
 in the alimentary canal. Its mode of formation is unknown, 
 the microscope affording no information on this point. 
 
 Turning now again to the question of the storage of reserve 
 carbohydrates in the body and the possibility of their trans- 
 location by means of diastase, we find our information is not 
 so complete as that concerning the secretions of the glands 
 described. The most prominent organ in the transformation of 
 such reserve-carbohydrates is the liver, in which it is easy to 
 detect large quantities of glycogen which vary considerably 
 from time to time. Glycogen in the animal organism appears 
 to take the place of starch in the vegetable one, being the 
 form in which carbohydrate material, largely derived from the 
 alimentary canal, is stored till needed in the processes of 
 nutrition. 
 
 It has been known since the time of Claud Bernard that 
 the blood coming from the liver contains a variable amount of 
 sugar, and that during a period of fasting it contains more than 
 does the blood of the portal vein which carries to that organ 
 the products of digestion. When the liver of an animal is 
 removed from the body, the post-mortem changes which take 
 place in it are always accompanied by the formation of sugar, 
 
Ill] ANIMAL DIASTASE. 39 
 
 and as the glycogen gradually disappears it is an indication 
 that the latter is the source of the former. 
 
 The question of the existence of a diastasic enzyme in the 
 liver cells is one which has excited a great deal of controversy. 
 Most of the older experimenters failed to extract such a body, 
 and the change was supposed therefore to be effected by the 
 protoplasm of the cells, both during life and during the post- 
 mortem changes. 
 
 In considering whether the evidence that has been ac- 
 cumulated points to the existence of an enzyme it should be 
 borne in mind that in many cases it is by no means easy to 
 extract such a body from the protoplasm of a cell by the mere 
 process of maceration with water or other extracting fluid. In 
 many cases it has been ascertained that the enzyme is held 
 by the substance of the protoplasm with very great tenacity. 
 This appears very evident on comparing the results of Wort- 
 mann on the diastase of foliage leaves with those of Brown and 
 Morris which have already been alluded to. Another instance 
 of this difficulty is found in the recent experiments of Buchner, 
 in which he has extracted an alcohol-producing enzyme froin the 
 body of the yeast cell by the employment of strong pressures. 
 These will be discussed in a subsequent chapter, but the 
 difficulty of extraction is apparently not sufficient proof that 
 an enzyme may not be present, though simple maceration fails 
 to extract it. 
 
 Of the earlier writers on the subject of the metabolism of 
 the liver, Von Wittich and Claud Bernard both claim to have 
 extracted an enzyme from the hepatic cells. Tiegel on the 
 other hand failed to obtain one, and Seegen and Kratchmer, 
 after many experiments, say that it is impossible to obtain 
 satisfactory evidence of its existence. 
 
 An extended series of observations was made by Miss Eves 
 in 1882. This author dried the livers of various animals at a 
 moderate temperature and then reduced them to a powder in a 
 mortar. The dried powder was subsequently extracted with 
 salt solution and the liquid filtered. Extracts so prepared were 
 found to be capable of effecting starch transformation, but the 
 diastasic powers they possessed were very small in comparison 
 
40 ANIMAL DIASTASE. [CH. 
 
 with those of saliva ; so small indeed that her conclusion was 
 that certainly the post-mortem formation of sugar and dis- 
 appearance of glycogen could not be attributed to an enzyme 
 in the cells. She attributed the small amount obtained in her 
 extracts to the blood which had remained in the organ. 
 
 The subject has been investigated by many experimenters 
 during the present decade, and there is a growing opinion that 
 a diastase is present in the liver, though there is not yet an 
 agreement as to the part which it plays in the metabolism of the 
 organ. About the year 1890 Kaufmann published the results 
 of some experiments made with the bile secreted by various 
 animals. He says that the bile of the pig, the sheep, and the 
 ox are all strongly diastasic, that no enzyme can be detected in 
 that of the dog, while that of the cat contains a relatively 
 small amount. Kaufmann infers from these results that the 
 liver cells do normally form diastase. 
 
 Very exhaustive experiments upon this subject have been 
 carried otxt by Pavy. He removed the liver from rabbits 
 immediately after death and rapidly reduced them to pulp by 
 passing them through a mincing machine and subsequently 
 pounding them in a mortar. The pulp was stirred up with a 
 large volume of absolute alcohol and allowed to stand under 
 this liquid for six months, by which treatment the proteids of 
 the tissue were completely coagulated. At the expiration of 
 this time, the alcohol was strained off and the liver substance 
 washed successively with fresh alcohol and with ether. It was 
 then dried, powdered, and passed through a fine sieve. Two 
 grams of this liver powder were thrown into boiling water, while 
 another portion of the same weight was incubated for 4 hours 
 at 46° C. with 20 c.c. of 1 % sodium chloride solution. Both 
 were then titrated with Fehling's fluid. The boiled digestion 
 contained '46 per cent, of reducing-sugar, the incubated one 
 4'27 per cent. As the original liver contained both glycogen 
 and a small amount of sugar, the increased quantity found after 
 incubation clearly shows the transformation of the glycogen 
 under the influence of liver diastase. 
 
 Pavy has shown further that the diastase so detected will 
 survive prolonged heating to 80° C. if perfectly dry. He 
 
Ill] ANIMAL DIASTASE. 4] 
 
 heated some of the liver powder suspended in absolute alcohol 
 for 5 minutes over boiling water, then dried it again. Experi- 
 ments with this material carried out on the lines of the one 
 just quoted gave the same result, except that the portion not 
 incubated but boiled at once in water caused no reduction of 
 Fehling's fluid. This shows that the reduction found in this 
 portion in the first experiment was due to sugar present at 
 death in the liver tissue; in the second experiment this had 
 been extracted by the alcohol in which the powder was heated. 
 Hot alcohol is of course much more efficacious than cold in 
 removing sugar from the tissue, which holds it with some 
 tenacity. 
 
 Miss Tebb has also been successful in preparing a diastasic 
 extract from liver. She dried the liver of the pig at 35 — 40^ C, 
 shredded it finely and dialysed away the sugar which it con- 
 tained. The liver substance was then dried and powdered. She 
 found it capable of hydrolysing both starch and glycogen. An 
 extract of the dried powder was prepared with a 5 per cent, 
 solution of sodium sulphate and dialysed till free from sugar. 
 It was then digested with a 4 per cent, solution of glycogen 
 for 21 hours at 37° C, at the end of which time phenyl- 
 hydrazine showed the presence of sugar in some quantity. 
 
 Bial holds that both during life and during post-mortem 
 changes the conversion of. the glycogen into sugar is due to an 
 enzyme. He found that the process was not prevented by a 
 1 per cent, solution of sodium fluoride, and he considers this 
 fact to prove the presence of diastase, as this salt is fatal to the 
 vital action of the cell. He does not decide whether the liver 
 cells secrete this enzyme, or whether it is derived by them 
 from the blood and the lymph which bathe them. He agrees 
 that the sugar which is formed is glucose, and on this ground 
 says that the diastase which is formed is the same as that 
 existing in the blood and lymph. There are reasons however 
 for thinking that this is not due to the diastase being of a 
 different kind from that of the saliva, but that there is another 
 enzyme also present which converts maltose into glucose. 
 
 Salkowski, from the results of an independent investigation, 
 coincides with the views of Bial. 
 
42 ANIMAL DIASTASE. [CH. 
 
 Schwiening also has made some experiments upon the post- 
 mortem changes in the liver, especially upon the influence of 
 chloroform upon them. As already mentioned, this reagent 
 kills protoplasm but does not interfere with the action of 
 diastase. He made an extract of liver with chloroform water 
 and boiled half of it as a control. In both his preparations liver 
 cells were present ; in one they had been killed by chloroform, 
 leaving any diastase unafifected ; in the other not only were the 
 cells killed but the diastase was destroyed by the action of the 
 high temperature. Both contained the glycogen originally 
 present in the tissue. After a certain time of incubation the 
 unboiled extract was found to contain no glycogen, but a 
 considerable quantity of sugar, while the boiled one contained 
 glycogen with only traces of sugar. He concluded in con- 
 sequence that the action was due to an enzyme, the direct 
 action of the protoplasm being impossible under the conditions 
 of the experiment. The traces of sugar found in the boiled 
 extract may very well have been due to a small quantity 
 existing in the liver at the time of its excision. 
 
 Schwiening says further that in the boiled extract there 
 was a conversion of glycogen into sugar after a prolonged 
 incubation, a fact which he holds to point to a continuous post- 
 mortem formation of diastase. This however seems a little 
 difficult to understand, as the boiling not only killed the liver 
 cells, but presumably destroyed any antecedent of diastase as it 
 did the enzyme itself. 
 
 The liver however is not the only seat of stored or reserve 
 carbohydrate, for the muscles also contain a considerable amount 
 of glycogen. Nasse has shown that these organs are also the 
 seat of an amylolytic or diastasic enzyme which can be detected 
 in the juice extracted from them by pressure. Halliburton 
 confirms Nasse's observation. He has found that a watery 
 extract of the dried precipitate produced by treating muscle 
 juice with alcohol can change both starch and glycogen into a 
 reducing-sugar with the concomitant production of an inter- 
 mediate body possessing the characters of a dextrin. He finds 
 the action on starch very slow, no sugar being noticeable till 
 the digestion has proceeded for 5 or 6 hours. 
 
Ill] ANIMAL DIASTASE. 43 
 
 Boudourg has observed the presence of diastase in the 
 pyloric tubes of certain Teleostean fishes, 
 
 Among the Invertebrata diastase appears to be of very 
 general occurrence; it can be detected even in unicellular 
 organisms, although from their structure it exists side by side 
 with other enzymes in the general protoplasm. 
 
 Fredericq has extracted a juice from the cells of several 
 sponges, which acts upon starch, fats and proteids. He obtained 
 a similar extract from several of the Echinodermata, which had 
 the same properties but was slower in its action. In Uraster 
 this secretion was found in the pyloric CiBca, situated in each 
 ray of the animal. An extract of that part of the alimentary 
 canal of Lumbricus contained in the anterior portion of the body, 
 up to the 6th segment, will convert starch into sugar. Its 
 intestine is almost surrounded by a glandular tissue which has 
 the same properties, but the conversion of starch by this 
 tissue will take place only in neutral solutions. Other worms 
 also form diastase in similar positions. In Rotifers two large 
 glandular tubes open into the anterior portion of the stomach, 
 and yield a secretion which acts on starch and on proteids. 
 
 It is very common in all the higher Invertebrata to find 
 both diastasic and proteolytic enzymes yielded by the same 
 gland, just as they are in the pancreas of vertebrates. The 
 so-called liver of the Crustacea and Mollusca is apparently a 
 hepato-pancreas and both enzymes can be prepared from it. 
 
 The glands which are situated towards the anterior region 
 of the alimentary canal in many of the lower forms are 
 only diastasic. Among the Insecta Blatta is known to be 
 furnished with well-marked salivary glands, secreting an alka- 
 line fluid which converts starch into sugar with great facility. 
 The enzyme can be extracted by precipitating an infusion of 
 the glands with dilute phosphoric acid and lime water, when it 
 is carried down by the calcic phosphate. The mixed precipitate 
 when washed with distilled water gives up the enzyme to the 
 solvent and it can be thrown down in a fairly isolated condition 
 by the addition of a large excess of alcohol. 
 
 In the Lepidoptera salivary glands are present in the 
 imago of many species; they possess also glandular follicles 
 
44 ANIMAL DIASTASE. [CH. Ill 
 
 in the stomach, the juice of which behaves like that of a 
 vertebrate pancreas. Biedermann has found in the intestine 
 of the larva of Tenehrio Molitor, the meal worm, a secretion 
 which contains diastase. 
 
 The spiders are similarly supplied with salivary and pan- 
 creatic glands. 
 
 As has been mentioned the "liver" of MoUusca has pan- 
 creatic functions. In Helix aspera there are true salivary 
 glands as well, which open into the mouth. In Helix poTnatia 
 the diastasic function disappears from the pancreatic organ 
 during the winter sleep. In the Cephalopoda the secretion of 
 the salivary glands is diastasic only, while the " liver " besides 
 hydrolysing starch, emulsifies fats, and saponifies them, makes 
 milk transparent and acts proteolytically on albumin. The 
 Lamellibranchiata are stated to possess no special salivary 
 glands. Diastase has been said to exist in the mantle of the 
 oyster so long as it is uninjured and to occupy different cells 
 from those which contain glycogen. 
 
 Abelous and Heim demonstrated the presence of diastase 
 in the eggs of certain Crustaceans, among which may be 
 mentioned Maia squinado, Platycarcinus pagurus, Portunus 
 puher and Galathcea strigosa. They were able to prepare it 
 from either fresh eggs or such as had been preserved for some 
 time in 95 per cent, alcohol, using as a solvent either water 
 or glycerine. They found it acted most energetically at a 
 temperature of 3-5° C. and in a medium containing "l per cent, 
 of hydrochloric acid. 
 
 Mtiller and Masayama state that the yolk of the ordinary 
 hen's egg contains an enzyme capable of transforming starch 
 into dextrin and sugar. 
 
 There is no doubt that the diastase which occurs in the 
 animal organism is identical with that which is of vegetable 
 origin. Brown and Heron have shown that the course of the 
 action of pancreatic and malt diastase is the same, that they 
 work under identical conditions and are influenced by various 
 external circumstances in precisely the same manner. 
 
45 
 
 CHAPTEE IV. 
 
 PREPARATION OF BIASTASE AND ITS COURSE OF ACTION. 
 
 The isolation of diastase in a state of purity has not yet 
 been accomplished. Indeed so little is known about its com- 
 position that no test of its purity has been satisfactorily 
 established. Beyond saying that it is undoubtedly a nitro- 
 genous body, and that it has much in common with the proteids, 
 it is at present difficult to go. This indeed is the case with 
 all the enzymes at present discovered. 
 
 The usual method of preparing a diastasic extract is to 
 mince or triturate in a mortar the tissue in which it exists and 
 to macerate the debris for some time with an appropriate 
 solvent, after which the solid matter is removed by filtration. 
 Such an extract of course can only be regarded as a preparation 
 containing diastase, together with such constituents of the 
 tissue as are soluble in the extracting fluid. 
 
 Many attempts have been made to prepare the enzyme free 
 from such admixture, and though several methods purify it from 
 many of the more general contaminations none can be regarded 
 as wholly satisfactory. 
 
 One of the earliest of these methods was employed by 
 Cohnheim, who took advantage of one of the properties of the 
 enzymes as a class, viz. their tendency to be carried out of 
 solution by any inert precipitate formed in the liquid in which 
 they exist. His method was the following : — he added to saliva 
 a certain quantity of phosphoric acid and then neutralised the 
 mixture with lime water, which caused a copious precipitate of 
 phosphate of calcium. This precipitate carried down with it a 
 
46 PREPARATION OF DIASTASE [CH. 
 
 large proportion of the proteids of the saliva together with the 
 diastase or ptyalin. The precipitate was then collected by 
 filtration and extracted with a volume of water equal to that of 
 the saliva originally used. The diastase is slightly more soluble 
 than the proteids and accordingly passed into solution first. 
 He repeated this process several times and finally precipitated 
 the last extract by the addition of alcohol. This precipitate 
 was collected, washed with alcohol and. dried over sulphuric 
 acid, appearing then as an amorphous powder, white in colour 
 and freely soluble in water. Cohnheim held it to be free from 
 proteids as its solution did not yield the usual reactions 
 characteristic of these bodies. 
 
 Another method of preparation of salivary diastase has 
 been described by Krawkow. Saliva is diluted with an equal 
 volume of water, and saturated with neutral ammonium sul- 
 phate. The precipitate which is caused by the saturation is 
 collected on a filter and washed for a short time with strong 
 alcohol. It is then allowed to stand under absolute alcohol for 
 one or two days, and finally dried at a temperature of 30° G. 
 On extraction with water it yields a solution which is strongly 
 diastasic and which gives no proteid reactions. 
 
 Von Wittich adopted the plan of treating a diastase-secreting 
 tissue with glycerine and subsequent precipitation with alcohol, 
 purifying the product by repetition of the process. 
 
 Mialhe obtained it from saliva by merely precipitating it by 
 means of alcohol. Probably neither he nor v. Wittich ever 
 obtained it in anything like a pure condition. 
 
 From several animal tissues a larger yield has been ob- 
 tained by using for the first extraction a solution of common salt 
 of about 10 per cent, concentration instead of water. 
 
 Lintner has prepared a relatively pure diastase from malt 
 by the following method. One part of either green or air-dried 
 malt was extracted for 24 hours with 2 to 4 parts of alcohol of 
 20 per cent, strength. The spirit was then filtered off and mixed 
 with two and a half times its volume of absolute alcohol. A 
 precipitate settled out, which was coljected on a filter and washed 
 with absolute alcohol. It was then transferred to a mortar and 
 well washed, with stirring, with a mixture of absolute alcohol 
 
IV] ' AND ITS COURSE OF ACTION. 47 
 
 and ether. The liquid having been decanted off, the residue was 
 dried in vacuo over strong sulphuric acid, and remained as a very 
 active yellowish-white powder. To purify it, it was redissolved 
 in water and again precipitated with alcohol, after which it was 
 submitted to dialysis. 
 
 Analysis of it in this condition showed it to contain nitrogen 
 and a certain amount of ash, which was chiefly composed of 
 calcium phosphate. The process of purification of the first 
 product increased the percentage of nitrogen, while it diminished 
 the amount of ash. At the same time it increased the diastasic 
 activity. A sample which on first preparation contained 8'3 
 per cent, of nitrogen, after purification by two reprecipitations 
 contained 9'06 per cent., and after subsequent dialysis, 99 per 
 cent, while the percentage of ash went down from 106 to 
 4-79. 
 
 The diastasic activity was determined by allowing 3 c.c. of a 
 solution of 1 gramme of the diastase in 250 c.c. of water to 
 act on 10 c.c. of a 2 per cent, solution of starch for one hour 
 at the ordinary temperature of the laboratory, comparing it 
 with a control made of a standard diastase and used in the 
 same proportions and under identical conditions. The same 
 sample as that mentioned above had a diastasic activity of 96 
 before purification and of 100 afterwards. 
 
 Another method of preparing diastase has been used by 
 Loew, which is not however so trustworthy as the last-mentioned 
 one. The tissue which contained the enzyme was extracted by 
 water, and soluble calcium salts and salts of lead were added to 
 it. Then it was made alkaline with caustic soda, which produced 
 a precipitate, and the latter carried down the diastase with it. 
 The precipitate was filtered off and dissolved in dilute acetic 
 acid, and the lead removed by a current of sulphuretted 
 hydrogen. Alcohol of 95 per cent, strength was next used to 
 precipitate the enzyme from the filtrate from the sulphide of 
 lead. This precipitate was filtered off, dried over sulphuric acid 
 and extracted with glycerine. 
 
 Musculus employed a method which was a modification of 
 that of Payen and Persoz. A quantity of powdered germinated 
 barley was macerated for an hour with twice its volume of water 
 
48 PREPARATION OF DIASTASE [CH. 
 
 and strained. To the turbid liquid its own volume of alcohol 
 was added, and the bulky precipitate filtered off. To the filtrate 
 its own volume of alcohol was again added, when a much less 
 bulky precipitate fell. This was collected on a filter and dried 
 at a gentle heat. The paper retained the precipitate, which was 
 found to be strongly diastasic. So prepared it could be preserved 
 for a considerable period. 
 
 Loew prepared the enzyme in a condition which he con- 
 sidered relatively pure by the following process : — A measured 
 quantity of germinating barley, softened by a little water, was 
 digested for two days with 40 per cent, alcohol, with frequent 
 stirring. After filtration, the liquid was precipitated by a 
 mixture of two volumes of alcohol and one volume of ether, 
 and the precipitate collected and washed with absolute alcohol. 
 It was then dried over sulphuric acid and extracted with water. 
 Sub-acetate of lead was added, which yielded a precipitate ; 
 this was filtered off and suspended in water. A current of 
 sulphuretted hydrogen was then passed through it to remove 
 the lead, and to the filtrate was added a mixture of absolute 
 alcohol and ether, which precipitated the diastase. This was 
 collected on a filter, washed with absolute alcohol and then with 
 ether and dried. This process was attended with a loss of a good 
 deal of the diastase and it did not yield a pure product. 
 
 Wroblewski ha.s recently published an account of some 
 researches which he carried out with a view to ascertaining the 
 nature of diastase, in the course of which he has adopted a 
 somewhat different method of preparation. He ground some 
 malt to a very fine powder and washed it repeatedly with alcohol 
 of 68 per cent, concentration, in which diastase is not soluble. 
 The enzyme will dissolve in a mixture of alcohol and water 
 which only contains about 45 per cent, of spirit. He therefore 
 extracted the powder twice with an alcoholic solution of that 
 concentration, mixing the two extracts after filtering them 
 separately ; a further quantity of alcohol was then added to the 
 mixed filtrates till the concentration again reached 68 per 
 cent, when the diastase which had been dissolved was pre- 
 cipitated. It was collected on a filter, redissolved in 45 per 
 cent, spirit, and again reprecipitated. The precipitate was then 
 
IV] AND ITS COURSE OF ACTION. 49 
 
 dissolved in water, and the solution saturated with magnesium 
 sulphate, which again threw down the diastase. The latter was 
 again dissolved in water and the solution freed from the salt by 
 dialysis. A mixture of alcohol and ether was then added to 
 precipitate the enzyme. Prepared in this way it was found to 
 be associated with a carbohydrate body, araban, yielding ara- 
 binose on hydrolysis. This substance has been found associated 
 also with emulsin by H6rissey. Bertrand observed the occur- 
 rence of arabinose as one of the products of the hydrolysis of 
 laccase, an enzyme which will be described later. 
 
 Wroblewski has described another process for the prepara- 
 tion of diastase which avoids the use of alcohol till the last 
 stages. To a malt extract or other aqueous solution of the 
 impure enzyme, ammonium sulphate was added till a turbidity 
 leading to a precipitate was reached. This consisted mainly 
 of the araban, and the diastase was almost entirely left in 
 solution. After filtration, more of the salt was added to the 
 filtrate till another precipitate fell. This was composed mainly 
 of pentosan, but some diastase was mixed with it. It was 
 filtered off and the filtrate saturated with the salt, when a 
 third precipitate fell, which was almost pure diastase. This was 
 dissolved in water and freed from the ammonium sulphate by 
 dialysis, after which the enzyme was thrown out of solution 
 by addition of a mixture of alcohol and ether. 
 
 Action of diastase on starch. 
 
 When a thin starch-paste, preferably containing 1 — 2 per 
 cent, of starch and prepared with boiling water, is mixed with 
 a small quantity of a diastasic extract a definite sequence of 
 changes can be noticed. In a few minutes the liquid loses 
 the opalescence due to the suspended starch, which goes into 
 solution and the whole becomes limpid. A few drops removed 
 at this stage become blue on the addition of iodine, showing 
 that the starch is chemically unaltered. After a little longer 
 interval the iodine reaction changes, a sample removed from the 
 digestion becoming purple with that reagent. Later the colour 
 given is a deep red-brown. Finally iodine ceases to produce 
 any colour. Periodical examination with Fehling's solution 
 
 G. F. 4 
 
50 PfiEPABATION OF DIASTASE L^H. 
 
 during this process shows that a power of reducing cupric oxide 
 which is not possessed by either starch or diastase is rapidly 
 developed and increases continuously as the action proceeds. 
 
 The series of changes so set up in the starch-paste by 
 the enzyme are thus evidently of a complicated character. 
 The action is one of hydrolysis or the decomposition of starch 
 together with the incorporation of water into its molecule, 
 leading ultimately to the formation of sugar. But the process 
 is a long and gradual one, and starch and sugar are not the 
 only two bodies which are met with daring the transformation. 
 Certain other products known as deoctrins are found to occur, 
 and one of these is the cause of the brownish-red colour which 
 the liquid gives on the addition of iodine at a particular stage. 
 
 The first attempt to ascertain the stages of the action was 
 made by Payen and Persoz in 1833. These authors recognised 
 one of the dextrins and described it as a body soluble in water 
 and in weak alcohol and not coloured by iodine. In 1860 
 Musculus recognised that dextrin and sugar are produced 
 simultaneously by diastasic action. He described the dextrin 
 a few years later as not colourable by iodine and having no 
 power of reducing cupric oxide. He thought it was not further 
 acted on by the diastase, but remained as a final product. 
 Payen says on the contrary that diastase saccharifies it easily. 
 
 About the years 1871-2 Griessmeyer, O'Sullivan and 
 Briicke, working independently, ascertained that there are at 
 least two dextrins formed, one of which is uncoloured by iodine 
 while the other becomes brownish-red with that reagent. 
 Briicke gave them the names of achroodextrin and erythrodex- 
 trin. O'Sullivan at that time thought they were really identical, 
 and describes them as having no power of reducing cupric 
 oxide and possessing a specific rotatory power of [a]j = + 213°. 
 He said that malt extract converted them both into maltose. 
 
 Some years later Musculus and Griiber showed that achroo- 
 dextrin was not a single body, but that there were at least 
 three such dextrins, none of which reacted with iodine. They 
 give for the specific rotatory powers of the three [a]j=-l-159°, 
 -I- 190°, and + 150° respectively. Based upon their experiments 
 these observers advanced a theory of the starch transformation 
 
IV] AND ITS COURSE OF ACTION. 51 
 
 which has only been slightly modified by subsequent workers. 
 This was that the starch molecule breaks down by a series 
 of hydrations and subsequent decompositions, maltose being 
 formed at each splitting, together with a dextrin of less mo- 
 lecular weight. 
 
 In 1879 the subject was investigated by Brown and Heron. 
 From their experiments they came to the conclusion that the 
 molecule of starch has the formula 10 CiaHjjOio, and that under 
 the action of diastase groups of C12H20O10 are successively 
 removed, the residue in each case being a dextrin whose mole- 
 cule becomes less complex at each stage. They suggested that 
 there are eight of these possible dextrins. Each successive 
 group removed then becomes hydrolysed to maltose. 
 
 O'SuUivan in the same year came to the conclusion that 
 there are probably four dextrins formed, erythi'odextrin and 
 three achroodextrins. He says that these when pure have no 
 power of reducing cupric oxide and have a specific rotatory 
 power of [a]j = + 222°. 
 
 Another view of the process was suggested about the same 
 time by Herzfeld. According to this the transformation of starch 
 by diastase is not a splitting up of the molecule, but there is 
 consecutively a conversion of it into soluble starch, erythro- 
 dextrin and achroodextrin, and the last is subsequently con- 
 verted into a body which he calls maltodeostrin and maltose. 
 When starch is transformed at a temperature lower than 65° C. 
 maltose and achroodextrin are formed ; above 65° C. erythro- 
 dextrin and maltodextrin in addition. Herzfeld suggested for 
 the latter body a composition corresponding to two groups of 
 dextrin united to one sugar group of the formula CsHijOb. 
 Herzfeld's views have not been generally accepted, the theory of 
 Musculus and Grllber meeting with more favour. Probably his 
 maltodextrin was by no means pure. 
 
 In 1885 a modification of the views of Musculus was put 
 forward by Brown and Morris. They found that in a starch 
 transformation the usual result was the formation of about 80 
 per cent, of maltose and 20 per cent, of dextrin, the latter 
 being further hydrolysed with great difficulty. Among their 
 products they found constantly occurring a certain amount of a 
 
 4—2 
 
52 PREPARATION OF DIASTASE [CH. 
 
 body intermediate in properties between maltose and dextrin, 
 and they gave to this the name maltodextrin, a term originally 
 used by Herzfeld. The substance however differed consider- 
 ably in its behaviour from that described by Herzfeld under 
 the same name. Brown and Morris conclude however that the 
 differences are due to the impurity of Herzfeld's preparation. 
 
 Starting with the observation that apparently only four-fifths 
 of the starch is readily convertible into maltose they put for- 
 ward the following theory. Starch has at leEist a formula of 
 5 [(Ci2H2oOio)s] and consists of five amylin or dextrin-like groups, 
 four of which are arranged symmetrically round the fifth. By 
 the action of diastase the decomposition takes place in suc- 
 cessive stages, one group of (CiaHjoOio), being split off at each 
 step, leaving a dextrin residue. Each group so split off is then 
 hydrolysed by the addition of water, forming maltodextrin 
 
 P H O 1 
 
 /'n TT n \ 1 ' ^^^ *'^^^ ^^ further hydrolysed by diastase below 
 
 65° C. into maltose, two molecules of water being taken up. 
 The last group, around which the others are arranged, under- 
 goes hydrolysis with great difficulty and consequently remains 
 as a dextrin at the conclusion of the transformation. The 
 maltodextrin being composed of maltose and dextrin they 
 term an amylo'in. 
 
 The reactions of the amy loin are given by them as follows: — 
 
 (1) It gives numbers on analysis which allow its com- 
 position to be expressed in terms of a mixture of maltose and 
 dextrin. 
 
 (2) It cannot be separated into maltose and dextrin and 
 is therefore not a mixture but a compound body. 
 
 (3) It is completely converted into maltose by the 
 action of diastase, while a mixture of the two in the same 
 proportion always leaves a residue of dextrin. 
 
 (4) It is unfermentable during the primary fermentation 
 by yeast. 
 
 In a later paper in 1889, while adhering to their theory of 
 the grouping of the starch molecule and the general course of 
 the transformation, they come to the conclusion that instead of 
 one maltodextrin there are several of them formed. They 
 
IV] AND ITS COURSE OF ACTION. 53 
 
 think that the groups in the starch molecule are considerably 
 larger than their first experiments led them to suggest. The 
 first decomposition may be expressed by the equation 
 
 6 [(Ci,H„Oi„)^] = (Ci,H^O„),o + 4 [(Ci,H^O„)^]. 
 
 starch stable dextrin amylin groups 
 
 The 4 amylin groups are capable of gradual and complete 
 hydrolysis to maltose, a series of amyloins of varying degrees 
 of complexity being formed, there being a general tendency for 
 the molecular aggregations to become smaller as the hydrolysis 
 proceeds, until finally maltose is reached. The extreme stages 
 of this hydrolysis can be represented by the following equa- 
 tions : — 
 
 and (Q,H,0,„),o + 19 H,0 = /(^-^"n"'*'' • 
 
 As the hydrolysis proceeds these complex amyloins are 
 broken up into smaller ones, two of which have been prepared 
 
 f O TT O 
 
 by them; these they call maltodextrin \,ri^'Ti^r^\ ^^^ 
 
 f O H O 
 
 amylodextrin ],r^^-r/'ri^-. ■ The latter body was first observed 
 
 by W. Nageli in 1874. He considered it erroneously to be 
 identical with soluble starch. 
 
 A curious difference in the action is noticed when diastase 
 is allowed to act on starch-paste in the cold. The optical 
 activity of the final result of the transformation is lower than 
 it should be on the assumption that only maltose and dextrin 
 are present. After standing for some time, or after boiling, 
 this discrepancy disappears, and the optical and cupric-oxide- 
 reducing powers show the same relationship that they do when 
 the starch transformation is carried out at higher temperatures. 
 The explanation appears to be that at the lower temperature 
 the maltose formed does not possess its full power of rotating 
 the polarised ray, being liberated in a state of " half-rotation." 
 Freshly prepared solutions of maltose always exhibit this pecu- 
 liarity. When freshly dissolved the optical power of maltose 
 
54 PREPARATION OF DIASTASE [CH. 
 
 bears the relation to that of an old or a boiled solution of about 
 133° to 150°. Brown and Morris have shown that this relation 
 holds good in the case of freshly prepared products from cold 
 starch-paste. 
 
 From these researches there seems little doubt that the 
 hydrolysis of starch is a gradual and continuous process. An- 
 other view has been recently put forward by Mittelmeier which 
 differs in many of its details from that of Brown and Morris. 
 According to Mittelmeier the action of diastase on starch occurs 
 in two stages; during the first a small portion of the starch 
 is rapidly converted into amylodextrin, erythrodextrin and 
 achroodextrin, and sugar, but these dextrins are not identical 
 with those formed during the second stage. The author speaks 
 of them respectively as primary and secondary dextrins. He 
 claims to have isolated primary erythrodextrin and primary 
 achroodextrin. The former is insoluble in water, has a specific 
 rotatory power [a]j, = about + 170°, does not react with phenyl- 
 hydrazine, reduces cupric oxide, and yields dextrose and maltose 
 when treated with diastase. He gives no reactions for the 
 other primary dextrin. The sugar from the primary dextrins 
 yields maltosazone, while that from the secondary ones gives an 
 osazone which is oily in character, and melts at 145 — 148° C. 
 Mittelmeier thinks it is derived from a new sugar for which he 
 suggests the name metamaltose. 
 
 His results have not at present been confirmed. 
 
 The nature of the sugar formed by the action of diastase is 
 according to most observers maltose when the transformation is 
 allowed to take place under the ordinary conditions of the 
 laboratory. Many writers have pointed out however the occur- 
 rence of glucose among the products under particular conditions, 
 and doubts as to the identity of the enzyme in different secre- 
 tions have been based upon these discrepancies in the final 
 results of their activity. 
 
 These two sugars differ considerably in their composition, 
 maltose being the most complex, and indeed in certain circum- 
 stances one molecule of it splits up into two molecules of 
 glucose. Glucose, or as it is often called dextrose, is colourless 
 and crystalline. The crystals when deposited from water occur 
 
IV] AND ITS COURSE OF ACTION. 55 
 
 in six-sided tables or prisms or are agglomerated into warty 
 lumps. They are soluble slowly in cold, rapidly in hot water ; 
 slightly soluble in cold alcohol, more readily in warm. A 
 solution of this sugar possesses a specific rotatory power of 
 («)r = + 52'5°. When heated with a solution of cupric sulphate 
 in the presence of an excess of caustic alkali, it reduces the 
 cupric oxide formed, to the cuprous condition. This power is 
 made use of in estimating the quantity of glucose present in a 
 solution, the latter being in a definite proportion to the 
 amount of cupric oxide reduced. 
 
 Glucose when warmed with phenylhydrazine acetate forms 
 an osazone which crystallises in yellow needles. It is almost 
 insoluble in water and melts at about 205° 0. Glucose is 
 capable of undergoing alcoholic fermentation by yeast without 
 previous decomposition. It has the composition CsHiaOg and is 
 represented by the formula [COH - (CH0H)4 - CHaOH]. 
 
 Maltose crystallises with difiBculty from its watery solution, 
 the crystals being in the form of fine needles. It is very 
 soluble in water, but not so readily dissolved by alcohol as is 
 glucose. Its specific rotatory power is much greater than that 
 of the latter, being (o)j) = + 140°. Its power of reducing cupric 
 oxide is on the other hand considerably less, being only about 
 two-thirds that of glucose. 
 
 Maltose forms with phenylhydrazine acetate an osazone which 
 crystallises in fine yellow needles resembling those of glucosazone, 
 but they are soluble in about 75 parts of water at 100° C. Its 
 melting point is 205° C. 
 
 There is a general agreement among chemists that the 
 action of vegetable diastase on starch results in the production 
 of maltose and not glucose. Somewhat conflicting statements 
 have been made about the action of the animal enzyme. 
 Musculus says that the vegetable diastase and the animal 
 ptyalin produce the same sugar. Nasse on the other hand 
 maintains that saliva forms a third sugar, differing from both 
 glucose and maltose ; he calls it ptyalose, and says its reducing 
 power is only half that of glucose. The changes that take 
 place in the liver both during life and after death lead to the 
 production of glucose in its cells, yet the action of diastase on 
 
66 PREPARATION OF DIASTASE [CH. 
 
 glycogen forms maltose. Nasse and some other observers 
 maintain that it is ptyalose. Bial says that the serum of 
 blood produces glucose and not maltose. 
 
 The discrepancy can be explained however in the light 
 of more recent researches. The liver has been shown by 
 Miss Tebb to contain in addition to the hypothetical diastase 
 another enzyme, glucose or maltase, which converts maltose into 
 glucose. The other animal tissues, which have been alluded to, 
 also contain this enzyme, as do blood and lymph. The action 
 of the diastase in all these cases g;ives rise to maltose, which is 
 subsequently but rapidly converted into glucose by the maltase. 
 This enzyme which has only comparatively recently been dis- 
 covered will be discussed in a subsequent chapter. 
 
 There has been considerable discussion as to whether there 
 are not at least two kinds of maltose formed during starch 
 transformation by diastase. Fischer has prepared a sugar by 
 the action of hydrochloric acid on glucose which possesses special 
 characteristics, and he has given to it the name isomaltose. 
 
 In 1891 Lintner claimed to have identified this sugar in 
 certain beer-worts. He said that on warming them with 
 phenylhydrazine acetate an osazone was formed which in 
 melting point and crystalline appearance corresponded to that 
 of Fischer's new sugar. Later, in conjunction with Dull, Lintner 
 stated that he had isolated this body, and the two authors 
 published an account of its preparation from the products of 
 the action of diastase on starch. According to them isomaltose 
 is a sugar which ferments with yeast very much more slowly 
 than maltose ; it is very hygroscopic, and possesses a very sweet 
 taste. It has a specific rotatory power like that of maltose 
 (°')i) = + 140°, but its reducing power is only 83 per cent, of that 
 of the latter. It is convertible into maltose by the action of 
 diastase. With phenylhydrazine acetate it forms an osazone 
 which melts at 150°— 153°. 
 
 In a later paper in 1895, Lintner says that the conversion 
 into maltose is incomplete, sometimes only 30 per cent, being 
 so changed. He puts forward the further hypothesis that 
 there may be two stereoisomeric isomaltoses. Lintner's views 
 have been challenged by several observers. Ulrich repeated 
 
IVl AND ITS COURSE OF ACTION. 57 
 
 the experiments on which they were founded, but failed to 
 prepare an osazone melting below 159°, which is several degrees 
 higher than that of Lintner's body. 
 
 Ling and Baker prepared the latter, and from a consideration 
 of its behaviour under different conditions came to the con- 
 clusion that it is not homogeneous. 
 
 Brown and Morris made a careful study of it and agreed 
 with Ling and Baker that it is not a chemical entity, and does 
 not correspond to Fischer's isomaltose at all. In their paper 
 they say that it can be further split up by careful fractionation 
 with alcohol and by fermentation with yeast, in such a way as 
 to show that it is a mixture of maltose and dextrinous com- 
 pounds of the maltodextrin or amyloiin class. The crystallisable 
 osazone which Lintner describes as isomaltosazone, and on which 
 he bases his views to a very large extent, is only maltosazone 
 modified in its crystalline habit and melting point by the 
 presence of small but varying quantities of another substance. 
 Brown and Morris have shown this by recrystallisation of pure 
 maltosazone in the presence of the non-crystallisable products of 
 the action of phenylhydrazine acetate on the maltodextrin ; 
 they thus obtained an osazone having the properties of 
 Lintner's body. Ling and Baker endorse this view, but they 
 think that the substance mixed with the maltose is a simple 
 dextrin having the formula OiaHjoOio -I- H2O. 
 
 Jalowitz confirms Brown and Morris, showing that pure 
 maltose, when mixed with varying quantities of dextrin, yields 
 osazones which not only differ in melting point, but also in their 
 general appearance and crystalline form. With equal quantities 
 of maltose and dextrin an osazone is obtained which melts at 
 150° — 155° C, but the melting point alters after several recrys- 
 tallisations. 
 
 Ost also controverts Lintner's views. 
 
 The balance of evidence is therefore very much in favour of 
 the view that the only sugar which results from the action of 
 diastase on starch is maltose. 
 
 The preparation of isomaltose by the action of animal 
 enzymes has also been suggested, though not very clearly 
 proved. Indeed it is probable that further investigation will 
 
.58 PREPARATION OF DIASTASE L^^- 
 
 show that isomaltose has not been isolated in this case either. 
 Kijlz and Vogel have published the results of investigations 
 into the action of the chief animal diastasic secretions, with 
 special reference to the sugar formed. They used a five per cent, 
 solution of rice starch, and identified the sugar by the phenyl- 
 hydrazine reaction, analysing the osazones. They give as the 
 results of their several experiments : — 
 
 Human parotid saliva — Isomaltose. 
 
 Mixed human saliva — Isomaltose and later maltose, 
 
 with traces of glucose. 
 Dogs' saliva — Isomaltose. 
 
 Ox pancreas — Isomaltose. 
 
 Using glycogen instead of starch they found : — 
 
 With liver glycogen, human parotid saliva gave a mixture 
 of isomaltose and maltose in the proportion of 1 : 2. 
 
 With muscle glycogen, the same saliva gave maltose only 
 when the saliva was in excess ; with less saliva they 
 obtained isomaltose, with a little maltose and a little 
 glucose. 
 
 With liver glycogen, ox pancreas gave isomaltose, with a 
 trace of maltose. 
 
 With muscle glycogen, the same enzyme gave isomaltose, 
 with a little glucose. 
 
 The products of the starch transformation by diastase in 
 the animal body probably differ somewhat from those which 
 are obtained in the laboratory. We have seen that in the 
 latter case the final bodies are generally maltose and dextrin in 
 the proportion of four parts of the former to one of the latter ; 
 the dextrin being the intractable group around which the four 
 more easily hydrolysable groups are placed. In the body the 
 final product seems to be maltose only. Lea, who definitely 
 ascertained this fact, attributes it to the removal of the maltose 
 from the region of diastasic activity as fast as it is formed. He 
 says that when in the laboratory starch transformations are 
 carried out in dialysing tubes, made of parchment paper and 
 placed in water which is frequently renewed, the final product 
 
IV] AND ITS COURSE OF ACTION. 59 
 
 is maltose only. This is in accordance with the statement 
 which many observers have made that any enzyme is much 
 impeded if not actually inhibited in its action by the accu- 
 mulation of the products of its activity. 
 
 The products which are formed in a plant during the 
 action of diastase have not been at all fully investigated. 
 There is no doubt that sugar is the most prominent of them if 
 not the only final one, as the dextrins are as indiffusible as starch 
 itself The researches of Brown and Morris on foliage leaves 
 leave no doubt that in Tropaeolum at any rate this sugar is 
 maltose. 
 
 The writer in the course of some researches on the ger- 
 mination of the pollen grain was able to trace certain changes 
 in the starchy reserves contained therein as the pollen tube 
 developed. The plant whose pollen gave the most satisfactory 
 results was Lilivmi pardalinum. The ripe pollen grains 
 when treated with a solution of iodine in chloral hydrate, 
 which renders them transparent, were found to be charged 
 with starch-grains which stained the usual blue colour. Mixed 
 with them here and there were a few grains staining like 
 erythrodextrin. As the tube was put out from the grain 
 these granules were gradually carried over into the protruding 
 portion, and they flowed slowly down the tube as it extended. 
 When the tube was as long as twice the diameter of the grain, 
 if the chloral-hydrate-iodine solution was added they were 
 found to be somewhat dififerent in colour, becoming slightly 
 purple with the iodine. With longer tubes, the grains in which 
 were still travelling forward, this change was more and more 
 marked, particularly near the tip of the tube. When a tube 
 which had attained a length of 20 — 30 times the diameter of 
 the grain was treated in the same way, the general efifect of the 
 iodine was markedly different. There were but few blue 
 granules and those in the part nearest the pollen grain. The 
 greater part of the length of the tube was studded thickly with 
 purple grains and towards the tip they became nearly red. 
 The starch was evidently in process of digestion under the 
 action of diastase, which other experiments had shown the 
 same pollen to contain. The granules did not change their 
 
60 PREPARATION OF DIASTASE [CH. 
 
 shape and showed no sign of corrosion even when magnified 
 very highly. The alteration of the starch was apparently 
 uniform throughout the grain. 
 
 Du Sablon has examined the transformations accompanying 
 the digestion of starch in several bulbs and other underground 
 reservoirs, especially those of the Lily, Tulip, Hyacinth, Arum, 
 and Colchicum. He finds it transformed into a sugar, dextrin 
 being an intermediate product. 
 
 These experiments, though far from exhaustive, appear to 
 show that the course of starch conversion inside the plant is 
 the same as that which has been demonstrated in the labora- 
 tory. 
 
 Before leaving the subject of the transformation of starch 
 some work published in 1890 by Wijsman calls for a passing 
 notice. In all the foregoing experiments it has been taken for 
 granted that diastase is a single enzyme. Wijsman holds that 
 this is incorrect and that under the one name two bodies have 
 been included; one of them he calls maltose, which is not 
 however the enzyme several times alluded to above, but more 
 nearly corresponds to diastase itself in that it converts starch 
 into a mixture of maltose and erythrodextriu ; the second one 
 he terms dextrinase, and says that the formation of the 
 maltodextrin of Herzfeld and of Brown and Morris is due to 
 its action. He agrees with the other authors that maltodextrin 
 is converted into maltose by his maltase, but says that erythro- 
 dextriu is converted by dextrinase into a dextrin which he 
 calls leucodextrin, which does not reduce cupric oxide and is 
 not coloured by iodine. To show the presence of two enzymes 
 in the diastasic solution prepared from malt extract he adopted 
 the method of allowing it to diffuse into a gelatinous mass 
 made by adding gelatin to a solution of Lintner's soluble 
 starch. When a little diastasic solution was placed upon a 
 layer of the gelatin mixture the progress of the hydrolysis 
 could be traced by subsequently moistening it with iodine 
 solution. After 1 — 2 days' action, the area so treated formed 
 a colourless zone bordered by a violet ring, while the gelatin 
 with unaltered starch was coloured blue. From this Wijsman 
 concludes that the two enzymes diffuse into the gelatin at 
 
IV] AND ITS COURSE OF ACTION. 61 
 
 dififerent rates according to their concentration. The violet- 
 coloured ring showed where the maltase had penetrated beyond 
 the dextrinase, while in the uncoloured area both were present. 
 When a portion of the violet-coloured zone was removed and 
 placed on a fresh film of the starch-gelatin, and the enzyme 
 allowed to diffuse, no non-coloured zone was observed, but all 
 was coloured violet by iodine. 
 
 Wijsman's results appear however to be quite in keeping 
 with the view that only true diastase is concerned in the 
 transformation ; its progressive action would explain why the 
 centre of the zone of influence was uncoloured, while the violet- 
 coloured margin would indicate the earlier stages of the 
 hydrolysis. The portion of such a zone removed would contain 
 relatively little diastase and hence the violet reaction would 
 be given for a considerable time when such a piece was allowed 
 to act on the starch gelatin. 
 
 There appears therefore very little evidence to support his 
 hypothesis. 
 
 Duclaux also holds the view that the ordinary diastase of 
 malt is a mixture of two enzymes. Of these the first, which 
 he terms amylase, liquefies starch paste, converting the starch 
 into dextrin. The second, dextrinase, transforms this by hydro- 
 lysis into sugar. There is not much evidence again in favour 
 of this view, except that, as will appear later, the power of 
 liquefying starch paste and that of hydrolysing soluble starch 
 do not always go together. Duclaux' dextrinase does not ap- 
 pear to be identical with the hypothetical enzyme of Wijsman 
 bearing the same name. 
 
 Action of diastase on glycogen. 
 
 The hydrolysis of glycogen by dilute acids appears to 
 resemble somewhat closely the corresponding process in the 
 case of starch. According to the recent work of Miss Tebb 
 when glycogen is heated, not necessarily to boiling point, with 
 1 per cent, of hydrochloric acid, the opalescent liquid rapidly 
 becomes clear and limpid. If the heating is stopped at that 
 stage, and the liquid neutralised, the latter is found to give 
 
62 PREPARATION OF DIASTASE [CH. 
 
 a red colour with iodine and to reduce Fehling's solution. The 
 sugar can then be removed by dialysis, and subsequent satura- 
 tion with ammonium sulphate throws out a gummy precipitate 
 which consists of soluble glycogen. After complete removal of 
 this body, dialysis for several days is necessary to remove the 
 ammonium salt. Concentration and subsequent addition of 
 excess of alcohol leads to the precipitation of an erythro- 
 dextrin. 
 
 More prolonged action of the acid, extending over 1 — 2 
 hours, transforms both the soluble glycogen and the dextrin so 
 far that iodine ceases to give a colouration. Addition of alcohol 
 then causes the precipitation of another dextrin, belonging to 
 the achroo-group, which like the erythrodextrin first formed is 
 not precipitated by ammonium sulphate. 
 
 Miss Tebb gives the following table, showing the percentages 
 of alcohol necessary to precipitate .''tarch and glycogen and the 
 intermediate products of their hydrolysis. 
 
 Percentage of alcohol necessaiy to 
 
 Starch and its products 
 
 eommenoe 
 precipitation 
 
 complete 
 precipitation 
 
 Starch-paste 
 
 5 
 
 27 
 
 Soluble starch 
 
 12 
 
 60 
 
 Erythrodextrin 
 
 45 
 
 90 
 
 Glycogen and its products 
 
 
 
 Glycogen 
 
 35-5 
 
 55 
 
 Soluble glycogen 
 
 44 
 
 50 
 
 Erythrodextrin 
 
 44 
 
 90 
 
 Achroodextrin 
 
 65 
 
 90 
 
 The action of the diastase of saliva or malt-extract differs 
 in some particulars from that of dilute acids. There is no 
 appearance of soluble glycogen or of erythrodextrin, but 
 achroodextrin is always formed. This may only mean that 
 the action is so rapid in the early stages that the first two 
 are directly hydrolysed further. In addition, a new dextrin 
 belonging to the achroo-group appears, which is peculiar in 
 resisting conversion into sugar. This body Miss Tebb has called 
 
IV] AND ITS COUESE OF ACTION. 63 
 
 dystropodewtrin, identifying it with a substance previously ob- 
 tained by Seegen from glycogen and called by him by the same 
 name. It appears to differ from the other achroodextrin by 
 requiring greater concentration of alcohol for its precipitation. 
 
 The enzyme prepared from the liver acts similarly. 
 
 The sugar formed from glycogen by diastase alone appears 
 to be maltose, according to Miss Eves and other observers. 
 The liver enzyme yields dextrose as it does with starch, but as 
 already mentioned this is probably due to glucase being present 
 as well as diastase in the extracts of this orgfiii. 
 
64 
 
 CHAPTER V. 
 
 CONDITIONS OF THE ACTION OF DIASTASE. 
 
 Throughout the foregoing discussion of diastasic hydrolysis 
 we have considered the action of diastase on starch in a general 
 sense, but there are many points which need a further exami- 
 nation. Comparing the action of the two varieties of vegetable 
 origin we have seen that diastase of translocation dissolves the 
 starch grain without corrosion, while the so-called secretion- 
 diastase disintegrates the grain before hydrolysing it. When 
 these two kinds of diastase are compared with regard to their 
 action on starch-paste and on a solution of soluble starch we 
 find this difference of behaviour is emphasised. The enzyme 
 of the scutellar epithelium is capable of liquefying starch- 
 paste with great rapidity, while that of the general vegetative 
 body of the plant has very little power of doing so, though it 
 can hydrolyse soluble starch with the greatest ease. The 
 power of liquefying starch-paste and that of eroding the starch 
 grain seem to go hand in hand and to be a property pre- 
 eminently of secretion-diastase. This power can be estimated 
 separately therefore from the hydrolytic action which is common 
 to both varieties. We may indeed speak of the liquefying 
 power and the power of hydrolysing starch as in a way separate 
 from each other, though we have no instance of a digestive 
 solution which will carry on the former process and fail to 
 secure the latter. Both these properties of diastase are capable 
 of being modified by variation of the conditions under which 
 the action is carried out. 
 
CH. V] CONDITIONS OF THE ACTION OF DIASTASE. 
 
 65 
 
 The progress of the hydrolysis is further dependent to a 
 considerable extent upon the kind of starch undergoing de- 
 composition. Baranetzky has carried out many investigations 
 upon this point, with the result of establishing the fact that 
 ungelatinised potato-starch is highly resistant to diastase, while 
 most of the starches of seeds are readily acted upon. Buck- 
 wheat starch appears to be perhaps the most easily hydrolysed. 
 
 Diastases from diiferent sources appear to possess very 
 different powers of inducing hydrolysis. Roberts has compared 
 equivalent quantities of many preparations, with the results 
 given in the following table, taking the pancreatic diastase of 
 the pig as a standard. 
 
 Pancreatic secretion of pig 
 
 ... 100 
 
 Human saliva 
 
 ... 10-17 
 
 Pancreatic juice of sheep 
 
 ... 12 
 
 J, „ ox 
 
 ... 11 
 
 Malt diastase at 60° C 
 
 ... 10 
 
 40° 
 
 4-5 
 
 Human urine 
 
 •03-13 
 
 The susceptibility of diastase to differences of temperature 
 is a feature which it shares, as already pointed out, with most 
 other enzymes. In each case the action of the ferment is not 
 perceptible at 0° C. ; gradually rising with elevation of the 
 temperature it reaches an optimum point, or point at which it 
 works with greatest freedom; beyond this its activity falls with 
 further warming until at another point, generally rather high, 
 it ceases to work and is destroyed. We note a minimum, an 
 optimum and a maadmum temperature therefore in each case. 
 These can be expressed in the form of a curve, the abscissae of 
 which represent degrees of temperature and the ordinates 
 the hydrolytic power possessed at each temperature, estimated 
 by the amount of cupric-oxide-reducing power possessed by the 
 products of digestions carried on at such temperatures for the 
 same amount of time. 
 
 If we compare the curves of the two varieties of vegetable 
 diastase we find that of translocation diastase rises more 
 
66 CONDITIONS OF THE ACTION OF DIASTASE. [CH. 
 
 sharply from its minimum point than that of the other form, 
 being as high at 4° C. as is the latter at 14' 5° C. It reaches 
 its optimum point at 45° — 50° C, and thence declines. The 
 curve of secretion-diastase, though rising slowly, shows that 
 the optimum point of action is not reached till 50° — 55° C. 
 Complete curves of the action of the two varieties have been 
 published by Lintner and Eckhardt. 
 
 Mtiller has quoted figures representing the relative starch- 
 transforming powers of diastase at different temperatures. He 
 carried out digestions at 0° C, 10° 0., 20° C, 30° C. and 40° C, 
 and found the relative rates of sugar formation were respec- 
 tively 7, 20, 38, 60, and 98. 
 
 The optimum point of the action of the enzyme of the 
 saliva and pancreatic juice differs again from those of both the 
 vegetable diastases, being apparently about 38° — 40° C, the 
 temperature of the body. 
 
 Some investigations made in 1892 and subsequent years by 
 Efiront make it evident that the action of diastase is capable 
 of being materially modified by changes in the medium in which 
 it is at work. He has ascertained that the enzyme of malt is 
 favoured in its action by weak traces of mineral acid, a fact 
 which was noted earlier by Baranetzky. Slightly larger quan- 
 tities of neutral salt, such as sodium chloride, have the same 
 effect. Efifront has found further that there are three classes 
 of bodies which have the power of markedly increasing this 
 diastasic activity. These are salts of aluminum, compounds 
 of phosphoric acid, and various amide bodies, among which 
 asparagin is conspicuous. In his first paper Effront details 
 some experiments with these bodies which are extremely 
 striking. 
 
 The method he adopted was that of preparing a malt 
 extract by macerating a given weight of ground malt with 
 40 times its weight of water. The filtered solution was then 
 allowed to hydrolyse a solution of soluble starch, the various 
 bodies influencing the digestion being added at the same time. 
 The following table gives the comparative results of several of 
 them : — 
 
V] CONDITIONS OF THE ACTION OF DIASTASE. 67 
 
 . . , , o , Percentage of Maltose formed 
 
 Malt Starch foreign body per 100 c c 
 
 extract solution used starch solution 
 
 1 C.C. 200 C.C. 8-63 gms. 
 
 1 » 200 „ -7 Hydric ammonic 51-63 „ 
 
 phosphate 
 
 1 » 200 „ -5 Calcic phosphate 46-12 „ 
 
 1 n 200 „ -25 Ammonia alum 56-3 „ 
 
 1 » 200 „ -25 Potash alum 54-32 „ 
 
 1 ., 200 „ -25 Acetate of 62-4 „ 
 
 aluminium 
 
 1 ,, 200 „ -02 Asparagin 37-0 „ 
 
 1 » 200 „ -05 Asparagin 61-2 „ 
 
 The same relative results were obtained whatever was the 
 temperature of the digestion. 
 
 It made no difference whether the diastase was submitted 
 to the action of the reagents before being added to the starch, 
 or whether they were added to the digesting mixture. 
 
 In a later paper Effront says that an infusion of raw grain, 
 made in the cold, filtered, and boiled to destroy any diastase 
 derived from the grain, has a slighter but still similar effect, 
 often tripling the diastasic power of the malt. 
 
 In these experiments the difference which has been already 
 alluded to between the two effects of diastase comes out very 
 conspicuously. The reagents employed affect the hydrolytic 
 action in the manner described, but they have little or no 
 effect upon the power of liquefying starch-paste. 
 
 The influence of these reagents can only be detected in 
 the earlier stages of the transformation, the maximum being 
 reached when about 25 per cent, of the starch has been 
 converted into maltose. Beyond this point it is much less and 
 in the presence of such a quantity of diastase as will rapidly 
 cause the sacch^rifi cation of 60 — 70 per cent, of the starch it 
 becomes very slight. 
 
 Their utility has consequently by no means such a practical 
 application as at first seemed probable. 
 
 Effront points out that the liquefying and saccharifying 
 powers of malt extracts are not in fixed proportion, but vary 
 
 6—2 
 
68 CONDITIONS OF THE ACTION OF DIASTASE. [CH. 
 
 very much in different cases, depending upon the methods of 
 manufacture of the malt. 
 
 It has been noticed by other observers that addition of 
 proteids to malt extracts increases their hydrolytic powers. 
 Yeast cells, after their vitality has been destroyed by boiling, 
 have a considerable intensifying action, which is presumably 
 due to their proteid constituents. 
 
 A similar influence has been observed in the case of the 
 diastase or ptyalin of human saliva. Chittenden and Ely 
 ascertained that peptone has a remarkable effect upon starch 
 transformation. In a series of experiments carried out in 1881 
 these observers worked with quantities of 100 c.c. of liquid 
 containing 1 per cent, of starch, 1 per cent, of peptone and 
 25 c.c. of filtered saliva. Controls were digested simultaneously 
 in the preparation of which the peptone was omitted. The 
 mixtures were maintained at 40° C. for 4.5 minutes and then 
 boiled to stop the action of the' enzyme. After being diluted 
 with water to 500 c.c. they were filtered and an aliquot part 
 of each was titrated with cupric-potassium-tartrate. The 
 percentage of starch converted into sugar was 4 per cent, 
 greater in the presence of the peptone. Similar experiments 
 with glycogen instead of starch yielded the same results. The 
 quantity of peptone most favourable to the digestive process 
 was found to be 1 per cent. ; increase of the amount had little 
 or no additional effect. 
 
 Chittenden and Ely found further that the addition of 
 •025 per cent, of hydrochloric acid to the digestion containing 
 1 per cent, of peptone was still more advantageous; the 
 quantity of starch transformed amounting to 48 per cent, as 
 against 41 per cent, when saliva alone was used. 
 
 Langley and Eves have endorsed with a certain reserve the 
 statement of the influence of peptone, but they came to an 
 opposite conclusion to Chittenden's when they added acid in 
 addition. In their experiments they found that the maximum 
 effect of peptone was reached with a rather low percentage of 
 the proteid; when the saliva was diluted 10 times the most 
 advantageous amount of peptone was "1 per cent. They state 
 in opposition to Chittenden and Ely that peptone combined 
 
V] CONDITIONS OF THE ACTION OF DIASTASE. 69 
 
 ■with hydrochloric acid retards the action of the diastase, and 
 the retardation is greater the nearer is the point of saturation 
 of the peptone by the acid. Langley and Eves found also that 
 myosin, alkali-albumin, and acid-albumin act like peptone and 
 that albumin probably has the same effect. 
 
 It was mentioned above that according to Baranetzky small 
 traces of neutral salts accelerate the action of malt diastase. 
 Nasse states that this influence of certain neutral salts is 
 maintained till the proportion of the salt reaches 4 per cent. 
 Chittenden and Ely found that by using -024 per cent, of sodium 
 chloride the diastasic activity of saliva was increased about 
 6 per cent., and with -03 per cent, of calcium phosphate it rose 
 3 per cent. With hydric-disodic phosphate there was on the 
 other hand a slight diminution of the activity. 
 
 Osborne and Campbell also state that sodium chloride is 
 beneficial, sometimes increasing the diastasic activity of a 
 solution seven-fold. 
 
 Mllller points out an interesting circumstance connected 
 with the action of diastase in the vegetable cell. Under 
 ordinary conditions the cell-sap in the vacuole of such a cell 
 contains a considerable quantity of COj, corresponding in some 
 cases to a pressure of several atmospheres. Even at ordinary 
 pressures saturation of the liquid of a digestion with CO2 may 
 triple the energy of the diastase. In presence of CO2 diastase 
 can act on unboiled starch. 
 
 The evidence respecting the action of traces of free acid 
 upon diastase is conflicting. Baranetzky pronounced the pre- 
 sence of such traces to be favourable. He has been confirmed 
 by Effront and by Brown and Morris. Chittenden and Griswold 
 have stated that the ptyalin of saliva acts most energetically in 
 the presence of '005 per cent, of hydrochloric acid. Astachewsky 
 has pointed out that human parotid saliva is sometimes acid 
 and says that then it is more active than the alkaline parotid 
 saliva collected at other times. 
 
 On the other hand Langley and Eves found that the action 
 of neutralised saliva was retarded by the presence of free 
 hydrochloric acid, as little as '001 5 per cent, having a distinct 
 effect in this direction, while '005 per cent, was very strongly 
 
70 CONDITIONS OF THE ACTION OF DIASTASE. [CH. 
 
 inhibitory. The retarding effect increased rapidly as the per- 
 centage of the acid was increased. 
 
 When the proteids of saliva were saturated with acid, they 
 found a diminution of diastasic action, though no free acid was 
 present. This diminution was made more marked by the 
 addition of the smallest quantity of free acid. 
 
 Chittenden and Ely came to the same conclusion with 
 regard to the influence of hydrochloric acid. With '025 per 
 cent, of the latter present in a digestion, they found that the 
 amount of sugar produced was only about 8 per cent, of 
 that formed in a similar digestion without the acid. Osborne 
 and Campbell state that the same thing is true of citric acid, 
 as well as hydrochloric. 
 
 Langley and Eves suggest that the beneficial effect of the 
 small traces of acid ('005 per cent.) noticed by Chittenden and 
 Griswold may be explained by the fact that saliva is normally 
 faintly alkaline, and the acid would probably merely neutralise 
 this alkalinity. Saliva, according to them, works most favourably 
 in a completely neutral solution. Chittenden says however that 
 its reaction is due to alkaline phosphates and not to free alkali. 
 The influence of the free acid appears not to be merely 
 inhibitory, but the enzyme is destroyed by it. If acid is al- 
 lowed to remain in contact with the diastase for some time and 
 is then removed by dialysis, the enzyme has afterwards no 
 more power to hydrolyse starch than it has when the acid is 
 added to the starch solution in which digestion is going on. 
 
 According to Kjeldahl, malt diastase is made more active 
 by the addition of 20 milligrammes of sulphuric acid to each 
 litre of solution. This quantity must not be exceeded, for 30 
 milligrammes and upwards affect it prejudicially. Fernbach 
 makes a similar observation, but he attributes the improvement 
 caused by the smaller quantity to the action of the acid on the 
 phosphates present in the malt extract. Under normal con- 
 ditions the latter contains bibasic phosphates which inhibit 
 the action of diastase. The acid converts these into monobasic 
 phosphates, which accelerate it. After suflScient acid has been 
 added to effect this change in the phosphates, any further 
 quantity remains free and inhibits the diastase, as the other 
 observers quoted have pointed out. 
 
V] CONDITIONS OF THE ACTION OF DIASTASE. 71 
 
 Weak alkalis also have a deleterious influence on the action 
 of diastase. Chittenden and Ely found that when -6 per cent, 
 of sodium carbonate was added to a starch digestion, the 
 diastasic activity of the saliva was only about one fourth what 
 it was in the absence of the salt. With "3 per cent, the 
 power was about half and with "1 per cent, about two thirds of 
 that possessed by the unaffected saliva. 
 
 Langley and Eves found a distinctly inhibitory action to be 
 manifested by the presence of as little as '0015 per cent, of 
 sodium carbonate. Sodium hydrate was still more deleterious 
 than the carbonate. 
 
 Comparing the action of acids and alkalis, the rate of 
 decrease of the action of ptyalin under the influence of the 
 latter is slow compared with that of the free acid. 
 
 Another difference between the two is that while both are 
 strongly inhibitory, the weak alkali is not destructive of the 
 enzyme. If it is removed by dialysis the diastase is found to 
 possess nearly its original starch- transforming power. Sodium 
 carbonate therefore works by impeding the action of the enzyme, 
 while hydrochloric acid destroys it. 
 
 Chittenden and Ely have ascertained that the inhibitory 
 effect of both acids and alkalis is diminished by the coincident 
 presence of peptone. Langley and Eves, while disputing this 
 in the case of acid, agree with the other observers in the case 
 of alkali. They say that the favourable influence of peptone 
 makes itself felt with very high percentages of the alkaline 
 salt. 
 
 Brown and Morris state that the presence of tannin is very 
 prejudicial to the action of diastase ; this fact has no doubt 
 considerable importance when an attempt is made to ascertain 
 the amount of diastase in such vegetable tissues as contain 
 tannin. It is not only inhibitory of the diastasic action, but its 
 presence is a formidable obstacle to its extraction from the 
 tissue. 
 
 Loew has indicated hydrocyanic acid in 25 per cent, solution, 
 and atropine in small doses, as both deleterious to diastase. 
 
 It has been mentioned in a preceding chapter that it is only 
 with considerable difficulty and delay that the whole of the 
 starch in a digestion is converted into maltose. Different 
 
72 CONDITIONS OF THE ACTION OF DIASTASE. [CH. 
 
 observers agree in noticing this, though they differ as to tlie 
 amount which resists the change. Musculus only succeeded in 
 transforming 33 per cent, of the starch he used; Payen was 
 able to convert 52"7 per cent., while Brown and Morris give 
 as the usual result of a starch transformation 80 per cent, of 
 maltose, and 20 per cent, of dextrin. 
 
 As the digestion proceeds more and more difficulty is found 
 to attend its progress. This is possibly due to an inhibitory 
 action exerted by the products of the hydrolysis. Sheridan 
 Lea has thrown some light upon this question by carrying 
 out starch transformations simultaneously in flasks and in 
 dialysing tubes suspended in a stream of running water. In 
 the former apparatus the difficulty noted by other observers 
 was met with, but in the latter nearly all the starch was con- 
 verted into sugar. Lea states that under such conditions the 
 rate at which the digestion takes place is increased, and the 
 total amount of starch converted into sugar is much greater, 
 while the residue of dextrin is much less than under conditions, 
 otherwise similar, when the products are not removed. Brown 
 and Morris deny that the inhibition is dae to the presence of 
 maltose. They found that when maltose was added to a 
 mixture of starch and diastase, the course of the action was not 
 affected but that 80 per cent, of the starch still underwent 
 complete hydrolysis. 
 
 When a digestion is prolonged, this inhibition is seen even 
 in glass vessels to be only a retarding of the action. Brown and 
 Morris quote one experiment in which digestion was continued 
 for 20 days, chloroform being used to prevent putrefactive 
 changes. At the end of that time 92*4 per cent, of the starch 
 dissolved had been converted into maltose. 
 
 Another fact of great interest is that diastase is not ex- 
 hausted by continued activity. This we have seen to be a 
 property of enzymes in general. It was first established by 
 Foster in the course of an examination of the properties of 
 saliva. Moritz and Glendinning have noticed the same pe- 
 culiarity in the case of malt diastase, which they say is as 
 potent at the end of a fermentation as at the beginning, so long 
 as the temperature is at or below the optimum point. If the 
 digestion is carried out at a point higher than this, the enzyme 
 
V] CONDITIONS OF THE SECRETION OF DIASTASE. 73 
 
 loses power, but they consider this to be due to the temperature 
 and not to anything of the nature of exhaustion. 
 
 Conditions of the secretion of diastase. 
 
 In the simplest organisms in which the secretion of diastase 
 occurs, such as bacteria or moulds, or among the higher plants in 
 such simple structures as pollen grains, the formation of the 
 enzyme is found to have a direct bearing upon the question of 
 the nutrition of the organism. Lauder Brunton and Mac 
 Fadyen found that a microbe upon which they were experi- 
 menting secreted diastase when it was cultivated upon starch 
 paste, but did not do so when the culture medium was meat- 
 broth. In this case it gave origin to a peptonising enzyme. 
 Pfeffer has found that in the case of several of the mould fungi 
 the secretion of diastase depends upon similar conditions. In 
 the case of the pollen grains of several species of plants the 
 appearance of diastase only becomes noticeable after the 
 absorption of a trace of sugar by the grain. During the 
 growth of the pollen tube the writer has noticed that the 
 quantity of diastase is continuously increased, presumably in 
 consequence of the growing amount of nutritive material acting 
 as a stimulus to the protoplasm of the pollen. This is very 
 noticeable in the case of special species of Lilium. In Zamia 
 no diastase can be detected in the ungerminated pollen grains, 
 but on the absorption of either cane sugar or glucose, even 
 before visible extrusion of the pollen tube has taken place, a 
 small amount of the enzyme can be found to have been 
 secreted. The secretion of diastase by pollen is not altogether 
 dependent upon such absorption, for in the case of Lilium there 
 is a considerable formation when the grains are germinated in 
 pure water. Here the necessary stimulus for the increased 
 secretion may have been afforded by the transformation of a 
 small quantity of the reserve starch existing in the resting 
 grain by the diastase also present in its ungerminated condition. 
 
 The secretion of diastase by the scutellar epithelium of the 
 grasses does not appear to be caused by similar conditions. 
 Barley embryos cultivated by Brown and Morris upon gelatin 
 containing starch grains did not form more diastase than similar 
 embryos cultivated upon gelatin alone. The influence of the 
 
74 CONDITIONS OF THE SECRETION OF DIASTASE. [CH. 
 
 culture medium in this case does not appear to be the same as 
 in the case of the micro-organisms already mentioned. 
 
 In some other experiments the same authors found that 
 while certain barley embryos attacked and dissolved starch 
 grains suspended in gelatin, others, precisely similar, left the 
 starch untouched when a small amount of an assimilable sugar 
 had been added to the gelatin-starch medium. In this case, so 
 long as any sugar remained unabsorbed by the young plantlet, 
 the starch grains escaped disintegration. They were attacked 
 however as soon as the supply of sugar was exhausted. 
 
 The authors ascertained that in these cases there was no 
 question of inhibition of the action of diastase by the sugar, 
 but that in the presence of the latter, the epithelial cells of 
 the scutellum actually did not secrete the enzyme. This in- 
 hibition of secretion was only caused by such carbohydrates as 
 were directly assimilable by the embryos, other carbohydrates, 
 especially mannitol and lactose, which are of no use in 
 nutrition, being incapable of exerting any inhibitory power. 
 
 Not only the complete embryo but sections of the scutellum 
 covered by the epithelium showed this inhibition of secretion 
 by assimilable carbohydrates. 
 
 The phenomenon of secretion in the case of the scutellar 
 epithelium appears to be one associated with starvation. As 
 long as readily assimilable substances are supplied the secretion 
 of diastase does not take place, but when no such substances 
 are available, diastase is formed at once. 
 
 When barley is examined during the process of germination, 
 the epithelium is found not to begin immediately to secrete 
 the enzyme. Petit, who has studied the question, has ascer- 
 tained that diastase does not appear in germinating barley till 
 the fourth day. It reaches a maximum almost at once and then 
 gradually diminishes till the ninth day, when the grain contains 
 about one twentieth part of what it possesses at the maximum. 
 This fact supports Brown and Morris's view of the stimulus to 
 secretion being one of starvation. During the early part of the 
 period of germination the embryo is feeding upon the carbo- 
 hydrates it contains ; its needs at that period are small, and it 
 is not till growth has become vigorous that it needs to draw 
 upon the reserves deposited in the endosperm. After the ninth 
 
V] CONDITIONS OF THE SECRETION OF DIASTASE. 75 
 
 day these reserves are considerably depleted, and the young 
 embryo having by this time developed its rootlets is able to 
 draw in nutriment from without. 
 
 Brown and Morris also found that the secretion gradually 
 increases during germination. They differ from Petit however 
 in saying that the maximum secretion is not reached so soon. 
 They found a decided increase from the fourth to the seventh 
 
 day- 
 Evans obtained slightly different results during the process 
 of malting a Californian barley. The diastasic power at the 
 end of 24 hours from steeping was 27'8 ; it rose gradually for 
 three days, when it was 95-4 ; it then sank for three days, falling 
 to 50; from this time it rose till the ninth day, when it was 
 86"5 and then gradually fell till germination ceased, being 62 
 on the eleventh day and 28"3 on the fourteenth, when the 
 malting was completed. 
 
 The stimulus of starvation has been observed also by 
 Wortmann, who has found that a certain microbe has the power 
 of excreting a starch-dissolving enzyme when starch grains are 
 the only available food, but that no secretion takes place if 
 sugar or tartaric acid is offered to the organism along with the 
 starch. 
 
 The same thing has been observed in connection with other 
 enzymes. 
 
 In the animal body the formation of diastase is largely 
 dependent on nervous impulses. The salivary glands are those 
 most easily examined. Each in the higher mammals is supplied 
 by nerves derived from the cerebro-spinal and from the sym- 
 pathetip systems. Stimulation of either nerve is followed by 
 an increased flow of saliva and consequently by an increased 
 formation of the characteristic enzyme. The stimulation may 
 be either direct or reflex, the latter method being that which is 
 called into play during normal secretion. Sapid substances 
 placed on the tongue, or even the smell of food, will cause an 
 increased flow of saliva. A detailed examination of these 
 phenomena would however be beyond the scope of the present 
 work. It will suffice to say that if salivary glands are examined 
 before, during, and after excitation of the nerves with which 
 
76 CONDITIONS OF THE SECRETION OP DIASTASE. [CH. V 
 
 the glands are supplied, the appearances lead to the conclusion 
 that such stimulation produces the changes in the gland cells 
 which indicate a formation of the solid constituents of the 
 secretion from the protoplasm of the cells, and a subsequent 
 extrusion of them together with a certain flow of fluid derived 
 from the lymph with which the secretory tubules are bathed. 
 
 The physiological object of the secretion of diastase must in 
 nearly all cases be the digestion of solid starch or glycogen with 
 a view to the absorption and utilisation of the soluble and 
 diifusible products which the enzyme forms. This digestion 
 may be extracellular, as in the cases of the secretions of saliva, 
 pancreatic juice, secretion-diastase as supplied by the scutellar 
 epithelium, and certain bacterial diastases. Again it may be 
 intracellular as in the cases of the liver, and the ordinary 
 vegetable reservoirs, such as potato tubers. The translocation 
 of starch in the cells of foliage leaves and other green parts of 
 plants belongs to the same class of phenomena. The immediate 
 result is in both cases one of nutrition, either of the whole 
 organism, or of the cell in which the changes may be seen. 
 
 In the guard-cells of stomata the transformation of starch 
 appears to have a different meaning. The function of these 
 cells is primarily if not entirely the regulation of transpiration, 
 or the escape of watery vapour from the interior of the plant. 
 When the guard-cells are turgid from the absorption of water 
 into their interior, they become convex and separate slightly, 
 allowing the aperture between them to be enlarged. The 
 working of the stomata consequently depends upon their 
 power of absorbing water from the cells on which their guard- 
 cells abut. This water is attracted into them by osmosis, and 
 this physical process depends upon their containing substances 
 which have an attraction for water, or an osmotic affi/nity. The 
 aflSnity possessed by starch is practically nil, but sugars and the 
 acids derived from the latter possess a considerable power of 
 attracting water. Digestion of the starch by the diastase in 
 these cells consequently leads to the development in them of 
 osmotic powers which must be of great value in causing their 
 change of shape in the ordinary course of the transpiration 
 process. 
 
77 
 
 CHAPTER VI. 
 
 INULASE. 
 
 Besides starch and glycogen carbohydrate reserve-material 
 occurs in the vegetable kingdom in the shape of inulin. It 
 usually exists in a condition of solution in the sap of the cells 
 ■which constitute the reservoir, and may be precipitated there- 
 from by the addition of alcohol. 
 
 Inulin as a reserve- material has a wider distribution than 
 was till recently known to be the case. It has long been 
 recognised in a group of plants which are prominent among 
 the Compositae, the chief of which are the genera Dahlia, 
 Helianihus, Inula and Atractylis. These plants possess either 
 tubers, or tuberous or fleshy roots, and in these the stores of 
 inulin are located. More recently Parkin has ascertained that 
 inulin is a very common reserve material in many Monocoty- 
 ledons, occurring in great abundance in many of the bulbous 
 Liliaceae and Amaryllidaceae as well as in other Natural Orders 
 related to them. Here it may be found in the leaves and 
 other parts concerned in active vegetative metabolism as well 
 as in the seats of storage of reserve materials, the bulbs or 
 corms which are so characteristic of these Natural Orders. 
 
 Inulin can be detected in such structures as the tuber of 
 the artichoke {Helianthus tuberosus) by steeping pieces of the 
 tissue in alcohol for some time and then immersing sections of 
 the hardened material in water, when it is found to crystallise 
 out in the form of sphaerocrystals, sometimes of large size. 
 These are seen to consist of needles symmetrically arranged so 
 as to radiate from a common centre. They may be found in 
 
78 INULASE. [CH. 
 
 the interior of the cell, or may extend through several cells, 
 forming a comparatively large aggregation, visible in such a 
 case to the naked eye. The fleshy roots of the Dandelion 
 {Taraxacum), the Helicampane {Inula Heleniwm), also yield 
 these crystals on similar treatment. 
 
 In the cells of the tissue of the bulb-scales of the snowdrop 
 {GaloMthus) and other Amaryllidaceae the crystalline appearance 
 is not so readily presented, but the inulin may be detected in a 
 somewhat amorphous form. 
 
 Inulin is insoluble in cold water, but may be prepared from 
 these plants by boiling the tissue containing it with large 
 quantities of water, concentrating the decoction so obtained, 
 and allowing it to stand till it deposits a sediment. This must 
 then be redissolved in a small quantity of hot water, decolour- 
 ised by boiling with animal charcoal and again concentrated, 
 when fairly pure inulin is gradually deposited. It can be further 
 purified by redissolving it and again evaporating, when the 
 inulin is once more precipitated; finally it should be well 
 washed with cold water containing a little alcohol. 
 
 Inulin so prepared is a white powder which is readily 
 soluble in warm water and which remains in solution when tbe 
 liquid cools. Very little can be dissolved by cold water, and it 
 is insoluble in alcohol. A solution of it can be precipitated by 
 the addition of the latter reagent, the precipitate occurring 
 when the percentage of alcohol in the liquid reaches 65. 
 
 The composition of inulin, like that of starch, cannot yet be 
 said to be definitely ascertained. The formula usually given 
 for it is C12H20O10 2H2O. Under the action of hydrating agents 
 such as mineral acids it is converted into fructose, a sugar 
 which has a laevo-rotatory power [a]2, = — 106°. Inulin itself 
 has a specific rotatory power of [0]^) = — 39'9°. 
 
 Inulin can be converted into fructose by boiling its watery 
 solution under pressure, or by allowing it to remain in contact 
 with cold water for a lengthened period, care being taken to 
 prevent the access of micro-organisms. 
 
 In the process of germination of the tuber, or in the 
 resumption of growth of the plant from the fleshy roots, the 
 transformation of inulin into sugar is brought about by the 
 
VX] INULASE. 79 
 
 action of an enzyme to which the name inulase has been given. 
 The existence of this enzyme was first demonstrated by the 
 writer in 1887, when the germination of the tuber of the 
 artichoke was made a subject of examination. 
 
 Examination of the young shoots and roots arising from the 
 germinating tuber showed that while inulin was present in 
 them, they contained also a far larger proportion of sugar than 
 was present in the resting tuber of the plant. The sap of the 
 tuber when expressed was almost neutral; very sensitive litmus 
 paper showed a faint trace of acid, but too little to account for 
 the conversion of inulin into sugar by its instrumentality. 
 
 Tubers of the artichoke were allowed to germinate till the 
 young plants arising from them had attained a height of about 
 six inches above ground. The tissue of the tuber by this time 
 had been considerably altered, the interior having become 
 spongy and the cells almost empty, while on the outside and 
 for some distance inwards it was firm and succulent. Tubers 
 in this condition were minced finely and extracted with 
 glycerine, in which inulin is insoluble. After twenty-four 
 hours the glycerine was strained off and dialysed in several 
 changes of water, till the dialysate failed to reduce Fehling's 
 solution. 
 
 A small quantity, about 10 c.c, of such an extract was then 
 mixed with about 4 times its volume of a one per cent, solution 
 of inulin and put into parchment dialysing tubes, which were 
 suspended in about 200 c.c. of distilled water, thymol being 
 used to prevent the action of micro-organisms. A control 
 experiment was set up at the same time, in which the extract 
 of the tubers employed had been boiled to destroy any enzyme 
 that might be present. 
 
 The dialysates were changed at intervals of 24 hours and 
 each as it was removed was tested for sugar. 
 
 Hydrolysis of the inulin in the tubes containing the unboiled 
 extract, began gradually and proceeded regularly for several 
 days, the difference between the dialysates of the boiled and 
 unboiled tubes becoming more and more emphasised as the 
 digestion proceeded. That in which the boiled extract was sus- 
 pended contained no sugar after one or two changes, while that 
 
80 INULASE. [CH. 
 
 suri'ounding the unboiled extract continued to give a copious 
 reduction of Fehling's solution. 
 
 Similar results were obtained when the digestions were 
 carried on in glass vessels, a continuous formation of sugar 
 being indicated. 
 
 Coincidently with the appearance of sugar in the dialysates, 
 the amount of inulin in the parchment tubes underwent a 
 regular diminution. The amount of precipitate thrown down 
 by alcohol from the fluid containing the unboiled extract of the 
 tubers became less and less as time went on, measured quantities 
 being withdrawn for each observation. Similar quantities 
 taken from the controls showed no such diminution. 
 
 From these results it is evident that the germinating 
 artichoke contains an enzyme by whose instrumentality inulin 
 is transformed into some form of reducing-sugar. The time 
 taken up in the experiments is possibly to be accounted for by 
 the very small quantity of the enzyme present and its dilution 
 in the process of extraction. In the artichoke it is probable 
 that it only exists at any particular time in the cells whose 
 contents are actually being hydrolysed, and as it takes several 
 weeks for the conversion into sugar of the inulin present in any 
 tuber, there must be very little of the enzyme to be found at 
 any one moment. 
 
 This enzyme, inulase, cannot be found in the tuber in the 
 resting condition, nor is it easy to demonstrate its presence 
 until the young stems begin to emerge. Once found, however, 
 its presence can be demonstrated so long as any inulin remains 
 in the germinating tuber. 
 
 During the progress of the growth of the young stems, they 
 may be found to contain inulin, which occupies the centre of the 
 shoot and leaves the circumference free. Inulin possesses a 
 feeble power of dialysis, and it is no doubt by means of this that 
 it passes from the tuber into the shoot. The inulin cannot be 
 traced up to the growing point, but stops abruptly just behind 
 the actively growing zone. It is accompanied in its progress by 
 sugar, which extends further forwards towards the growing point. 
 The transformation under the action of inulase is not therefore 
 confined to the tuber, though it originates there. The enzyme 
 
Vl] INULASE. 81 
 
 is present in the growing shoot, and continues to hydrolyse the 
 inulin as growth proceeds. 
 
 The occurrence of inulin in this region may be due to 
 another cause than dialysis or transport of the store in the 
 tuber. It may be brought about by the supply of sugar being 
 in excess of the needs of the growing cells, the surplus being 
 temporarily converted again into inulin. Such a reconversion 
 is a matter of constant occurrence in the case of supplies of 
 starch in other plants where the latter is the form in which 
 the reserve store of carbohydrate material occurs. 
 
 The occurrence of inulase has been observed by Bourquelot 
 in the fungus Aspergillus niger. When cultivating its spores 
 in different media during the year 1885 he found that they 
 were capable of growing in a solution containing inulin as 
 freely as in others which contained glucose or cane-sugar. 
 At that time the enzyme had not been described, but on 
 returning to the subject in 1893 Bourquelot was able to prepare 
 it from the mycelium of the fungus. Aspergillus contains 
 several enzymes, but he was able to convince himself that 
 inulase was present and was a distinct entity, quite unlike 
 diastase. 
 
 Chevastelon has shown that the inulin which can be pre- 
 pared from the Natural Orders of Monocotyledons already 
 alluded to is capable of hydrolysis by the inulase derived from 
 Aspergillus. He worked especially with the inulin contained 
 in garlic. 
 
 The presence of this carbohydrate in such abundance in 
 Monocotyledons suggests that inulase is present in them also. 
 Up to the present year however it had not been identified there. 
 Recently the writer has ascertained that it can be obtained 
 from the bulbs of Leucojwm and Sdlla. Its existence is most 
 easily demonstrated in the germinating bulb, at the time when 
 the flowers are just formed and before they have expanded. 
 The method used by the author was exactly the same as that 
 described above, and the enzyme behaves in the same way 
 as that of the artichoke. It is more diflScult to extract on 
 account of the mucilage present in the cells. 
 
 The enzyme appears in all cases to be mainly if not entirely 
 
 G. F. 6 
 
82 INULASE. [CH. 
 
 confined to the cells in ■which the actual conversion of the 
 inulin is taking place. 
 
 The action of inulase can best be studied by carrying out 
 the digestion in dialysing tubes. In the author's experiments 
 such digestions were carried on under antiseptic precautions for 
 several days, the dialysates being changed every 24 houi-s. 
 The first three days' dialysates were rejected, to ensure that 
 whatever might be found in the later ones besides inulin should 
 be only the products formed by the action of the enzyme. The 
 fluid was of course bulky and contained only a relatively small 
 quantity of carbohydrates. The later dialysates were mixed 
 and concentrated over water baths till of small volume, when 
 they had a syrupy appearance and consistency. The syrupy 
 liquid was found to contain three bodies which could be 
 separated from each other by treatment with alcohol. The 
 first of them was a sugar which was separated by extracting 
 the residue with absolute alcohol. About half of the syrup 
 dissolved, and on concentrating the alcoholic liquid over a 
 water bath, it again became syrupy and remained so, refusing 
 to crystallise, even when exposed over strong sulphuric acid. 
 
 After separation from the sugar the residue was treated 
 with cold and subsequently with hot water. A good deal of it 
 dissolved in the cold water, which indicated that it was not 
 composed of inulin, as this is insoluble until the water is 
 warmed. The residue insoluble in gold, but soluble in hot 
 water, was found to consist of inulin, which having a feeble 
 power of dialysis had passed through the membrane during the 
 later stages of the digestion. 
 
 The two solutions were mixed and alcohol added gradually. 
 When the liquid contained 65 per cent, of alcohol the inulin 
 separated out as a crystalline precipitate. This was filtered off 
 and more alcohol added. When 82 per cent, of spirit was 
 present the liquid became opalescent and gradually a very 
 finely-granular precipitate settled out. In 100 parts of the 
 residue there were about 62'5 parts of unchanged inulin and 
 37"5 parts of this second body, which appeared to be an inter- 
 mediate body resulting from the action of the inulase. 
 
 On concentration of the watery solution of this substance it 
 
Vl] INULASE. 83 
 
 deposited a quantity of crystalline matter. The crystals differed 
 remarkably from the sphaero-crystals of inulin, appearing 
 generally as plates, sometimes pentagonal, sometimes rhom- 
 boidal or oblong, with here and there needle-like prisms 
 forming part of a rosette. 
 
 On examining several commercial samples of so-called 
 inulin, it was found that variable quantities of this inter- 
 mediate body were present. It can be separated from inulin 
 by fractional precipitation by alcohol or less easily by dialysis. 
 It has a greater power of dialysis than inulin; one sample of 
 the commercial inulin examined contained 12 — 14 per cent, 
 of it as ascertained by the alcohol method ; after dialysis 
 for five days the dialysate when concentrated contained both, 
 but the intermediate body formed sixty-six per cent, of the 
 total precipitate thrown down from it by alcohol. 
 
 This product then differs from inulin in the following 
 particulars : — 
 
 (1) It is more soluble in cold water. 
 
 (2) It has a greater power of dialysis. 
 
 (3) It has a different crystalline form. 
 
 (4) It is soluble in alcohol of sixty-five per cent, 
 strength, not being precipitated by less than eighty-two per 
 cent. 
 
 It is clear that this body occurred in the dialysates of the 
 digestions in consequence of its formation by the inulase and 
 not from having been present in the inulin used, for the 
 dialysates of the first three days were rejected. If any had 
 been mixed with the inulin taken for experiment this would 
 have escaped during that time, as its dialysing power is so 
 great compared with that of inulin. Its occurrence as a 
 consequence of the activity of the enzyme recalls the occurrence 
 of dextrins during the action of diastase on starch. It some- 
 what resembles dextrin in its behaviour with alcohol, being 
 soluble in stronger percentages of this reagent than the original 
 carbohydrate, but not soluble in a greater percentage than 
 eighty-two, at which point dextrin also is precipitated. 
 
 The sugar formed has been investigated by Bourquelot. He 
 quotes a series of observations made with a solution of inulin 
 
 6—2 
 
84 
 
 INULASE. 
 
 [CH. 
 
 containing 1-32 per cent., which was mixed with inulase and 
 left at the temperature of the laboratory till digestion was 
 complete. As it proceeded, the rotation of a ray of polarised 
 light by the digesting liquid was observed at definite intervals 
 of time, the temperature being noted at each observation. 
 The results are expressed in the fallowing table. 
 
 Duration of 
 digestion. 
 
 Botation 
 observed. 
 
 Temp, of 
 liquid. 
 
 Percentage of 
 
 reducing-sngar 
 
 calculated from the 
 
 observed rotation. 
 
 
 -1-06 
 
 17° C. 
 
 
 
 12 hours 
 
 -2-03 
 
 17° C. 
 
 0-871 
 
 36 „ 
 
 -2-43 
 
 17-5° 0. 
 
 1-283 
 
 64 „ 
 
 -2-5 
 
 19° C. 
 
 1-371 
 
 84 „ 
 
 -2-53 
 
 19-5° C. 
 
 1-403 
 
 The inulin used had a specific rotatory power of (a)j=— 39'9°. 
 
 From this calculation Bourquelot concludes (1) that the 
 proportion of reducing-sugar actually formed attained nearly 
 that which could theoretically be yielded by the inulin originally 
 present ; (2) that, reckoning the influence of the temperatures 
 noted on the rotatory power of levulose (fructose), the observed 
 rotation in the last observation corresponded very closely to 
 that which would be given by a solution containing a propor- 
 tion of levulose equal to that of the reducing-sugar found. 
 
 It thus appears that under the action of inulase, the inulin 
 used (prepared from Atractylis) was converted almost entirely 
 into levulose (fructose). 
 
 Bourquelot calls attention to a further fact of some interest. 
 When inulase is added to a solution of inulin, alcoholic fer- 
 mentation by yeast can be induced in the mixture. Now 
 inulin does not undergo the alcoholic fermentation, while 
 levulose does so readily. 
 
 Inulase is not present in the resting tuber of the artichoke, 
 as we have seen above. It can however be prepared from 
 such tubers before germination begins. If they are minced 
 and kept for 24 hours at a temperature of 35° C. the extract 
 prepared from them possesses the power of hydrolysing inulin^ 
 Without the preliminary warming such an extract is quite 
 
Vl] INULASE. 85 
 
 inert. The inulase appears to exist in such tubers in the form 
 of an antecedent body or zymogen. 
 
 Inulase has no action on starch ; it exists however in some 
 plants in the same region as diastase, with which there is some 
 danger of confusing it. In Aspergillus it is found associated 
 with several other enzymes, with one of which, trehalase, it may 
 be confounded. It can be distinguished from the latter by the 
 temperature at which it is destroyed. 
 
 Inulase works most advantageously in a neutral or very 
 faintly acid medium, the best proportion of acid being about 
 •001 per cent, of hydrochloric acid. Stronger acids than this 
 are prejudicial, and exposure for an hour at 40° C. to an acidity 
 equal to '2 per cent, of hydrochloric acid destroys it altogether. 
 Alkalies are also deleterious, no hydrolysing power surviving an 
 hour's exposure to a strength equal to 1"5 per cent, of sodium 
 carbonate. The rapidity with which the destruction of the 
 enzyme by acid takes place is dependent on the temperature 
 at which it is kept during the time the two are in contact. 
 At a low temperature it is much less affected than at 40° C, 
 but after an hour's exposure at only 10 — 15° 0. its working 
 power is much impaired. 
 
 The energy of inulase shows the same variation with the 
 temperature, being much greater at 40° 0. than at the ordinary 
 temperature of the laboratory. It survives exposure to a tem- 
 perature of 64° 0., at which point trehalase is destroyed, but it 
 loses its activity if heated to about 70 — 75° 0. 
 
86 
 
 CHAPTER VII. 
 
 CYTASE, AND OTHER CELLULOSE-DISSOLVING ENZYMES. 
 
 In the seeds of many plants, especially among the Mono- 
 cotyledons, the carbohydrate reserve food-material for the 
 nutrition of the embryo during germination takes the form of 
 extremely thickened cell-walls, usually those of the endosperm in 
 which the young plantlet is embedded. In other cases cell- walls 
 of comparatively little thickness serve the same purpose, though 
 to a much smaller extent, as may be seen in the barley and 
 other grasses. During the germinative processes these cell- 
 walls are dissolved and disappear, undergoing transformations 
 comparable with those of starch so far as to yield ultimately 
 some form of sugar. It has been usual to regard these cell- walls 
 as composed of cellulose, but recent researches show that they 
 are of a more complex nature than has been supposed. 
 
 The composition of the cell-wall varies very greatly in 
 different plants, and indeed in different parts of the same plant. 
 In addition to cellulose it contains bodies known as pectoses, 
 which include several very complex substances. The term 
 cellulose itself is now taken to cover a very large class of plant 
 constituents, the members of which show considerable differ- 
 ences in properties and in facility of decomposition. The 
 gradual utilisation of the cell-walls of different endosperms 
 involves very complex changes, in which both celluloses and 
 pectoses are concerned. 
 
 The cellulose bodies according to Cross and Bevan fall into 
 three distinct categories as under : — 
 
 (1) Those which offer a maximum resistance to hydrolytic 
 action, and which contain in their molecule no directly active 
 
CH. VIl] CTTASE, ETC. 87 
 
 CO groups. These are represented by the cellulose of cotton 
 fibres. 
 
 (2) Those of less resistance to hydrolysis, which contain 
 active CO groups. These are perhaps best regarded as oxy- 
 celluloses. They appear to constitute the main mass of the 
 fundamental tissue of flowering plants, and they exist in 
 conjunction with lignin in the walls of wood-cells. 
 
 (3) Those which hydrolyse with some facility, being more 
 or less soluble in alkalies and easily decomposed by acids, with 
 formation of carbohydrates of low molecular weight. Included 
 among these is the cellulose of the walls of the cells of seeds. 
 
 The members of these three groups behave very differently 
 with hydrolysing agents, and the ultimate products of their 
 decompositions vary considerably. 
 
 The pectose group of the constituents of the cell-wall has 
 been investigated by many writers, among whom may be 
 mentioned Payen, Vauquelin, Mulder, Fremy, Kutsch, Vogl, 
 and Wiesner. 
 
 The most recent observations have been made by Mangin, 
 who has given the fullest account of them and their chief 
 reactions. According to him they fall into two series, one 
 comprising bodies of a neutral reaction, while the others are 
 feeble acids. In each series there are probably several members, 
 which show among them every stage of physical condition 
 between absolute insolubility and complete solubility in water, 
 the intermediate bodies exhibiting gelatinous stages, charac- 
 terised by the power of absorbing water in a greater or less 
 degree. 
 
 Of the neutral series the two extremes are presented by 
 Pectose and Pectine. The former is insoluble in water and is 
 closely associated with cellulose in the substance of the cell- 
 membranes ; the latter is soluble in water and forms a jelly 
 with more or less facility. In the other series the two most 
 noteworthy members are Pectio and Metapectic acids. The 
 former generally exists in the cell-membranes in combination 
 with the metals of the alkaline earths, especially calcium ; when 
 in the free state, it is insoluble in water. Metapectic acid is 
 soluble in water, but does not form a jelly. 
 
88 CYTASE, AND OTHER [CH. 
 
 The two series are closely related to each other, for by the 
 action of heat, acids, and alkalies, the various members of both 
 can be prepared from peotose. The final product of the action 
 of the reagents is the freely soluble metapectic acid. 
 
 Mangin gives their characteristic peculiarities as under : — 
 
 Pectose. The actual properties of this substance are not at 
 all easy to ascertain, nor can they be said to be well known. 
 The material is so closely associated with cellulose that it has 
 not yet been obtained pure. The reagents which separate it 
 from cellulose convert it either into pectine or into pectic acid, 
 the former being soluble in water, the latter in alkalies. The 
 cell-wall can be shown to contain the two constituents by the 
 action of Schweizer's reagent (ammonio-cupric sulphate) which, 
 when used with proper precautions, dissolves out the cellulose 
 and leaves the framework of the cell apparently unaltered ; it 
 then consists, however, not of pure pectose, but of a compound 
 of pectic acid with the copper of the reagent. 
 
 Pectine. This body swells up and dissolves in water, forming 
 a viscid liquid which soon becomes a jelly. It exists in con- 
 siderable quantity in many ripe fruits and in some mucilages. 
 It gives no precipitate with neutral acetate of lead, but is 
 thrown down by the basic acetate in the form of white flocculi. 
 If boiled for several hours in water, it is converted into an 
 isomer, parapectine, which is precipitated by neutral lead 
 acetate. Further boiling with dilute acids converts it into 
 metapectme, which is precipitated by barium chloride. 
 
 Pectic acid. This body is insoluble in water, alcohol, and 
 acids ; it forms soluble pectates with alkalies and insoluble ones 
 with the metals of the alkaline earths, of which calcic pectate 
 is most widely distributed. It dissolves in solutions of alkaline 
 salts, such as the carbonates of sodium and potassium, stannates, 
 alkaline phosphates, and most organic ammoniacal salts, forming 
 with them double salts, which gelatinise more or less freely 
 with water. Its solution in alkaline carbonates is mucilagi- 
 nous, butSvhen ammonic oxalate is the solvent it is perfectly 
 limpid. 
 
 Metapectic acid. This is a body with an acid reaction, 
 freely soluble in water, and forming soluble salts with all 
 
VIl] CELLULOSE-DISSOLVING ENZYMES. 89 
 
 bases, especially those of calcium and barium, which precipitate 
 pectic acid. Metapectates warmed with an excess of alkali are 
 coloured yellow. This body and its compounds approach the 
 gums in their composition. When acted upon by dilute 
 sulphuric acid it splits up into a dextro-rotatory crystallisable 
 sugar, apparently identical with ardbinose, and into a little- 
 known organic acid, indicating by this behaviour some relation- 
 ship to the group of the glucosides. 
 
 The cellulose and pectose constituents of the cell-wall show 
 considerable differences of behaviour. The former are soluble, 
 the latter insoluble in Schweizer's reagent ; when oxidised with 
 nitric acid the former yield oxalic, the latter mucic acid. The 
 celluloses when partially hydrated stain blue with iodine; the 
 pectoses give no coloration with this reagent. They behave 
 differently also to staining reagents, and to dilute acids and 
 alkalies. According to Tollens and Ganz the sugar produced 
 by hydrolysing cellulose with dilute sulphuric acid is a dextro- 
 rotatory mannose. 
 
 Under the action of strong acids and other hydrating 
 reagents, a thickened cell-wall that has not been lignified or 
 cuticularised swells up strongly and shows traces of lamination. 
 The successive laminae correspond to the layers or shells of 
 thickening matter which have been deposited upon the original 
 membrane. Each layer contains a mixture of the substances 
 described, and they differ somewhat from each other in the 
 proportions of the constituents. 
 
 Between contiguous thickened cell-walls a very conspicuous 
 layer frequently appears, known as the middle lamella. This 
 can with difficulty be ascertained to exist in unthickened 
 cell-membranes. It gives characteristic reagents, which show 
 that it differs markedly from the rest of the cell-wall. Its 
 composition will be discussed in a succeeding chapter. 
 
 Gytase. 
 
 The composition of the cell-walls that ultimately yield 
 nutritive material for the young embryo during the germination 
 of the seed is thus extremely complex, and the products of their 
 digestion cannot be ascertained as easily as those which are 
 
90 CYTASE, AND OTHER [CH. 
 
 derived from the transformation of starch. During germination, 
 such transformations are brought about by the action of an 
 enzyme, which has been called cytase, though how far this is a 
 single enzyme, and if so which of the constituents it attacks, 
 cannot at present be said to be fully ascertained. A similar if 
 not identical enzyme has been discovered to exist in certain 
 fungi. It may be that we have several such enzymes, or there 
 may be only one. 
 
 The existence of cytase has not been known for many 
 years, our acquaintance with it dating back only to 1886, when 
 it was discovered by De Bary. In some investigations into the 
 life-history of a peculiar fungus, Peziza sclerotioriwm, he 
 found that while normally a saprophyte, it could under certain 
 conditions become a parasite, and that then it frequently 
 attacked reservoirs of reserve-materials, such as the roots of 
 carrots and turnips. In its behaviour it appeared to live 
 saprophytically, though infesting the living tissue. Its hyphae 
 or some of them excreted something which poisoned the cells, 
 the latter then yielding a nidus in which the hyphae spread and 
 multiplied. In its attack upon the living tissues this Peziza 
 formed dense masses of mycelia in the interior of the roots, 
 softening them as if they had been boiled. Besides assailing 
 reservoirs like the roots, it was capable of infesting the stems 
 of living plants, and produced there the same curious softening 
 of the substance. Microscopical investigations showed that the 
 hypha grew especially between the cells, destroying the middle 
 lamella and converting the cell-walls into a semi-mucilaginous 
 material. On studying cultures of the ascospores of this fungus 
 De Bary observed that each developed a mycelium of branched 
 septate hyphae, and that these formed curious organs of 
 attachment composed of branchfed hyphae in the shape of a 
 kind of tassel, which eventually gained the power of penetrating 
 through cellulose. If the culture was made upon a young 
 seedling. Petunia inolacea being peculiarly susceptible to its 
 attacks, these organs of attachment ultimately killed certain 
 cells of the epidermis and efiected an entrance into the sub- 
 jacent tissues, in which the hyphae subsequently ramified. The 
 cells in the neighbourhood of the hyphae were curiously 
 
VIl] CELLULOSK-DISSOLVING ENZYMES. 91 
 
 affected, losing their water and collapsing. The destruction 
 appeared to be caused by something excreted from the hyphae 
 and to prepare the way for their subsequent growth. 
 
 De Bary then examined the tissues attacked by this 
 excreted material; he expressed the juice from them, and 
 discovered that when pieces of the intemodes of the stem of 
 the bean, or fragments of fresh carrot, or of seedling cabbages, 
 were steeped in it, they underwent rapid destruction, the cells 
 becoming plasmolysed, and their walls swollen, while later the 
 middle lamellae were dissolved. 
 
 In the sap of the carrot expressed after the fungus had 
 attacked it De Bary thus discovered the presence of something 
 having the property of causing cell-walls to swell and of 
 dissolving a certain portion of them. When the expressed sap 
 was boiled it was found to lose this power. The fluid which 
 possessed the peculiarity of dissolving the cell-walls could also 
 be expressed from the sclerotia of the Peziza, so that the 
 substance in question was a secretion of the fungus and not a 
 product of the disintegration of the tissue of its host plant. 
 This substance was the enzyme now under discussion, to which 
 the name cytase has been given by subsequent observers. 
 
 In 1888 this enzyme was discovered by Marshal] Ward in a 
 fungus belonging to the genus Botrytis, which infests certain 
 lilies, especially lAlium candidum, and which presents many 
 features of resemblance to that investigated by De Bary. The 
 germinating spores of this Botrytis may be cultivated in a 
 hanging drop of culture-fluid suspended on a cover-slip over a 
 moist chamber in such a way as to be easily observed under a 
 microscope. The spore puts out hyphae which develop into a 
 mycelium ramifying through the culture-fluid ; from this short 
 branches grow out at right angles to the mass of the mycelium, 
 and run up to the lower surface of the cover-slip. As each 
 branch comes in contact with the glass it swells and softens, 
 and its outer contour becomes surrounded by a glairy film, 
 which is derived from some deliquescent constituent of the cell- 
 wall. In many cases these erect hyphae branch, either at or 
 near the extreme tip, or further behind. In some cases branches 
 from the mycelium grow from the surface of the hanging drop 
 
92 CYTASE, AND OTHER [CH. 
 
 towards the cavity of the moist chamber, and coming into 
 contact with the sides of the latter attach themselves to the 
 glass just as do those first described. These attaching organs 
 in many respects resemble those described by De Bary. 
 
 It may frequently be noticed that when attachment is not 
 made, the tips of the hyphae exude small drops of a translucent, 
 more or less viscous fluid, which contains a number of minute 
 brilliant granules, the protoplasm of the hypha becoming 
 extremely vacuolated. The exudation may go on for a few 
 hours, the drop becoming more and more granular, and its colour 
 changing to a pale brownish-yellow. The drops when tested 
 by microchemical reagents appear to contain quantities of 
 proteid matter, suggesting, in connection with the vacuolation, 
 that they are composed either of the protoplasm itself, or some 
 substance derived immediately therefrom. 
 
 These drops contain the enzyme cytase, which thus appears 
 to be a secretion of the protoplasm of the hyphae. Such 
 granules as have been described are often found to be associated 
 with the formation of enzymes in other cases. The secretion 
 is not the pure enzyme, but is complex in composition, con- 
 taining various proteid bodies, as already demonstrated. The 
 evidence which points to the existence of the enzyme in the 
 drops is twofold. The tips of the hyphae which extrude the 
 drops have been observed to penetrate into and through the 
 cell-walls of thin sections of parenchymatous tissue placed with 
 them on a glass slide on the stage of a microscope. Further, a 
 watery extract of such a mass of hyphae will cause unchanged 
 cell-walls to swell up when thin sections of parenchyma are 
 placed in drops of it. 
 
 The localisation of the enzyme in the tips of the hyphae 
 suggests that the softening of those tips when they come into 
 contact with the glass of the cover-slip, or the side of the moist 
 chamber, may itself be due to the action of the ferment, the 
 deliquescent matter which has been described as surrounding 
 the tips of the attached branches being transformed cellulose, 
 made mucilaginous in order to adhere to the surface. 
 
 The action of the cytase may be seen also in the process of 
 the growth and branching of the hyphae. The ferment is 
 
VIl] CELLULOSE-DISSOLVING ENZYMES. 93 
 
 secreted mainly at or near the ends of the filaments ; ac- 
 cumulating there, it causes a softening of the cell-wall either at 
 the apex or just behind it, enabling the hydrostatic pressure 
 within the hypha to originate a new branch, or to continue the 
 apical growth of the original filament. 
 
 When the mycelium is allowed to grow upon the surface of 
 a piece of tissue such as a leaf or bud of a lily, contact of the 
 erect branches with the epidermis of the latter creates a stimulus 
 to which the fungus responds by secreting the enzyme. It is 
 then discharged from the tips as in the cases described above, 
 but it does not attach the hypha to the epidermis as it does to 
 the glass slip. On the contrary it softens the walls of the tissue 
 and, slowly dissolving the cellulose, allows the hypha to make 
 its way into the interior ; subsequent continuous formation and 
 excretion of the enzyme enables the mycelium to develop at 
 the expense of the cell- walls of what has now become the host 
 of the parasite, together with whatever nutrient matter the 
 cells themselves contain. 
 
 It is interesting to notice in the behaviour of this fungus, 
 that the stimulus of contact with a living leaf seems to lead to 
 an increased formation of the enzyme. The secretion appears 
 to be a starvation phenomenon in the main, the fungus 
 appreciating the presence of food-material in the substance 
 touching it, and at once endeavouring to appropriate it. If 
 in a culture carried on under microscopic examination, a little 
 fresh soluble nutritive material is added to the culture fluid 
 when the mycelium has begun to attack a piece of vegetable 
 tissue by means of the exudation of these drops, the formation 
 and extrusion of the latter is at once suspended, and the 
 mycelium goes on growing and branching at the expense of the 
 new food supplied. When this store is exhausted the drops 
 are again formed and the tissue present in the culture fluid is 
 again attacked. 
 
 The cytase does not attack all parts of the cell-wall with 
 equal facility ; though it softens the outer layers of the epi- 
 dermal cells and penetrates transversely through the walls of 
 the subjacent parenchyma, it runs more frequently in the plane 
 of the middle lamella between the cells. The composition of 
 
94 CYTASE, AND OTHER [CH. 
 
 these three modifications of cell-wall we have seen to be very 
 varied, and it does not appear very evident what is the par- 
 ticular chemical change which is brought about. To this 
 point reference will be made later. 
 
 It was stated above that a watery extract of an active 
 mycelium possesses the power of causing the cell-walls to swell 
 up when a piece of tissue is placed in it. The enzyme can be 
 thus extracted from the fungus by artificial means; it is 
 moreover actually excreted from the mycelium into the 
 nutrient fluid in which the latter is cultivated. If either the 
 culture-medium or the expressed extract is boiled the power of 
 dissolving cellulose is lost, the cytase being destroyed. This 
 destruction of activity on heating may indeed be taken as 
 evidence that the action is really due to an enzyme. 
 
 Microscopic examination of a thick cell- wall exposed to the 
 action of cytase shows that the action of the latter resembles 
 that of dilute mineral acids, the successive laminae of the wall 
 becoming evident and each lamina in turn swelling up, and 
 ultimately dissolving. 
 
 Cytase may be prepared from such cultures as have been 
 described by grinding up the mycelium with sand, and pressing 
 out the fluid from the resulting pulp. It should then be 
 filtered and the filtrate allowed to fall into a large excess of 
 alcohol, when a precipitate will be thrown down, flocculent in 
 character, partly amorphous, and partly crystalline. In this 
 condition it is very impure, being mixed with several other 
 bodies. On dissolving the precipitate in water the solution will 
 be found to possess the property of the original liquid, and to 
 dissolve cell-walls with some facility. The middle lamella 
 between the cells of a piece of tissue placed in either is the 
 first part to disappear; the cells become separated and their 
 walls show evident lamination and swelling. 
 
 Cytase is seen from these observations of De Bary and 
 Marshall Ward to be secreted by fungi, and to play an im- 
 portant part in their development and in their nutrition. 
 Confirmation of their results has been obtained more recently 
 by Kean, who has separated a variety of cytase from Rhizopus 
 nigricans. What part of the fungus it resides in has not been 
 
VIl] CELLULOSE-DISSOLVING ENZYMES. 95 
 
 determined, nor is more said of its action than that it is capable 
 of softening cellulose cell-walls. In studying this fungus as 
 cultivated in gelatin media, Arthur has noticed that here and 
 there upon the sides of the hyphae there is an extrusion of 
 drops something like those of Ward's Botrytis, and further that 
 branches of the hyphae originate at the spots where the drops 
 had been observed. This is to a certain extent a corroboration 
 of Kean's statement. 
 
 Griiss has detected cytase in the mycelium of Penicilium. 
 
 Cytase has been discovered in the higher plants, but so far 
 as is known it has not the wide distribution of diastase. Its 
 detection is due chiefly to the investigations of Dr Horace T. 
 Brown, undertaken in some cases jointly with Dr Morris and 
 with Mr Escombe. 
 
 The first experiments which may be discussed were under- 
 taken by Brown and Morris in connection with a study of the 
 process of germination of the barley and other cereals. This 
 work has already been referred to as leading to the discovery of 
 the secretion of diastase by the epithelium of the scutellum. 
 It has already been said that cytase as well as diastase is 
 present in the material formed and excreted by these cells, 
 though not so much as by those of the aleurone layer. 
 
 Brown and Morris have described in detail the changes 
 which can be noticed in the endosperm immediately underneath 
 the scutellum. Between the starch-containing cells of the 
 former and the scutellar epithelium is a comparatively thick 
 layer of emptied and compressed cells forming a somewhat 
 transparent band of endosperm tissue, the starch these cells 
 originally contained having been utilised by the embryo 
 during its early growth and before it entered upon its resting 
 period. After 24 to 36 hours' germination the cell-walls of 
 this band of tissue are softened and partially dissociated. As 
 the germination proceeds, a dissolution of the walls of the 
 starch-containing cells also can be observed, which starts 
 under the aleurone layer and proceeds progressively from the 
 proximal to the distal regions of the endosperm. No action of 
 the diastase upon the starch is perceptible so long as these 
 walls are intact, indeed the diastasic digestion of the starch 
 follows continuously upon the disappearance of the cell-walls. 
 
96 GYTASE, AND OTHER [CH. 
 
 Brown and Escombe have observed similar changes taking 
 place in the endosperm of barley grains from which the embryo 
 has been carefully removed. The changes begin immediately 
 underneath the aleurone layer and spread thence into the 
 interior of the grain. The first effect of the germinative process 
 is the separation of the aleurone layer from the cells beneath it 
 by the solution of the cell-walls of that region. 
 
 If the progress of the dissolution of the cell-walls is 
 carefully followed, the course of events is seen to be the 
 following: at first the cell-wall swells up slightly and its 
 stratification becomes very apparent ; the separate lamellae are 
 then gradually disintegrated, the middle lamella being the 
 most resistant. Ultimately the whole of the cell- wall is broken 
 down into very minute spindle-shaped fragrnents, with their 
 longer dimensions arranged tangentially to the original cell- 
 wall. Finally these fragments also disappear. The whole 
 contents of the region of the endosperm affected become mealy, 
 even before the starch begins to be dissolved. 
 
 The same course of action can be traced in other grasses, 
 notably Bromus mollis, and B. asper, where the walls of the 
 endosperm cells are very considerably thickened. 
 
 The extent to which the dissolution of the cell-wall precedes 
 that of the cell-contents was measured by Brown and Morris in 
 some barley grains that had been germinated for 5 days. At 
 that period all traces of cell-walls had disappeared from most 
 of the tissue in the proximal region of the endosperm ; in- 
 dications of action on the starch-grains did not then extend 
 further than 0'15mm. from the scutellum. 
 
 The extraction of cytase from the germinated barley can 
 be effected with considerable ease. If an extract of air-dried 
 malt is prepared and sections of barley immersed in it, the 
 parenchymatous tissue becomes disintegrated in about 24 hours, 
 the cell-walls either disappearing or remaining in a much 
 swollen and altered form. Previous heating of the extract to a 
 temperature of from 60° — 70° 0. destroys its activity, the tissue 
 then remaining in it unaltered for an indefinite time. Nor is 
 the action confined to sections of barley, for pieces of potato 
 immersed in it lose their coherence in a few hours, and fall to 
 
VIl] CELLULOSE-DISSOLVING ENZYMES. 97 
 
 pieces with great readiness. The cell-walls may be seen to 
 swell up and become differentiated into numerous very thin 
 laminae, which later break up into spindle-shaped fragments 
 and ultimately disappear, with the exception of a thin layer 
 which probably represents the middle lamella. Brown and 
 Morris found that the parenchyma of different plants did not 
 all behave in the same way. The artichoke/ carrot and turnip 
 were like the potato m this respect, but the cell-walls of the 
 beet were affected only slightly, and those of the apple not at 
 all. Nor does cytase attack the thickened cell-walls of the 
 endosperm of Palms. These facts however need not surprise us 
 when we remember the probable differences in such cell- 
 membranes as stated at the commencement of this chapter. 
 
 Brown and Morris prepared the cytolytic enzyme by the 
 same method as they adopted in the case of diastase. They 
 precipitated an extract of malt by alcohol, dehydrated the 
 precipitate by absolute alcohol and dried it ia a vacuum. This 
 precipitate contained however both cytase and diastase. 
 
 Cytase is destroyed by heating its solution in water to a 
 temperature of 60° C, while diastase is unaffected at this 
 poiut. 
 
 The action of cytase is accelerated if the medium in which 
 it works is made slightly acid with formic or acetic acid. 
 
 Cytase only attacks unchanged cell-walls, having no action 
 on those which have undergone partial lignification or cuti- 
 cularisation. 
 
 Brown and Morris have shown that the secretion of cytase, 
 like that of diastase, takes place in the cells which constitute 
 the aleurone layer and to a less extent in those of the scutellar 
 epithelium. Their method was the same as in the case of the 
 diastase and has already been described in a previous chapter. 
 The secretion of both enzymes seems to be dependent on the 
 necessity for obtaining nutriment for the embryo; that is, it 
 is a phenomenon attending starvation. 
 
 In the paper by Brown and Escombe already quoted it has 
 been shown that the aleurone layer takes a much larger share than 
 the scutellum in the formation of cytase. In the earlier stages 
 of the germination the mealiness of the grain is due almost 
 
 G. F. 7 
 
98 CYTASE, AND OTHER [CH. 
 
 entirely to the enzyme secreted there, the secretion of the scu- 
 tellar epithelium being at that time of subsidiary importance. 
 
 Cytase exists not only in germinating barley, but in 
 certain varieties in the resting grain also. There appears 
 to be some relation between its occurrence and the climatic 
 conditions under which the cereal is cultivated. The more 
 perfect the latter, the less of the ferment is present. Brown 
 states that the presence of the enzyme can be demonstrated in 
 a cold-water extract of the grain, and its influence in the self- 
 digestion of the cell-walls is often distinctly noticeable when 
 the coarsely ground meal is macerated with water for a short 
 time at a temperature of 35° — 40° C 
 
 Brown has found evidence of the presence of cytase in rye 
 and in oats, the latter being especially rich in it. An aqueous 
 infusion of raw oats is in fact much more active in swelling and 
 dissolving cellulose than an extract made from an equal weight 
 of air-dried barley malt. 
 
 Cytase is not capable of resisting so high a temperature as 
 diastase, being destroyed at 60° — 65° 0., while diastase needs to 
 be heated to 70° 0. 
 
 Though the grasses have been shown to be especially rich 
 in C3rtase, this enzyme can be shown to be present in other 
 plants. It has been examined by Gardiner in the endosperm of 
 Tamus communis, where it can be observed to attack the 
 thickened cell- walls during the germination of the seeds. The 
 cell-membranes of this endosperm exhibit the continuity of the 
 protoplasm of the contiguous cells, threads of it being traceable 
 through the walls even before they become thickened. The 
 cytase is secreted in the interior of the cells and passes along 
 the connecting threads into the substance of the wall soon after 
 germination has begun. The penetration begins at several 
 spots in the wall, and when the cytase has once entered them 
 its action can be traced through the substance of the membrane, 
 quickly causing its complete disorganisation. The walls on 
 being attacked do not swell so markedly as in the cases already 
 mentioned, but their stratification or lamination becomes very 
 evident. The middle lamella in this case is the least resistant 
 to the action of the enzyme. 
 
VIl] CELLULOSE-DISSOLVING ENZYMES. 99 
 
 Newcombe has confirmed the results of Brown and Morris 
 completely, as to the occurrence and mode of action of cytase 
 in the barley grain. He has discovered its presence also in 
 germinating seedlings of Lupinus albus, and in the coty- 
 ledons of Pisum sativum and Fagopyrum esculentum (Buck- 
 wheat). 
 
 In his experiments he made a watery extract of the material 
 under investigation, and precipitated the cytase by the addition 
 of alcohol. He purified it to some extent by redissolving it in 
 water and reprecipitating it. Delicate sections of vegetable 
 tissue, generally starch-free sections of barley grains whose 
 protoplasm had been killed by heating them, were placed in 
 a faintly acidulated solution of the precipitate, and after a few 
 hours the cell-walls were found to have become hyaline, and 
 to be gradually dissolving. He found that the first part of 
 the wall to disappear was a strip on each side, intermediate 
 in position between the middle lamella and the free edge. 
 The middle lamella was the most resistant. 
 
 Newcombe also detected cytase in the so-called Taka- 
 diastase, which is a preparation of the fungus Aspergillus 
 (Eurotium) oryzce. The commercial product is a powder which 
 dissolves in thirty times its weight of water. Such a solution, 
 slightly acidulated with hydrochloric acid, was able to dissolve 
 the cell- walls of the barley endosperm morf rapidly than the 
 preparations made from the seedlings abeady alluded to. The 
 course of solution was however very different, the middle lamella 
 being the first part to be attacked. 
 
 All Newcombe's experiments were carried out in the 
 presence of chloroform. 
 
 There are several other plants in which . the presence of 
 cytase is probable, especially the Palms, in which the carbo- 
 hydrate reserve-material of the seeds takes the form of very 
 much thickened cell-walls. The thickening matter is composed 
 of a variety of cellulose mixed probably with pectic bodies. 
 Careful search has been made for an enzyme during the germin- 
 ation of the seeds of several species, but it is only recently that 
 satisfactory evidence of its existence has been obtained. The 
 process of germination is however so slow that if the enzyme 
 
 7—2 
 
100 CYTASE, AND OTHER [CH. 
 
 exists, it is in very small quantity at any time, and hence may 
 well escape the orc^inary process of extraction. 
 
 The first of the Palm seeds investigated was the Date 
 {Phcenix dactylifera), upon the germination of which Sachs 
 published some observations in 1862. The embryo lies em- 
 bedded in about the middle of the hard seed, and on germination 
 it remains attached to it by a portion of the cotyledon, which 
 becomes transformed into an absorbing organ. This continually 
 encroaches upon the endosperm as the young plant grows, 
 until the store of cellulose is exhausted. The surface of this 
 absorbing organ is covered by an epithelium which strongly 
 resembles the scutellar epithelium of the grasses. Sachs 
 suggested that the cells of this layer excrete a cytase, but he 
 did not definitely prove it. 
 
 Griiss states that there is an enzyme present in these seeds, 
 which acts on the reserve cellulose, but very slowly. It probably 
 yields mannose as the final product. 
 
 The writer made a series of observations in 1887 on the 
 germination of the seed of Livistonia humilis, which has a 
 similar structure to that of Phoenix. The progress of events 
 was the same as that observed by Sachs. The epithelium cells 
 were very granular and stained deeply with iodine and with 
 Hoffman's blue. By the use of these and other staining 
 reagents some additional facts were obtained bearing on the 
 behaviour of the cellulose. After two months' germination the 
 absorbing organ had penetrated about half-way through the 
 endosperm. The cells of the latter in contact with the cotyledon 
 were broken away at their edges, and when chlor-zinc-iodine 
 was applied to the affected area, the cells nearest the cotyledon 
 stained a deep violet, while those farther back were coloured 
 only a pale blue. Two well-marked zones thus became 
 evident, which differed in the chemical or physical constitution 
 of the cell-walls. An aqueous solution of iodine stained the 
 inner zone a pale blue, but did not affect the outer one. When 
 Hoffman's blue was used as a reagent the inner zone was 
 coloured very deeply towards its outer part, while the coloration 
 was gradually paler and paler as it was traced towards the 
 unaltered endosperm. The outer zone, slightly affected by 
 
VIl] CELLULOSE-DISSOLVING ENZYMES. 101 
 
 the chlor-zinc-iodine, was not coloured by the blue dye. The 
 inner deeply-staining part was much swollen and the outlines 
 of the cells were indistinct. The appearances led to the view 
 that the cellulose was being gradually hydrated, the change 
 starting from the absorbing organ and proceeding outwards. 
 
 When sections of the disintegrating endosperm were boiled 
 with Fehling's solution there was a copious deposition of 
 cuprous oxide on the surface of the inner zone and particles of 
 the same substance were seen to be embedded in the almost 
 deliquescent region abutting on the cotyledon. These gradually 
 became less numerous towards the interior of the umer zone and 
 did not extend beyond it. The cavity of the endosperm after 
 removing the absorbing organ showed a deposition of cuprous 
 oxide over its whole surface. There was an evident gradual 
 conversion of the cellulose into something that reduced Fehling's 
 fluid, presumably some form of sugar. 
 
 The appearances were consistent with the view that the 
 epithelium cells of the cotyledonary absorbing organ excreted 
 a cytasic enzyme, which gradually corroded and dissolved the 
 endosperm, forming a sugar capable of absorption. 
 
 More definite evidence has recently been secured by New- 
 combe. He cultivated date-seedlings in earth till the endosperm 
 was nearly half-consumed, and then collected the cotyledons 
 and the endosperm in which they were plunged. They were 
 separately either dried and ground in a mill and subsequently 
 extracted with water, or ground up with water and infusorial 
 earth while still moist. After a short extraction with water, 
 five times its volume of alcohol was added to each extract, when 
 a precipitate was obtained, which was filtered off and dried. 
 A very strong extract of the precipitate was made with a '05 
 per cent, solution of hydrochloric acid and a little chloroform 
 was added as an antiseptic. These extracts were found to be 
 capable of dissolving the cell-walls of barley endosperm after 
 the sections of the latter had been killed. The middle lamella 
 was found to be the most resistant layer to this preparation of 
 cytase. 
 
 The enzyme was found to be capable of dissolving the 
 cell-walls of the cotyledon of the lupin and those of the 
 
102 CYTASE, AND OTHER [CH. 
 
 endosperm of the date. In the latter case the process was one 
 of corrosion followed by solution, as in the case of Livistonia. 
 
 In some investigations upon various gums and the trees 
 yielding them Lutz has found that in Acacia gum-reservoirs 
 occur in the bark and in thepericycle. These consist of lacunae 
 caused by an enormous swelling and ultimate deliquescence of 
 the cell-walls. His experiments have led him to suggest that 
 this formation is due to a kind of cytase. 
 
 Elfving's observations on the germination of the pollen of 
 the grasses point to the existence of cytase in the pollen tube, 
 which was found to penetrate the tissue of the style by boring 
 its way through the middle lamellae of the cells, and not entering 
 their cavities. 
 
 De Bary describes the behaviour of a micro-organism. 
 Bacillus amylobacter, which is an agent in the destruction of 
 decaying parts of plants and which acts by destroying the cell- 
 membranes. He says it decomposes the cellulose, forming 
 dextrin and glucose, and that it does so by disengaging an 
 enzyme. It does not attack suberised membranes, nor those of 
 bast-fibres, of submerged water-plants, of Mosses and many 
 Fungi, but it rapidly decomposes the cell-walls of fleshy and 
 juicy tissues, such as those of leaves, herbaceous stems, and the 
 softer kinds of wood. 
 
 The digestion of cellulose in the intestines of animals 
 is probably brought about by similar organisms. No definite 
 evidence of the presence of cytase in these microbes is at present 
 forthcoming. De Bary does not quote experiments proving the 
 formation of the enzyme in the cases he mentions, and for the 
 present it can hardly be considered established. The course of 
 the decomposition too as suggested by him does not rest on an 
 experimental basis. 
 
 On the other hand certain experiments point to the exist- 
 ence of cytase in some animal secretions. Griffiths has found 
 that the digestive fluid which is present in the intestine of the 
 earth-worm dissolves cellulose. More recently Biedermann and 
 Moritz have shown that the secretion of the so-called liver of 
 the snail {Helix pomatia) contains a very active enzyme which 
 attacks not only the hemicelluloses of the cell-wall, but the 
 
VIl] CELLULOSE-DISSOLVING ENZYMES. 103 
 
 more insoluble constituents which have not been actually 
 Hgnified. The cytase is present in the liver as an antecedent 
 of the enzyme and is developed from the latter as the secretion 
 is poured out into the intestine. 
 
 Vignal has shown that Bacillus mesentericus vulgatus secretes 
 a cytase which dissolves the middle lamella of vegetable cells. 
 
 The products of the action of cytase have not been satis- 
 factorily examined up to the present time. The decompositions 
 are no doubt very complex, as we should infer from the varying 
 character of cell-walls. The cellulose constituents of the latter 
 ultimately yield some form of sugar, while the pectose is hydro- 
 lysed to metapectic acid, which also possibly forms a similar body. 
 By the action of acids, especially sulphuric acid, cellulose itself 
 can be hydrolysed through a series of dextrins to glucose. Some 
 of its modifications yield on hydrolysis pentose sugars as well 
 as hexoses. The pectoses yield a series of gelatinous hydrates, 
 resembling in this behaviour the mucilage-yielding constituents 
 of many seeds. Ultimately metapectic acid is formed, which 
 is apparently identical with the arable acid found in great 
 abundance in gum arable. From the latter by continued 
 hydrolysis, galactose (a hexose), and arabinose (a pentose), can 
 be prepared. 
 
 According to Tollens and Ganz cellulose can undergo trans- 
 formation into a sugar which they call seminose, which is 
 apparently identical with mannose. They prepared it by 
 warming mucilage with dilute acids. Reiss prepared the same 
 sugar from reserve-cellulose by the action of dilute sulphuric 
 acid. 
 
 Cross and Bevan say that when the oxycelluloses, such as 
 occur in lignified cell-walls, are boiled with sulphuric acid, at 
 first concentrated and subsequently diluted, they are resolved 
 into carbohydrates of low molecular weight, glucose and 
 sometimes also mannose being formed. The pecto-cellulose 
 group, of which the cell-walls of ordinary parenchyma consist, 
 yield in addition galactose and the two pentoses xylose and 
 arabinose. Galactose has been obtained from the cell-walls 
 of the seeds of Lwpinus luteus, Soja hispida, Goffea arabica, 
 Pimm sativum, Cocos nucifera, Phomioc dactylifera, etc. Man- 
 
104 CYTASE, AND OTHER [CH. 
 
 nose is obtained in relatively large quantity from many seeds, 
 especially those of Phytelephas, and pentoses from the seeds of 
 the cereal grasses and of several leguminous plants. 
 
 Reiss has stated that in the hydrolysis of cellulose obtained 
 from the seeds of Phoenix and Phytelephas, a body correspond- 
 ing to dextrin is first formed. It is tevo-rotatory. By further 
 hydrolysis it yields a dextro-rotatory sugar, identical with the 
 seminose of ToUens and Ganz. Schulze obtained both galactose 
 and a pentose, probably arabinose, from the thickened walls of 
 the cells of the cotyledons of Lupinus luteus. 
 
 Bourquelot and Hdrissey have recently described a variety 
 of cytase to which they have given the name pectinase, and 
 which they have found capable of acting on pectine, destroying 
 its power of gelatinising, and giving rise to a reducing-sugar. 
 They prepared it from germinated barley which had been slowly 
 dried at a temperature of 30° — 35° C, by extracting it with 
 chloroform water and adding alcohol till a precipitate fell. The 
 precipitate was collected and dried in vacuo. In three vessels 
 they prepared the following mixtures : 
 
 (1) 15 c.c. of a 1 per cent, solution of the precipitate, 
 
 and 15 c.c. of a 2 per cent, solution of pectine. 
 
 (2) A similar mixture, but the enzjone solution was boiled. 
 
 (3) A mixture like the first, with the addition of '05 grm. 
 
 of chalk. 
 These were then digested at the laboratory temperature for 
 42 hours. At the end of this time mixtures (1) and (3) had 
 lost the power of gelatinising, which was not affected in (2). 
 Alcohol gave an abundant gelatinous precipitate with (2) but 
 not with either of the others. Titrated with Fehling's solution, 
 (2) gave no reduction of the cupric oxide, while (1) showed the 
 presence of 11 mgrms. of sugar computed as glucose; (3) con- 
 tained 36 mgrms. of the same sugar. They attribute the 
 difference between (1) and (3) to the feeble acidity of the 
 pectine, which retards the action of the enzyme. This acidity 
 was neutralized in (3) by the chalk. 
 
 It is not clear that this enzyme is different from thau 
 discovered by Brown and Morris. 
 
 From a consideration of all these experiments there appear 
 
VIl] CELLULOSE-DISSOLVING ENZYMES. 105 
 
 to be two varieties of C3rtase, one attacking most readily the 
 middle lamella and the other the layers deposited upon it. 
 The first includes the enzymes discovered by De Bary, Marshall 
 Ward, Gardiner and Vignal; the second is the cytase of the 
 grasses and palms. Newcombe has prepared representatives of 
 both. 
 
 Carouhinase, 
 
 An enzyme which has much in common with cytase, if it is 
 not identical with it, has been described by Effront. It occurs 
 in the seeds of the Carob-bean (Geratonia siliqua) which are 
 sometimes used for fodder. These seeds contain a peculiar 
 carbohydrate, resembling a variety of galactan, which is differ- 
 ent from either starch or cellulose, though it seems to have a 
 composition similar to the latter. When the seeds, freed from 
 their coats, are digested with hot water over a water-bath, they 
 yield a transparent jelly which when diluted is somewhat 
 syrupy in consistence and can be filtered through silk. Addi- 
 tion of twice its volume of alcohol or baryta water precipitates 
 from this jelly a carbohydrate, which can be purified by re- 
 dissolving it and again precipitating it with alcohol. When 
 this product is dried at 100° 0. it is a white spongy, friable 
 substance, giving no coloration with iodine, and having the 
 formula CsHmOs. If soaked in water or weak solution of soda 
 it again forms a jelly or a viscid transparent mass: 3 — 4 grams 
 dissolved in a litre of water yield a viscous fluid of a syrupy 
 consistency. It is soluble in hydrochloric acid in the cold, and 
 the solution does not reduce Fehling's solution, and has no 
 action on polarised light. If oxidised by nitric acid, it gives only 
 small traces of mucic acid, showing that it is not of a pectic 
 nature. This new carbohydrate has been observed by Effront 
 not only in the Carob-bean, but also in the grains of rye and 
 barley. It appears to be a member of the group of celluloses, 
 and to resemble mucilage in its reactions. During the germi- 
 nation of the Carob-bean this body, to which Effront gives the 
 name caroubin, is readily hydrolysed by the action of an 
 enzyme, which he calls carouhinase. The hydrolysis takes place 
 
106 CYTASE, AND OTHER [CH. 
 
 feebly at first, but becomes vigorous as soon as chlorophyll is 
 developed in the young seedling. 
 
 In his experiments Effront proceeded as follows: — Seeds 
 of the Carob were bruised in a mortar, and extracted with 
 water to which a few drops of chloroform had been added, and 
 they were allowed to macerate for 24 hours at 30° C, after 
 which they were pressed and the extract filtered. The latter 
 was then examined for the enzyme. Glass vessels were taken 
 and in each were placed 50 c.c. of water, '1 c.c. of normal 
 formic acid and 1 gram of powdered caroubin. After mixing, a 
 measured quantity of the extract was added to each, and a little 
 chloroform used as an antiseptic. Controls were prepared in 
 each case, in which the extract used had been exposed for half- 
 an-hour to a temperature of 90° C. The activity of the extract 
 was estimated by the degree of gelatinisation remaining in each 
 case after a definite interval. The controls were found to 
 remain completely gelatinised, while the mixtures prepared 
 with unheated extract were liquefied more or less completely, 
 the amount of viscosity remaining being inversely proportional 
 to the quantity of extract used. Tested in this way, the latter 
 was found to liquefy the caroubin and subsequently to convert 
 it into a reducing-sugar. At the moment of liquefaction only 
 traces of the latter were present, but from that point onwards 
 the cupric-reducing power of the digestion continued to increase 
 as long as the experiment was continued. When an excess 
 of alcohol was added to the liquid at the moment of liquefaction, 
 it threw down a precipitate consisting of an intermediate pro- 
 duct, which was strongly dextro-rotatory and freely soluble in 
 water. This body was easily hydrolysed into sugar by the action 
 of dilute mineral acids. 
 
 The sugar resulting from the action was found to be un- 
 crystallisable, and to be a hexose. It was fermentable by yeast, 
 and with phenyl-hydrazine acetate it yielded an osazone which 
 melted at 183° 0. The sugar had the same reducing power as 
 glucose, but its specific rotatory power was (a)^=-|-24°. 
 
 Effront states that the enzyme can be precipitated by 
 addition of five times its volume of alcohol to the extract of the 
 seeds, in his experiments he was able by the method described 
 
VIl] CELLULOSE-DISSOLVING ENZYMES. 107 
 
 to compare the amount of caroubinase present in seeds at 
 different stages of germination. 
 
 Caroubinase acts energetically at 40° C, but its optimum 
 point is between 45° and 50° C. At 70° C. it acts with diflSculty, 
 and is destroyed at 80° C. It acts feebly in neutral solution, 
 and more energetically in the presence of '01 — '03 per cent, 
 of formic acid. 
 
 More recently Bourquelot and Hdrissey have examined the 
 reserve carbohydrate of the Carob-bean, and state that it is a 
 manno-galactan which on hydrolysis by sulphuric acid yields a 
 mixture of mannose and galactose. They have found similar 
 manno-galactans in the seeds of Trigonella fcenum-grcecum, and 
 of Medicago sativa, which yield similar products of hydrolysis. 
 There are probably several bodies of this sort in the different 
 seeds, as the relative amounts of mannose and galactose vary 
 in different cases. The lucerne (Medicago) yields 1'223 grm. 
 of mannose to 1'178 grm. of galactose ; the fenugrec gives a 
 proportion of 1'249 grm. of the former to •978 grm. of the 
 latter. Bourquelot and H^rissey have extracted the enzyme 
 from these germinating seeds. They suggest for it the name 
 seminase. 
 
 The sugar, called by Effront caroubinose, has been examined 
 also by Marlifere, who believes it to be a mixture of glucose, 
 fructose, and galactose. 
 
 Hadromase. 
 
 The varieties of cytase so far described act upon either the 
 pectose or the cellulose constituents of the unaltered cell-wall. 
 The more permanent tissues of the plant, ^specially the woody 
 ones, undergo profound changes of composition as they take on 
 their adult structure. Wood is marked by the presence of 
 lignin in the cell-wall. This new substance appears to arise 
 in consequence of a chemical change in the substance of the 
 ' membrane, which becomes marked as soon as the wall begins 
 to increase in thickness. The exact nature of the change is not 
 very definitely known, but it appears to be due to a chemical 
 alteration of some of the constituents of the wall, which are 
 
108 CYTASE, AND OTHER [CH. 
 
 converted into lignin. The change takes place gradually, and by 
 appropriate solvents the newly-formed matter can be separated 
 from the rest of the membrane, leaving the latter apparently 
 intact. Lignification is thus not a mere infiltration of lignin into 
 the body of the wall. The substance of the woody wall seems 
 to be a compound of cellulose and lignin having the composition 
 of an ether. The lignin can be recognised by turning red when 
 treated with phloroglucin in the presence of a weak mineral 
 acid. 
 
 Many fungi have been found to live upon lignified cells, 
 their mycelium penetrating the woody walls and deriving a 
 certain amount of their nourishment therefrom. Among these 
 fungi may be mentioned Merulius lacrymans, Pleurotus pul- 
 monarius, and Stereum hirsutum. 
 
 Czapek has found that an enzyme is present in the mycelia 
 of these plants, and that it can be extracted from them by the 
 ordinary methods. The extract so prepared can effect the 
 decomposition of woody substance if macerated with it for 
 some days, and can bring the lignin constituent into solution, 
 leaving the cellulose framework intact. He made a watery 
 extract of the tissue of the fungus, and after adding to it a 
 little chloroform and some shavings of wood, set it aside at 
 28° C. to digest. After about a week's exposure he found 
 the wood was undergoing decomposition. When a sample of 
 the digestive liquid was evaporated to dryness, the residue 
 gave up to alcohol the material which becomes red in colour 
 when treated with phloroglucin and hydrochloric acid. The 
 action went on for several days, and at the end of the experi- 
 ment the residue of the woody matter only gave the cellulose 
 reaction with iodine and sulphuric acid. 
 
 A boiled control showed the wood unaltered at the end of 
 the same time, and an alcoholic extract of it gave no reaction 
 with phloroglucin. 
 
 The enzyme, which Czapek has named hadromase, can be 
 precipitated by alcohol from a watery extract of the fungus as 
 a grey powder. A solution of this in water possesses the same 
 power as the original extract. 
 
 Hadromase thus separates the woody constituent of a 
 
VIl] CELLULOSE-DISSOLVING ENZYMES. 109 
 
 lignified cell-wall from the cellulose basis in which the lignin 
 is formed, dissolving the former and leaving the latter un- 
 attacked. Czapek found that it was associated in the fungi 
 with cytase, which subsequently dissolved the cellulose 
 
 Gum ferment of Wiesner. 
 
 In 1885 Wiesner described an enzyme existing in gum- 
 arabic, which he says transforms cellulose into gum or mucilage 
 and which has the power of converting starch into dextrin but 
 not iato a reducing-sugar. In his experiments he used 5 c.c. of 
 a 2 per cent, solution of gum-arabic and 1 c.c. of thin starch- 
 paste. After 6 hours' exposure to a temperature of 23° 0. the 
 solution of starch became limpid; after 24 hours it gave the 
 erythrodextrin reaction with iodine, and subsequently this 
 disappeared and achroodextrin only remained. No reducing 
 sugar was formed in 24 hours. 
 
 Reinitzer subsequently examined the action of gum-arabic, 
 and came to conclusions opposed to Wiesner's. He found that 
 the solution of gum had no action on cellulose, and that it 
 converted starch-paste into a reducing-sugar. He held it 
 therefore to be only diastase. 
 
no 
 
 • CHAPTER VIII. 
 
 SUGAR-SPLITTING ENZYMES. 
 
 The enzymes which we have discussed so far have been 
 shown to have one peculiarity in common; they set up different 
 series of transformations of various carbohydrates, and the final 
 product of each action is a sugar, although the same sugar is 
 not formed in every case. 
 
 Before Fischer started his classical researches on the sugars, 
 our acquaintance with them hardly extended beyond such as 
 contained within their molecule 6 carbon-atoms or a multiple 
 of them. Of those with 6 carbon-atoms we knew glucose, 
 fructose, galactose and sorbose. Kiliani showed that another 
 sugar, arabinose, has only 5 carbon-atoms. Fischer has shown 
 that the simpler sugars are to be regarded as aldehyde-alcohols, 
 or as ketone-alcohols, and that the simplest of them is glycoUic- 
 aldehyde G2H4O2. We can now distinguish a series of these 
 sugars, the successive members of which differ from each other 
 by CHjO. Fischer has shown that the synthesis of these can 
 be as easily accomplished as that of the organic acids and other 
 compounds. On the hypothesis of Le Bel and Van t'Hoff on 
 the asymmetric carbon-atom, there should be various isomerides 
 of each of these sugars except the first, the number varying 
 according to the configuration of the molecule. Sixteen dif- 
 ferent isomeric sugars should theoretically exist having the 
 formula CeHjaOo, and the synthesis of twelve of these has 
 already been accomplished. 
 
 Besides these sugars another series exists, the polysac- 
 charides, which may be regarded as derived from the first 
 series by the elimination of one molecule of water from two or 
 
CH. VIIl] SUGAR-SPLITTING ENZYMES. INVERTASE. Ill 
 
 more molecules of the simpler sugar. When they undergo 
 hydrolysis through the dilute action of mineral acids the 
 change which takes place is represented by the equation 
 CiaHjiiOn + H2O = C|jHi20e+ CsHijOs. The resulting hexoses may 
 be the same, or may be different ones ; cane-sugar yields glucose 
 and fructose; milk-sugar glucose and galactose; maltose gives 
 two molecules of glucose only. 
 
 In the living organism, whether animal or vegetable, we 
 find enzymes which are capable of effecting the hydrolysis of 
 these polysaccharides with considerable facility. Six of them 
 have been discovered up to the present time, and have been 
 named invertase or invertin, glucase or maltase, lactase, trehalose, 
 raffinase and melizitase. 
 
 Invertase (Invertin, Sucrose). 
 
 As long ago as 1847 Dubrunfaut discovered that cane-sugar 
 could be decomposed by the action of dilute mineral or organic 
 acids, which cause it to take up water, and split into equal 
 quantities of glucose and fructose, the former rotating a ray of 
 polarised light to the right, the latter turning to the left. The 
 process of the decomposition has been termed inversion and has 
 given its name to the enzyme under discussion. Cane-sugar 
 itself has a dextro-rotatory power, but the mixed sugars resulting 
 from its transformation turn the ray of polarised light to the 
 left. This inversion of the direction of the displacement of 
 the polarised ray is due to the fact that the levo-rotatory power 
 of fructose is considerably greater than the dextro-rotatory 
 power of glucose, and as the two hexoses are formed in equal 
 quantities, the resulting mixture of the two must deflect the 
 ray to the left. 
 
 In his experiments upon this process of inversion Dubrunfaut 
 succeeded in separating the two hexoses from each other by 
 treating a solution of the mixture, now commonly known as 
 invert-sugar, with a salt of calcium. This forms a compound 
 with both glucose and fructose, but that with the former is 
 much the more soluble of the two. When a mixture of these 
 sugars is treated in this way the fructose-lime compound 
 slowly crystallises out and can be separated from the other by 
 
112 SUGAR-SPLITTING ENZYMES. INVERTASE. [CH. 
 
 filtration. The crystals can be dissolved in an excess of water, 
 and the two sugars can both be freed from calcium by passing 
 a stream of COa through the respective solutions. The pre- 
 cipitate of calcium carbonate can be removed by filtration and 
 the separated sugars left in solution. 
 
 Dubrunfaut did not discover the enzyme which can set up 
 this change. The latter remained unknown until 1860, when 
 it was isolated from yeast by Berthelot. Its discovery is to be 
 associated with the fact that cane-sugar is not fermentable by 
 yeast, but that the alcohol produced by the latter arises from 
 the sugars into which cane-sugar splits on hydrolysis. The 
 earlier workers attributed this inversion to a certain acidity of 
 the yeast, Pasteur suggesting that it arose from the succinic 
 acid which he proved the latter to produce during fermentation. 
 Berthelot was able however to show that it was quite inde- 
 pendent of the reaction of the wort. He proved it to be due 
 to a soluble principle which is present in the water in which 
 yeast has been washed, and which is therefore presumably 
 excreted by the cells of the organism. Berthelot gave this new 
 body the name " ferment glucosique." 
 
 In his experiments he pressed the yeast till it was free from 
 the wort in which it had been fermenting, and after washing 
 the powder, allowed it to macerate for several hours with twice 
 its weight of water. After filtering he found that the filtrate 
 contained 1-5 per cent, of soluble matter. His first point being 
 the investigation of Pasteur's hypothesis that the active agent 
 in the hydrolysis of cane-sugar was the succinic acid of the 
 fermentation, he added some of his extract to a solution of cane- 
 sugar, containing 20 per cent, of the latter and 2 per cent, of 
 sodium bicarbonate. Under such conditions he found hydrolysis 
 took place and the alkalinity of the solution was not disturbed. 
 The change was therefore clearly not brought about by any acid. 
 
 Berthelot further isolated the enzyme, though he did not 
 prepare it in anything like a pure condition. His method 
 consisted in precipitating it from an extract of yeast by the 
 addition of an equal volume of alcohol. The precipitate' took 
 the form of white flocculent aggregations which sank to the 
 bottom of the flask. They were separated from the liquid by 
 
VIIl] SUGAR-SPUTTING ENZYMES. INVEETASE. 113 
 
 decantation, and washed with alcohol at the ordinary tempera- 
 ture, when a yellowish horny substance was obtained, weighing 
 about one-fiftieth of the original soluble matter. Berthelot 
 found that a repetition of this treatment considerably impaired 
 the hydrolytic power of the product. When prepared by a 
 single precipitation only, between 50 and 100 parts of cane- 
 sugar could be inverted by one part of the enzyme. 
 
 In 1864 Bechamp published the results of some investiga- 
 tions which considerably extended the state of knowledge of the 
 distribution of this ferment. Besides existing in yeast, he says 
 it is present in several moulds and in certain micro-organisms, 
 though he does not specify them. If these moulds are bruised 
 in solutions of cane-sugar and the resulting mass filtered, the 
 filtrate can soon be found to contain glucose. When sufficient 
 quantity of the mould can be obtained, an extract can be 
 prepared of it in the same way as Berthelot's extract of yeast, 
 or the active principle can be precipitated from an infusion of 
 the mould by the method adopted by Payen and Persoz in 
 the case of diastase. In either case it is found capable of 
 hydrolysing solutions of cane-sugar of various degrees of con- 
 centration, but it loses the power if heated to a temperature 
 of 60°— 70° C. 
 
 Besides investigating its occurrence in fungi, Bechamp 
 sought for it in the higher plants. Supposing that the presence 
 of chlorophyll indicated a radical difference between the nature 
 of the ultimate metabolism of the green plants and that of 
 the colourless fungi, he formed the idea that those parts of 
 the higher plants which had no chlorophyll would probably 
 show the same phenomena as the fungi. Acting on this idea, 
 he sought for the hydrolysing principle of the moulds in the 
 coloured parts of various flowers. Though his fundamental 
 position was unsound, he was nevertheless fortunate in his 
 pursuit of resemblances of behaviour, for he discovered the 
 enzyme in the petals of Robinia viscosa, R. pseudo-acacia, 
 Papaver Rheas, and some varieties of Rosa; also in the 
 coloured bracts of Bougainvillea spectahilis. He has described 
 his experiments with the petals of Robinia viscosa in detail. 
 Taking a quantity of flowers, he bruised the petals and 
 
 G. F. 8 
 
114 SUGAR-SPLITTING ENZYMES. INVERTASE. [CH. 
 
 expressed the sap from them; dividing the liquid into two 
 parts, he added to one a certain weight of cane-sugar and a 
 definite quantity of water and observed the rotation of a ray of 
 polarised light sent through a length of 200 mm. of the 
 mixture. The other half was then boiled and made up to the 
 same strength of cane-sugar as the first and its rotatory power 
 observed. The two coincided, showing that the boiling of the 
 juice was not accompanied by any change that would interfere 
 with subsequent comparison of the two digestions. He pro- 
 tected himself against disturbance by micro-organisms by the 
 addition of two drops of creosote to each 100 c.c. of the 
 digesting liquids. The two preparations were then set aside at 
 the ordinary temperature and the rotatory power was examined 
 at intervals. The results were as under : — 
 
 10 c.c. sap, 20 gms. cane-sugar, 2 drops creosote, Boiled 
 
 made up to 100 c.c. with distilled water. control. 
 
 Initial rotation + 29-52° + 29-52° 
 
 Botation after 24 hours' digestion + 28-8° + 29-52° 
 
 „ 20 days „ - 4-8° + 29-28° 
 
 Bechamp failed to find the enzyme in the leaves of plants, 
 a fact which seemed to lend a certain plausibility to his 
 hypothesis. The optimum temperature for its working he 
 determined to be 40°— 50° C. 
 
 Bechamp thought that the enzyme from fungi was not 
 quite the same as that existing in the flowers, the latter in his 
 opinion not being so active as the former. He gave to the 
 first the name zymase, which a little later was changed to 
 zyihozymase, while he called the second anthozymase to indicate 
 its localisation. 
 
 He identified a similar body in the fruit of the Mulberry, 
 but finding that the juice of the latter was at the same 
 time capable of saccharifying starch solutions, he thought it 
 contained a new enzyme possessing both properties. He 
 named this new ferment morozymase. In the light of sub- 
 sequent discoveries we can say with tolerable certainty that it 
 was a mixture of his zymase and diastase. 
 
 The name zymase was soon abandoned, as it began to be 
 
Vlll] SUGAR-SPLITTING ENZYMES. INVERTASE. 115 
 
 applied to enzymes in general. The term invertin was sub- 
 stituted for it by Donath in 187-5, and this name has been used 
 in connection with the enzyme until quite recent years. It is 
 indeed still employed by writers on animal physiology to denote 
 an identical enzyme which has its origin in animal secretions. 
 In vegetable physiology it is becoming replaced by invertase. 
 
 The recognition of the existence of this enzyme among the 
 secretions of the alimentary canal of animals was first made by 
 Claud Bernard. He ascertained that cane-sugar when present 
 in the blood of an animal is of no value for purposes of 
 nutrition. When injected into a vein it is almost immediately 
 excreted from the body by the kidneys and appears unchanged 
 in the urine. When taken into the alimentary canal by the 
 mouth, however, it has a distinct nutritive value. Bernard was 
 able to localise the region in which it undergoes digestion, and 
 he discovered that it is hydrolysed in the course of its progress 
 through the small intestine, and gives rise to the same reducing- 
 sugars as are formed by the extract of yeast. If a solution of 
 cane-sugar is placed in a loop of the intestine, or if it is mixed 
 with an infusion of the mucous membrane of that region, after 
 a very short interval the liquid is found to have acquired the 
 power of reducing Fehling's fluid, which cane-sugar is unable 
 to accomplish. The power of rotating a ray of polarised light 
 possessed by the original solution at the same time becomes 
 changed, and a comparison with a similar cane-sugar solution 
 treated with a dilute mineral acid shows that the products 
 of the action are the same in both cases. Bernard showed the 
 transformation thus taking place in the intestine to be due to 
 an enzyme identical with that extracted by Berthelot from 
 yeast. He established the existence of invertin in the in- 
 testines of rabbits, dogs, birds, and frogs. Other subsequent 
 investigators have shown that it exists also in the alimentary 
 canal of some of the Invertebrata ; Balbiani proving its presence 
 in the digestive apparatus of the silkworm. Von Planta says 
 the stomach of the bee contains a juice in which invert-sugar is 
 present, while its food consists of cane-sugar. 
 
 Bernard's discovery that the animal organism is not able to 
 assimilate cane-sugar as such, led him to the view that this 
 
 8—2 
 
116 SUGAR-SPLITTING ENZYMES. INVERTASE. [CH. 
 
 form of carbohydrate is probably not of immediate nutritive 
 value to vegetable protoplasm either, a similar parallelism 
 having been established in the case of starch. With the object 
 of testing this hypothesis he instituted a series of researches 
 upon the chemical processes involved in the resumption of 
 development of the Beet after the formation and the resting 
 period of the fleshy root. In the life-history of this plant two 
 well-marked periods can be observed. During the first year of 
 its life it produces only leaves ; at the end of the summer the 
 root becomes enormously enlarged and the sub-aerial parts die 
 down. The succulent tissue of the root is found to contain a 
 very large amount of cane-sugar which has been transferred to 
 it from the leaves during their summer activity. After the 
 winter, growth is resumed, and a new stem is put out which bears 
 leaves and gives rise during the next summer to the flowers, 
 fruit, and seeds. This renewed growth takes place at the 
 expense of the reserve-materials stored in the root, the chief of 
 which is the cane-sugar. Bernard discovered that at the onset 
 of this second period of active life the cane-sugar in the root 
 begins to diminish and gets continually less and less as the 
 growth proceeds. Glucose at the same time makes its appear- 
 ance there and can be traced upwards along the stem to the 
 growing region. Glucose also appears in the leaves, While 
 cane-sugar is altogether absent from them. Bernard further 
 ascertained that a similar enzyme to that he had discovered in 
 the animal intestine could be extracted from the beet-roots on 
 which he was experimenting and he consequently proved that 
 his hypothesis was well-founded, and that in the vegetable, as 
 well as the animal organism, cane-sugar needs to undergo a 
 process of digestion or hydrolysis before it is capable of 
 affording nutritive material to the active protoplasm. The 
 agent in both cases can be extracted from the seat of the 
 digestion and is undoubtedly the same enzyme. 
 
 In recent years a very important modification of the 
 generally received ideas of the primary formation of carbo- 
 hydrates in leaves has been advanced by Brown and Morris, in 
 the course of a discussion of a series of experiments they 
 carried out on the physiology of these organs. These researches 
 
VIIl] SUGAR-SPLITTING ENZYMES. INVERTASE. 117 
 
 have already been referred to in connection with the occurrence 
 of translocation diastase in the cells of the mesophyll. The 
 authors suggest that cane-sugar and not glucose is the earliest- 
 formed sugar and that the starch which rapidly appears in the 
 chloroplasts is the expression of the production of an excess of 
 this carbohydrate. As it is almost certain that cane-sugar is 
 not immediately available for the nutrition of protoplasm, and 
 as the cells in which the carbohydrate material is being elabo- 
 rated are engaged in a very active metabolism, it is probable 
 that some other form of sugar is produced to supply their 
 immediate needs. There are two possible sources of such 
 sugar, — the starch, which can be converted into maltose, and 
 the cane-sugar, which may undergo hydrolysis as in other parts 
 of the plant. It appears most probable, as Brown and Morris 
 suggest, that the newly formed cane-sugar supplies this nutri^ 
 ment, the surplus only being stored away as starch. On 
 this assumption, invertase should be present in the cells to 
 hydrolyse the cane-sugar, so as to present a nutritive sugar 
 to the protoplasm. It is far from unlikely, too, that such 
 preliminary hydrolysis takes place in the case of that portion 
 of the cane-sugar which is the antecedent of the starch, 
 as the latter is more nearly related to the simpler hexoses than 
 to the complex polysaccharide. The hypothesis of the first 
 formation of cane-sugar, with the subsequent transformations 
 suggested above appears to call therefore for the demonstration 
 of the existence of invertase in foliage leaves. 
 
 Brown and Morris made some experiments to test this 
 point. They took some air-dried leaves of a species of Tropoe- 
 olum and powdered them in a mortar. Two grammes of the 
 powder were digested, with antiseptic precautions, with 100 c.c. 
 of a solution containing 4'881 grms. of cane-sugar and were 
 kept at a temperature of 30° C. for seven days. At the outset 
 the solution had no action on Fehling's fluid but at the end of 
 the week the 100 c.c. reduced 2-064 grms. of CuO. The 
 opticity changed during the time from 9'4i° to 7'2°. These 
 numbers indicate an invertive action of the leaf equal to the 
 production of 0'894 grm. of invert-sugar, an amount equal to 
 nearly 45 per cent, of the weight of the dry leaf used. 
 
118 SUGAE-SPLITTING ENZYMES. INVERTASE. [CH. 
 
 Kosmann also has detected invertase in the buds and 
 leaves of young trees, and Gonnermann has found it m the 
 leaves of the sugar beet. 
 
 The enzyme has also been discovered in the rootlets of 
 germinated barley, but its extraction presents considerable 
 difficulty. The discovery was first made by Kjeldahl, whose 
 work has been supplemented by the researches of J. O'Sullivan. 
 The latter observer removed weighed quantities of moist root- 
 lets from germinating barley and immersed them in. a solution 
 of cane-sugar, allowing them to digest for 24 hours at the 
 laboratory temperature. Controls were at the same time 
 prepared, in which water was substituted for the cane-sugar 
 solution. The amount of invert-sugar present in the latter at 
 the end of the experiment was a measure of the quantity 
 present in the rootlets themselves. Deducting this amount he 
 found in a series of three experiments : 
 
 (1) 1'7 grms of moist rootlets inverted 1"22 grms. of cane- 
 
 sugar. 
 
 (2) 0'5 grm. of moist rootlets inverted 1'37 grms. of cane- 
 
 sugar. 
 
 (3) 0*5 grm. of moist rootlets inverted 015 grm. of cane- 
 
 sugar. 
 
 While inversion took place in all, the activity was not the same 
 in each case, a fact possibly due to the material used containing 
 different proportions of the enzyme. 
 
 The existence of the enzyme and the difficulty of extracting 
 it are also established by the following experiment which 
 O'Sullivan quotes — 
 
 (1) 0"75 grm. of rootlets were digested in the cold for 
 two hours with 10 c.c. of water ; the extract was filtered and the 
 residue washed on the filter till filtrate and washings amounted 
 to 50 c.c. To 40 c.c. of this extract 25 c.c. of a 10 per cent, 
 solution of cane-sugar were added. (2) The residual rootlets on 
 the filter were added to another 25 c.c. of the same sugar 
 solution. (3) 0-75 grm. of the same rootlets were added to 
 a third 25 c.c. of the sugar solution. The three sets were then 
 digested at 55° — 57° C. for eight hours, and after standing at 
 the laboratory temperature for a further eight hours, all were 
 
VIIl] SUGAE-SPLITTING ENZYMES. INVERTASE. 119 
 
 made up to 100 c.c. The cane-sugar inverted was calculated 
 from the cupric-oxide-reducing power of each 100 c.c. and was 
 found to be as under: — 
 
 (1) Extract 0-75 grm. of rootlets inverted 0-19 grm. of 
 
 cane-sugar. 
 
 (2) Residual rootlets of (1) inverted 1*9 grams of cane- 
 
 sugar. 
 
 (3) Original 0'75 grm. of rootlets inverted 1-6 grm. of 
 
 cane-sugar. 
 
 The cupric-oxide-reducing power of the sugar in the rootlets 
 themselves was ascertained from the remaining 10 c.c. of the 
 original extract in (1), which was reserved for that purpose. 
 
 The invertase of the embryo of the barley was not confined 
 to the rootlets, but a certain amount was obtained from the 
 plumule. The endosperm of the grains did not contain the 
 enzyme. 
 
 These experiments taken in conjunction with those of 
 Brown and Morris, point to cane-sugar, and consequently 
 invertase, having an important part to play in the nutrition of 
 actively growing vegetable protoplasm. 
 
 Invertase has been described by Atkinson and by Kellner, 
 Mori and Nagaoko as existing in rice, and in Koji, a peculiar 
 preparation of that cereal which is much used by the Chinese 
 in the preparation of fermented liquids. The rice is treated 
 under certain conditions with the fungus Eurotium oryzoe. 
 It does not appear very evident whether the latter is not the 
 source of the enzyme, rather than the grain. 
 
 Frankfurt has detected the enzyme in isolated wheat- 
 embryos. Invertase is said by Mieran to be present in the 
 ripe fruit of the Banana. An aqueous extract of the fruit, made 
 at ordinary temperatures and allowed to stand for some time 
 at 55° C, was laevo-rotatory, the rotation being given by Mieran 
 as (a)2> = - 7'65° ; a similar extract made at 100° C. was dextro- 
 rotatory, the rotation being (a)2, = -l-l7'49°. When the ripe 
 fruit was digested with a solution of cane-sugar the rotation 
 of the polarised ray became reversed in direction. In one 
 experiment it was observed to change from -f99'6° to — 11'22°. 
 
120 SUGAE-SPLITTING ENZYMES. INVERTASE. [CH. 
 
 Mieran does not appear to have extracted the enzyme from 
 the fruit. 
 
 Invertase is associated as we have seen with cane-sugar in 
 reservoirs of reserve-materials in various parts of the plant. 
 Cane-sugar is sometimes stored in other places than those 
 mentioned, with a view to subsequent utilisation. Many pollen 
 grains are known to contain it in small quantity and it is 
 consumed in their germination. Van Tieghem first discovered 
 it in these bodies in 1886; he germinated the pollen of 
 various plants in a 10 per cent, solution of cane-sugar and 
 detected a reducing-sugar in the liquid which he filtered 
 oiF from his cultures. When the pollen-grains were placed 
 in the same solution and germination prevented by the 
 addition of a few drops of chloroform, the same result was 
 arrived at, so that he was able to conclude that the enzyme 
 was present in the pollen apart from germinative changes. 
 
 In 1893, the writer demonstrated its presence in the pollen 
 of Eucharis, Narcissus, ffelleboriis, Richardia, Liliwm and 
 Zamia, and was able to extract it from the resting grains by 
 bruising them in an agate mortar and extracting the resulting 
 powder by various solvents. He was able to ascertain further 
 that the quantity increased considerably during the process of 
 germination, and that the increase was much greater when the 
 pollen was allowed to germinate in cane-sugar solution than 
 when it was sown in water only. A most striking experiment 
 was made with the pollen of Narcissus poeticus. The pollen 
 was collected from 906 anthers, and weighed "3 grm. This was 
 divided into three parcels of "1 grm. each. One parcel (A) 
 was steeped at once in 10 c.c. of chloroform-water; another 
 (B) was made to germinate in water on a glass plate ; and the 
 third (C) similarly in 15 per cent, solution of cane-sugar. 
 When germination was well advanced the cultures were care- 
 fully washed from the plates and all three made up to 15 c.c. 
 with chloroform- water. They were then all filtered, each was 
 mixed with 25 c.c. of 10 per cent, cane-sugar solution and all 
 were allowed to digest for 93 hours at the ordinary laboratory 
 temperature. The chloroform was subsequently removed from 
 each digestion by boiling it for some time and finally all were 
 
VIIl] SUGAR-SPLITTING ENZYMES. INVERTASE. 121 
 
 titrated with Fehling's solution. The quantities of invert- 
 sugar in the three cases were found to be as under: — 
 
 A. (Ungerminated) '19 grm. 
 
 B. (Germinated in water) "5 grm. 
 
 G. (Germinated in cane-sugar solution) 1'37 grm. 
 
 The secretion or formation of the enzyme appears from 
 this experiment to be materially increased by the absorption 
 of the nutritive sugar, derived from the hydrolysis of the 
 original carbohydrate. 
 
 Besides this very wide distribution in the higher plants, 
 invertase has been found to exist in many fungi besides those 
 already alluded to. Hansen has found it in several of the 
 Saccharomycetes. Wasserzug discovered it in certain fungi 
 belonging to the genus Fusarium, which are capable of growing 
 in solutions of cane-sugar, and which lead to the formation of 
 glucose in the culture liquid. He was able to detect an 
 excretion of the enzyme by the mycelium by modifying the 
 culture medium. When the latter was the bouillon prepared 
 from veal, invertase was discharged into it in small quantity 
 just at the time when the fungus was developing its conidia. 
 
 In 1878, Gayon found that when Aspergillus niger was 
 cultivated in a medium containing cane-sugar a certain amount 
 of invert-sugar was produced, a discovery which was confirmed 
 by Duclaux in 1883. Fernbach in 1890 determined this to be 
 due to the presence of invertase, and ascertained that the 
 secretion of the enzyme was not confined to the period of 
 fructification, but extended over the whole period of the life of 
 the organism, the quantity yielded being approximately the 
 same at all times. Bourquelot three years later found that 
 an extract of the mycelium of Aspergillus effected complete 
 inversion of a 2'5 per cent, solution of cane-sugar when 
 digested with it for 24 hours at 22° C. 
 
 Bourquelot in 1886 detected the presence of invertase in 
 Peniciliwn glaucum and was able to extract it from the 
 mycelium, but only when the fungus had arrived at maturity. 
 An allied species, P- Duclauxi, when cultivated in cane-sugar 
 solution was found capable of inverting it, but the invertase 
 was not discharged into the culture medium. The enzyme is 
 
122 SUGAR-SPLITTING ENZYMES. INVERTASE. [cH. 
 
 apparently present in the latter fungus throughout its whole 
 life, as its spores germinate readily in cane-sugar solutions. 
 Fischer and Lindner have noticed a similar occurrence of the 
 enzyme in Monilia Candida, the fungus hydrolysing cane-sugar 
 but not yielding the ferment to an extracting fluid. 
 
 Invertase is not uniformly present in the fungi ; Polyporus 
 sulphureus has been found by Bourquelot not to contain it. 
 Its distribution throughout the group however has not been at 
 all fully investigated. 
 
 Invertase appears to be very seldom met with in Bacteria. 
 Fermi and Montesano found that Bacillus megatherium, B. 
 fluorescens liquefaciens, the red Kiel Bacillus and Proteus 
 vulgaris were capable of producing it in bouillon to which cane- 
 sugar had been added. Van Tieghem has shown that it is 
 secreted by Leuconostoc mesenterioides. The writer has detected 
 it in cultures of a bacillus which is associated symbiotically with 
 a yeast in certain jelly-like masses infesting the sugar-cane. It 
 is excreted by the microbe into the surrounding liquid. 
 
 It is not certain how far the action of invertase in these 
 lower forms is intracellular. Different observers differ as to 
 whether in normal life the invertase is excreted into the 
 surrounding medium. The opinion is however gaining ground 
 that when the organisms are growing under favourable conditions 
 the enzyme passes out through the thin cell-walls and works 
 extra-cellularly. Its power of diffusion is not very great and 
 frequently so little of it is sent out of the cells that it may 
 easily escape observation. This is not difficult when the 
 method of examination used is that of precipitation of the 
 liquid by alcohol. This as we have seen above is deleterious to 
 invertase and probably destroys it entirely when it is present 
 in small amount. This may explain the failure of some 
 observers to obtain evidence of its excretion and extra-cellular 
 action. 
 
 The occurrence of invertase in the alimentary canal of 
 Mammals is chiefly confined to the small intestine, in which, as 
 already mentioned, Bernard first discovered it. Miura has stated 
 that it is present also in small quantity in the tissue of the 
 colon, stomach, and pancreas. He detected it not only in the 
 
VIll] SUGAR-SPLITTING ENZYMES. INVEETASE. 123 
 
 digestive tract of living animals, but also in that of animals 
 which were still-born. Abelous and Heim found it with 
 diastase in the eggs of certain Crustaceans, and Biedermann 
 says it is present in the intestinal secretion of the larva of 
 Tenebrio molitor, the mealworm. 
 
 Various methods have been adopted for the preparation of 
 invertase from one or other of the sources already described. 
 Hoppe-Seyler killed yeast by ether and then extracted the 
 enzyme by water, subsequently precipitating it by alcohol. 
 Gunning extracted it from washed yeast by means of glycerine. 
 A more elaborate method was used by Zulkowsky and Konig ; 
 these observers washed yeast with alcohol, dried and powdered 
 it and extracted the powder with water. After filtration the 
 filtrate was shaken up with ether, which caused the invertase 
 to separate out in a mass resembling frog-spawn. This pre- 
 cipitate was again extracted with water and the enzyme 
 thrown out of solution by alcohol. So prepared it was when dry 
 a white substance, soluble in water, yielding a neutral solution. 
 It precipitated lead, copper, and mercurous salts, but had no 
 action on ferric perchloride or potassium ferrocyanide, and its 
 solution was not rendered turbid on boiling with dilute 
 acetic acid. According to Bechamp it has an optical activity 
 
 O'SuUivan and Tompson have investigated the action of 
 invertase in considerable detail. They prepared it in large 
 quantity from ordinary top fermentation pressed yeast. This 
 was allowed to stand at a temperature of about 15° C. for 
 a month, when it had become partially liquefied. The liquid 
 mass was then pressed in a screw filter-press and yielded 
 a clear yellow solution. Alcohol was next added until 47 per 
 cent, of the spirit was present and the mixture was allowed 
 to stand two days. There was a fairly bulky precipitate and 
 the supernatant liquid was quite inactive. The precipitate 
 was allowed to settle in tall vessels and water was added to 
 redissolve the invertase. When the strength of the alcohol 
 was reduced to 28 per cent, the enzyme had gone into solution 
 and a considerable amount of albuminous matter remained 
 undissolved. The liquid was then filtered and again made up 
 to 47 per cent, of alcohol. The precipitate reappeared, was 
 
124 SUgiAR-SPLITTING ENZYMES. INVERTASE. [CH. 
 
 separated and washed with absolute alcohol, and dried in vacuo 
 over sulphuric acid. So prepared the invertase was very active, 
 but not quite pure ; it contained about 6 per cent, of ash, which 
 consisted of phosphates of potassium and magnesium. The 
 authors regard the ash as an impurity and do not consider it 
 as entering into the composition of the enzyme. Efforts to 
 purify the latter beyond this point were futile, as it proved 
 to be a very unstable body and decomposed during the further 
 treatment. It possessed a specific rotatory power of {a)j = + 80°. 
 Yeast appears to contain an amount of invertase equal to about 
 2 — 6 per cent, of its dry weight. 
 
 The action of the enzyme is the same as that of dilute 
 mineral acids, a molecule of cane-sugar taking up a molecule 
 of water and splitting into two molecules, one of glucose, the 
 other of levulose (fructose). Evidence of the action is afforded 
 by the change in the power the solution has of rotating a 
 beam of polarised light and by the development of the property 
 of reducing cupric to cuprous oxide when boiled in the presence 
 of excess of alkali. Cane-sugar does not possess the latter 
 property. 
 
 When invertase is digested with a solution of cane-sugar 
 the action is most rapid at first and gradually diminishes as the 
 cane-sugar disappears. The rate of the inversion may always 
 be represented by a definite time-curve, which is practically 
 that given by Harcourt as being the one expressing a chemical 
 change of which no condition varies excepting the diminution 
 of the changing substance. So long as the conditions under 
 which the digestion is taking place remain unchanged this 
 curve expresses the course of the transformation. 
 
 Among conditions which materially influence the progress 
 of the action of invertase, we may mention the reaction of the 
 liquid, the concentration of the cane-sugar solution, and the 
 temperature at which the digestion is carried out. 
 
 Minute quantities of sulphuric acid accelerate the action in 
 a very remarkable manner, but the actual amount required to 
 produce the greatest effect varies with the amount of invertase 
 present and with the temperature at which the hydrolysis is 
 conducted. The more there is of the enzyme in the solution 
 the greater is the amount of acid required to obtain the 
 
VIIl] SUGAR-SPLITTING ENZYMES. INVERTASE. 125 
 
 maximum action. When the temperature of the digestion was 
 50° C. O'Sullivan and Tompson found that with -4 per cent, 
 of invertase present, the optimum amount of sulphuric acid was 
 12-5 parts per million of the solution; with 1-5 per cent, the 
 amount required for the greatest activity was 15 per million. 
 In another experiment, carried out at 15-5° C, when the 
 quantity of invertase amounted to 1-5 per cent., computed on 
 the weight of sugar used, the most advantageous quantity of 
 acid was 75 parts per million of the solution ; with ten times 
 as much invertase the optimum amount of acid was 250 
 parts per million. There appears to be no definite ratio be- 
 tween the quantities of invertase and acid which are necessary 
 to secure the maximum rate of hydrolysis. 
 
 If the amount of acidity was carried beyond the- most 
 favourable point, even to a very slight extent, its effects were 
 very detrimental. In one of O'Sullivan and Tompson's ex- 
 periments, conducted at 60° C, "34 per cent, of invertase being 
 used, an excess of only two parts of acid per million of the 
 solution lowered its activity elevenfold. 
 
 Miiller found that so feeble an acid as carbonic acid 
 accelerated the hydrolysis. 
 
 The action of the caustic alkalis, even if they are present 
 in very small proportion, is very detrimental; to vegetable 
 invertase they are instantly and irretrievably destructive of 
 the enzyme. This does not appear to be the case with the 
 invertin of the small intestine, which effects hydrolysis while 
 the reaction of the intestinal contents is still alkaline. 
 
 The most favourable concentration of the sugar solution has 
 been found by O'Sullivan and Tompson to be about 20 per cent., 
 the digestion being conducted at 54° C. Below this percentage 
 the speed of inversion rapidly declines ; stronger solutions are 
 hydrolysed at only slightly lower rates, until the concentration 
 reaches 40 per cent. In saturated solutions the hydrolysis is 
 very slow and feeble. 
 
 The activity of invertase can be detected at a temperature 
 a very little above 0° 0. but it then works very slowly. As the 
 temperature rises the action becomes more evident but rises 
 only gradually at first, and then more rapidly up to nearly 
 
126 SUGAR-SPLITTING ENZYMES. INVERTASE. [CH. 
 
 60° C. According to O'Sullivan and Tompson it doubles itself 
 approximately for each rise of 10° C. The optimum point 
 is somewhere between 55° C. and 60" 0. Muller found the 
 relative activities at 0°, 10°, 20°, 30°, and 40° C. to be respect- 
 ively 9, 19, 36, 63 and 93, and the optimum temperature to be 
 a little under 50° 0. Above 65° 0. the enzyme is slowly 
 destroyed and at 10° higher the destruction is immediate. 
 
 The action of alcohol is deleterious, particularly if much is 
 present. The enzyme is precipitated unaltered by 47 per cent, 
 of the spirit but larger proportions decompose it. When 
 alcohol is present in quantities too small to affect the com- 
 position of the enzyme it can diminish its hydrolysing power. 
 If 5 per cent, only is present in a digestion, the activity of the 
 invertase is reduced one half. 
 
 There is some difference of opinion as to the influence of 
 the products of hydrolysis on the action of the enzyme. 
 O'Sullivan and Tompson say that they have no influence on 
 the rate of the change, while Muller states that they are 
 distinctly inhibitory. 
 
 The power of the enzyme is practically inexhaustible; a 
 sample which had induced inversion of 100,000 times its own 
 weight of cane-sugar was found by O'Sullivan and Tompson to 
 be still active ; they showed moreover that invertase itself is 
 not destroyed or materially injured by its action on cane-sugar. 
 
 Fernbach noted that his extract prepared from Aspergillus 
 was less active in light than in darkness, and that the inhibitory 
 effect of the illumination was greater as the extract was 
 gradually made more acid. 
 
 A very remarkable feature of the invertase obtained by 
 O'Sullivan and Tompson was that when it was heated in a 
 solution of cane-sugar during a period of active hydrolysis it 
 was able to withstand without decomposition a temperature of 
 25° C. higher than was sufiicient to destroy it when in solution 
 in water and consequently inactive. A discussion of this very 
 interesting fact must be deferred however to a subsequent 
 chapter. 
 
 The importance of invertase in both the animal and 
 vegetable economy seems to be primarily what has already 
 
VIIl] SUGAR-SPLITTING ENZYMES. INVERTASE. 127 
 
 been indicated, viz. to convert cane-sugar into other sugars 
 which can be assimilated by protoplasm. This species of 
 digestion can take place either in the interior of the cells in 
 which the enzyme is secreted, as in most vegetable sources, or 
 outside them, as in the alimentary canal of the animal organism. 
 In the case of yeast the excretion of the invertase appears to 
 precede any digestion of sugar. Onimus has shown that when 
 a solution of yeast in distilled water is allowed to diffuse 
 through parchment paper into a solution of cane-sugar, in- 
 version can be observed in the latter after 15 to 20 minutes. 
 Yeast cells do not undergo rapid growth outside the dialyser 
 until nearly 12 hours later. He suggests that the secretion of 
 the invertase is necessary to prepare a nidus favourable to the 
 development of the yeast organism. On this view the inversion 
 of cane-sugar by yeast is extra-cellular. 
 
 Invertase has been shown to act (Jhiefly if not entirely on 
 cane-sugar. O'Sullivan however has stated that it is capable 
 of hydrolysing raffinose, but with considerably less rapidity. 
 The products appear to be galactose, glucose and fructose. 
 Bourquelot has recently found it capable of hydrolysing 
 gentianose, a peculiar sugar which is present in the roots of 
 various species of Gentians. Like cane-sugar gentianose does 
 not reduce Fehling's solution; it has a dextro-rotatory power 
 on polarised light. On inversion a reducing-sugar is produced 
 and the liquid becomes laevo-rotatory. Bourquelot thinks that 
 gentianose is a very complex polysaccharide containing in 
 its molecule cane-sugar and another polyglucose, and that 
 invertase only attacks the former group. 
 
128 
 
 CHAPTER IX. 
 
 SUGAR-SPLITTING ENZYMES (continued). 
 
 Glucase. (Maltase.) 
 
 In our discussion of the action of diastase on starch in an 
 earlier chapter, attention was called to a curious discrepancy in 
 the nature of the final product when diastase from different 
 regions of the alimentary canal is taken as the hydrolysing 
 agent. The enzyme of saliva converts starch into maltose ; 
 among the products of hydrolysis taking place in the intestine 
 glucose undoubtedly appears ; the sugar also which leaves the 
 liver is the latter variety. A good deal of uncertainty has 
 consequently been felt as to whether or no there might not 
 exist more than one variety of diastase, characterised by 
 the power of transforming starch into these different sugars 
 respectively. 
 
 The study of the fate of maltose in the economy has shown 
 that like cane-sugar, of which it is an isomer, it is not made 
 use of by the tissues without preliminary hydrolysis. Philips 
 showed in 1881 that when maltose is injected into the blood 
 of an animal, a good deal of it is excreted unchanged in the 
 urine, much as cane-sugar is, though not to so large an extent. 
 Glucose on the other hand is used up by the organism if 
 introduced into the body in a similar manner. Dastre and 
 Bourquelot found in 1884 that if the quantity of maltose 
 injected is not too large, a good deal of it is not voided by the 
 kidney, and that therefore its fate differs somewhat from that 
 of cane-sugar, part of it at any rate being utilised by the 
 organism. 
 
CH. IX] SUGAE-SPLITTING ENZYMES. GLUCASE. 129 
 
 The similarity of its composition to that of cane-sugar, 
 coupled with the ascertained fate of the latter and with the 
 discovery that glucose appears among the products of the 
 digestion of starch in the intestine, points to a process of 
 inversion taking place before absorption. Maltose is capable of 
 such hydrolytic conversion, for when boiled with dilute mineral 
 acids it splits up according to the equation 
 
 CxsH^Ou + H,0 = C,Hi,Oe + CeHi,0„ 
 
 two molecules of glucose being formed, instead of one of 
 glucose and one of fructose as in the case of the corresponding 
 hydrolysis of cane-sugar. Maltose differs from cane-sugar and 
 from glucose in its power of rotating a ray of polarised light, 
 its specific rotatory power being (a)^ = -1- 140° in ] per cent, 
 solution at 20° C, while that of glucose is (a)^ = + 52-5°. 
 Maltose reduces alkaline solutions of cupric oxide, but only 
 two-thirds as much as glucose. When hydrolysis takes place, 
 therefore, the optical activity of the solution diminishes while 
 the reducing power increases. 
 
 An investigation into the details of the digestive changes 
 in the small intestine was undertaken by Brown and Heron, 
 who published their results in 1880. They examined the be- 
 haviour of the pancreas of the pig and of the mucous membrane 
 of the small intestine of the same animal. Taking a pancreas 
 which was known to be in an active condition, they prepared 
 an extract from it, which they found capable of hydrolysing 
 maltose, with the formation of glucose, but which only acted 
 very feebly. In their experiments with the small intestine, 
 instead of using the fresh tissue, they dried the tube at 35° C. 
 in a current of air, and shredded it, using the shreds with a 
 solution of maltose kept at 40° C. They found that it was 
 capable of converting the maltose into glucose with considerable 
 ease, much more quickly indeed than the pancreas. They held 
 that the most active region of the intestine was the part in 
 which the so-called glands of Peyer occur, these structures 
 being really lymphatic follicles. A piece of dried intestine 
 from this region completely hydrolysed a solution of maltose 
 in seven hours. They were thus led to the view that the 
 
 G. F. 9 
 
130 SUGAR-SPLITTING ENZYMES. GLUCASE. [CH. 
 
 digestion of starch in the small intestine consists of two stages, 
 one, conducted by the pancreatic juice, being marked by the 
 transformation of starch into maltose, the other, taking place 
 subsequently, mainly under the action of the succus entericus, 
 but with some assistance from the pancreatic juice, consisting 
 of the further hydrolysis of the maltose into glucose. They 
 further put forward the view already suggested that maltose is 
 not directly assimilable by the living organism. 
 
 Brown and Heron further drew attention to the digestion of 
 maltose as being similar to, but not identical with that of cane- 
 sugar. They found the small intestine to be capable of in- 
 verting the latter, but not to be so active as in the case of 
 maltose. The extract of the pancreas, while feebly hydro- 
 lysing maltose, had in their experiments no power of inverting 
 cane-sugar, nor had the tissue of the gland itself 
 
 In 1881, von Mering confirmed the observations of Brown 
 and Heron on the part played by the pancreas. He found that 
 an extract of that organ taken from a dog was capable of slowly 
 hydrolysing maltose. 
 
 In 1883, Bourquelot carried out a series of researches on the 
 rabbit, in which he confirmed and extended the conclusions of 
 the previous observers. He used the fresh tissues and carried 
 out his experiments under antiseptic conditions. His results 
 may thus be summarised : (1) In the rabbit, during digestion, 
 the pancreas and the small intestine both produce an enzyme 
 which hydrolyses maltose. (2) This enzyme is much more 
 abundant in the intestine than in the pancreas. (3) It is 
 produced almost entirely in the median region of the small 
 intestine, portions taken on the one hand near the pylorus and 
 on the other near the large intestine being almost or entirely 
 without action. (4) The enzyme is a diflFerent one from in- 
 vertin, for the enzyme of the pancreas which hydrolyses maltose 
 has no action on cane-sugar and the invertase from yeast (which 
 is identical with invertin) has no action on maltose. 
 
 Bourquelot next turned his attention to the vegetable 
 kingdom, and selected for experiment two fungi, Aspergillus 
 niger and Penicilium glaucum, both of which thrive extremely 
 well when cultivated in a solution of maltose. He triturated 
 
IX] SUGAR-SPLITTING ENZYMES. GLUCASE. 131 
 
 the mycelia with sand in a mortar, and extracted the pasty- 
 mass with a little water. After filtering his material, he added 
 alcohol to the filtrate and obtained a precipitate which he 
 separated by a further filtration, and dried at a low tempera- 
 ture. This precipitate was soluble in water, and the solution 
 when mixed with maltose rapidly hydrolysed it. 
 
 On account of the fact that yeast can set up alcoholic 
 fermentation in maltose solutions, as well as in those in cane- 
 sugar, Bourquelot next directed his attention to this organism, 
 thinking it probable that in both cases hydrolysis of the polysac- 
 charide must precede the formation of alcohol. He failed how- 
 ever to obtain direct evidence of the presence of the new enzyme 
 in the yeast-cells, but some experiments led him nevertheless to 
 infer that it was present there. Adding yeast to a solution of 
 maltose and inhibiting the growth of the yeast cells by the 
 addition of chloroform, he found that there was very soon a 
 diminution of the rotation of the ray of polarised light and a 
 coincident increase of the power of reducing cupric oxide. 
 Both these phenomena we have seen accompany the hydrolysis 
 of maltose. Bourquelot concluded from these experiments that 
 when yeast is present in a solution of this sugar, it secretes an 
 enzyme which hydrolyses it and the resulting glucose is at once 
 split up with the formation of alcohol, the alcoholic fermenta- 
 tion taking place as fast as the glucose is formed. 
 
 Fischer also has detected this enzyme in yeast. 
 
 The discrepancy between the results of Bourquelot and those 
 of Philips, as to the fate of the maltose injected into the blood 
 was explained by the discovery of Dubourg in 1889 that 
 blood contains the enzyme under discussion. Dubourg took 
 blood from a rabbit which had been fed on an amylaceous diet 
 for 5 days, mixed 25 c.c. with 50 c.c. of a solution of maltose, 
 and digested the mixture at 37'5° C. for 2 days. He found 
 that after that period the optical activity was much diminished 
 and that the cupric-oxide-reducing power was considerably 
 increased. The action was not very energetic, but the experiment 
 showed reason to believe that when, as in Bourquelot's experi- 
 ments, a small quantity of maltose was injected into the blood 
 there was sufficient enzyme present to hydrolyse it, so that no 
 
 9—2 
 
132 SUGAR-SPLITTING ENZYMES. GLUCASE. [CH. 
 
 maltose was excreted by the kidney ; when, as in the work of 
 Philips, a good deal of the sugar was injected, part only was 
 hydrolysed and the rest appeared in the urine. 
 
 Bourquelot has given the name maltose to this enzyme. 
 
 In 1886, Cuisinier described an enzyme under the name of 
 glucase, which he claimed to have discovered in barley-malt and 
 in several of the cereal grains, which he said converted starch 
 into glucose. His conclusions were opposed a little later by 
 Lintner, but were reaffirmed in 1891 by Geduld, who extracted 
 from maize a soluble ferment capable of hydrolysing maltose. 
 Cuisinier's enzyme appears to have been a mixture of diastase 
 and glucase, which occur together as they do in the secretion of 
 the pancreas. 
 
 Geduld and subsequent writers apply the name gliicase to 
 the enzyme which hydrolyses maltose, and which was previously 
 termed maltase by Bourquelot. Geduld claims to have extracted 
 it from maize in a fairly pure condition. It contains 8"12 per 
 cent, of nitrogen, is only slightly soluble in water, and gives a 
 blue coloration with tincture of guaiacum and hydrogen 
 peroxide. It is capable of hydrolysing only about one hundred 
 times its weight of maltose, so that it is feeble compared with 
 the invertase prepared by O'Sullivan and Tompson. It is most 
 active at a temperature of 57° — 60° C. ; above that point it is 
 weakened and at 70° it undergoes decomposition. 
 
 Since Dubourg's discovery of the presence of glucase in 
 blood, in 1889, many observers have conducted researches on its 
 occurrence there and in the tissues and juices of the animal body. 
 
 In 1893, Bial ascertained that the serum of both blood and 
 lymph contains enzymes which convert starch into glucose and 
 which act also upon dextrin and maltose. The ferments are 
 not present in the corpuscles. When alcohol is added to serum 
 and the resulting precipitate is allowed to stand for a time and 
 then extracted with water, the extract transforms starch into 
 maltose only, and has no further action on the latter. The 
 action noted seems therefore to be due to the presence of 
 diastase and glucase together in the serum, and to the latter 
 being destroyed by contact with alcohol. 
 
 In 1894, Eohmann made a comparative examination of the 
 
IX] SUGAR-SPLITTING ENZYMES. GLUCASE. 133 
 
 digestive properties of serum, saliva, pancreatic juice and 
 succus entericus, and concluded that both diastase and glucase 
 exist in all four, but in very different proportions. Saliva 
 acting on starch forms mainly maltose and dextrin, but gives 
 also a trace of glucose. Pancreatic juice behaves similarly but 
 produces more glucose. Serum is more active in the latter 
 direction than either. On the other hand, while pancreatic 
 juice hydrolyses starch more rapidly than saliva, the latter is 
 much more efficient in this respect than serum. While all four 
 liquids contain both enzymes, diastase is most plentiful in 
 pancreatic juice and saliva, and in smallest quantity in serum, 
 but glucase is present in greatest amount in the latter and but 
 little of it occurs in the other fluids. 
 
 Bourquelot and Gley have also prepared glucase from serum. 
 
 Eohmann's results received confirmation in the succeeding 
 year by Hamburger, who compared the same four liquids. His 
 results show that when all four are allowed to act simultane- 
 ously on soluble starch, differences appear in two directions. 
 (1) The power of reducing cupric oxide attained in a digestion 
 of 24 hours' duration is different. (2) The time taken to attain a 
 maximum power varies. Taking the reducing power of glucose as 
 1, the reduction he obtained in 24 hours by the several fluids was 
 in the case of saliva 'SI, of pancreatic juice "36, of succus entericus 
 •26, of serum '8. The maximum reducing-power was obtained 
 by saliva in one hour ; by blood only after 24 hours' digestion. 
 Pancreatic juice acted more rapidly than saliva ; intestinal juice 
 was slower than blood. While saliva acts quickly the reducing 
 power of the product is low; blood on the other hand acts 
 slowly, but the reducing power is high, a phenomenon which 
 Hamburger suggests is due to the different sugar formed, which 
 in the case of saliva is chiefly maltose, while in that of blood it 
 is glucose. He concludes that glucase is especially abundant 
 in blood ; saliva contains more diastase than either blood or 
 succus entericus but scarcely a trace of glucase; pancreatic 
 juice contains more diastase than saliva, and glucase is present 
 in it in quite appreciable quantities. Succus entericus contains 
 less diastase than blood, and more glucase than saliva, but not 
 so much as either blood or pancreatic juice. 
 
134 SUGAB-SPLITTING ENZYMES. GLUCASE. \_CU. 
 
 A search for glucase in the various tissues of the body, 
 including several regions of the alimentary tract, was made in 
 1893 by Miss Tebb. Her mode of procedure was based on that 
 of Brown and Heron, and like them she experimented with the 
 tissues of the pig. 
 
 She sometimes used infusions made by steeping the dried and 
 disintegrated organs in saline solutions, and sometimes the dried 
 tissue itself The temperature of drying was 37 — 40° C. In 
 all cases she checked her results by control experiments in 
 which the dried tissue or its extract had been boiled. The 
 saline solutions were usually 5 per cent, solutions of sodium 
 sulphate, and the extracts were always made with antiseptic 
 precautions. She found the power of hydrolysing maltose to be 
 possessed by dried pancreas, the mucous membrane of the small 
 intestine, Peyer's patches, lymphatic glands, salivary glands, 
 liver, kidney, stomach, spleen, and striated muscle. The relative 
 activity possessed by equal weights of the dried tissue of these 
 organs was found to be very different. Her results may be 
 represented proportionately in the following table: 
 
 Mucous membrane of small intestine 
 
 3-21 
 
 Spleen 
 
 1-35 
 
 Lymphatic glands 
 
 0-93 
 
 Liver 
 
 0-80 
 
 Peyer's patches 
 
 0-64 
 
 Kidney 
 
 0-66 
 
 Stomach 
 
 0-45 
 
 The figures represent the proportion of glucose formed to 
 one part of maltose left unchanged at the conclusion of the 
 experiment. 
 
 Besides these various tissues Miss Tebb found glucase to be 
 present in the serum from pig's blood, and in bile collected from 
 the gall-bladder of the same animal. 
 
 Pregl found glucase in the succus entericus of a young 
 lamb and Pautz and Vogel in the mucous membrane of the 
 alimentary tract of dogs and new-born children. The most 
 active region was the jejunum, but some inversion of maltose 
 was effected by the stomach, ileum and colon. 
 
IX] SUGAR-SPLITTING ENZYMES. GLUCASE. 135 
 
 A good deal of interest attaches to the discovery of glucase 
 in the liver. As we have already seen, the sugar leaving this 
 organ is not maltose, but glucose. In the earlier work on the 
 diastase of the liver most writers agree in saying that if an en- 
 zyme exists there at all it differs from the diastase of the saliva 
 and pancreatic juice in forming glucose and not maltose from 
 glycogen. At any rate the sugar in the blood of the hepatic 
 vein is glucose. It is probable from Miss Tebb's work that the 
 so-called liver-diastase is a mixture of diastase and glucase, the 
 first hydrolysing glycogen to maltose, and the second com- 
 pleting the action by further hydrolysing the maltose. 
 
 There is some controversy still as to whether glucase is 
 present in germinating barley and other cereals. Morris holds 
 that it is an enzyme peculiar to maize, as he was unable to 
 obtain any evidence of the inversion of maltose when the latter 
 was mixed with a cold-water extract of either barley, malt, oats, 
 rye or wheat. On the other hand Ling and Baker prepared a 
 ferment-extract from kiln-dried malt, which formed a certain 
 amount of glucose as well as maltose when allowed to digest 
 with soluble starch. They suggest that the diastase was altered 
 during the process of kiln-drying. When a little of this so- 
 called diastase was allowed to act on maltose alone, it converted 
 some of it into glucose. Krober is of opinion that glucase 
 exists in normal malt. 
 
 Various species of yeast stand out conspicuously as sources 
 of glucase. Fischer and Lindner found it to be present in 
 Saccharomyces octosporus, the extract of which hydrolyses 
 maltose but has no action on cane-sugar. S. Maronanus on the 
 contrary hydrolyses the latter, but has no action on maltose. 
 C. J. Lintner found that in the case of other yeasts both 
 enzymes are present, glucase being less soluble than invertase. 
 Fischer has arrived at similar results in the case of Froberg 
 yeast. 
 
 Lintner and Krober have studied the action of glucase 
 prepared from yeast by extracting the dried cells with water 
 at ordinary temperatures. They have found its optimum 
 point to be 40° C, which indicates a difference between it and 
 the enzyme prepared by Geduld from maize. The latter works 
 
136 SUGAR-SPLITTING ENZYMES. GLUCASE. [CH. 
 
 most advantageously at 57°— 60° C. Yeast glucase differs also 
 in this respect from invertase, which Lintner and Krober 
 found to have an optimum point of 52°— 53° 0. Their glucase 
 was destroyed by heating it to 55° 0. At temperatures up 
 to 35° C. its hydrolytic activity was proportional to the tem- 
 perature, the quantity present and the time of digestion 
 remaining constant. An increase of the quantity of the en- 
 zyme did not proportionately accelerate the inversion. 
 
 Besides acting on maltose Fischer has found that glucase is 
 capable of effecting the decomposition of several artificial 
 glucosides which he prepared from various sugars, methyl and 
 other alcohols, and hydrochloric acid. He has based upon 
 these experiments a theory of the action of enzymes in 
 general, which will be discussed in a subsequent chapter. 
 
 He has ascertained also that glucase is capable of acting 
 upon certain natural glucosides which yield glucose on hydro- 
 lysis. Of these the most noteworthy is amygdalin, which occurs 
 in certain plants belonging chiefly to the Rosaceae. As we 
 shall see later, amygdalin undergoes hydrolysis under the 
 action of an enzyme known as emulsin, the change being ex- 
 pressed by the equation 
 
 C^H^NOu + 2H,0 = CeH„COH -t- HON + 2(C6H,A) 
 
 Amygdalin Benzoic Prussic Glucose 
 
 aldehyde acid 
 
 Glucase attacks this body in a different way from emulsin, and 
 instead of splitting it up as represented above, it only causes 
 the separation of one molecule of grape-sugar, leaving another 
 glucoside containing only one glucose group in its molecule. 
 The sugar of amygdalin appears to be maltose, and to be 
 capable of hydrolysis to glucose, either while in combination 
 in the glucoside, or when the latter is completely decomposed. 
 
 Trehalose. 
 
 The sugars which exist in the members of the group of 
 Fungi include representatives of the polysaccharides as well as 
 of the simpler hexoses. Of the former the most characteristic 
 is that to which the name trehalose has been given. This 
 sugar was first described by Berthelot in 1857 as occurring in 
 
IX] SUGAR-SPLITTING ENZYMES. TEEHALASE. 137 
 
 Syrian manna. It was discovered almost at the same time by 
 Mitscherlich, who obtained it from a specimen of rye which was 
 infested by ergot (Glaviceps purpurea). MtLntz found it in 
 several species of Hymenomycetes in 1873 ; in recent years it 
 has been described by Bourquelot as present in many species 
 belonging to several different groups, though it is not of uni- 
 versal distribution throughout the fungi. 
 
 The formula assigned to it by Berthelot was C24H220j,2 ; he 
 described it as crystallizing easily, forming large crystals and 
 appearing much like sugar-candy, but not having such a sweet 
 taste as the latter. More recent investigations point to its 
 possessing the formula C12H22OU -|- 2H2O. It appears to re- 
 semble maltose by splitting up on hydrolysis into two molecules 
 of glucose. 
 
 It is not present throughout the life of the different fungi 
 in which it occurs, but is at a maximum just before the period 
 of fructification or spore-formation. If a fungus containing it 
 is plucked and allowed to remain in a fairly warm temperature 
 the sugar disappears. It is extruded in the sap of Lactarius 
 piperatus if this fungus is exposed in a closed vessel to the 
 vapour of chloroform. 
 
 Bourquelot found in the course of an extended research 
 into the sugars contained in fungi that the appearance of 
 glucose is always preceded by that of trehalose, and was thus 
 led to the hypothesis that the latter is hydrolysed by a definite 
 enzyme, just as cane-sugar is by invertase and maltose by 
 glucase. He tested the truth of the hypothesis by experi- 
 ments which were at first confined to three species, Aspergillus 
 niger, Penicilium glaucum, and Volvaria speciosa. The organisms 
 were cultivated for about four days in a fluid containing cane- 
 sugar, when they had put forth abundant sporangiophores. 
 They were then ground up in a mortar with dry sand and 
 allowed to stand under alcohol for about six hours. After 
 filtering, the residue was pressed between folds of filter-paper 
 and dried in vacuo. The mass was next macerated for some 
 time with water, filtered, and the filtrate mixed with strong 
 alcohol. A precipitate fell, which was collected on another 
 filter, washed with alcohol and dried in vacuo. 
 
138 SUGAE-SPLITTING ENZYMES. TREHALASE. [CH. 
 
 The powder so prepared was known from other experiments 
 to contain both invertase and glucase. 
 
 A simpler method of procuring the ferment was sometimes 
 used, which consisted in replacing the culture-fluid by distilled 
 water which was renewed after 12 hours. The fungus excreted 
 its enzymes into the water, which on filtration after two or 
 three days was found to possess a very decided power of hydro- 
 lysing cane- and malt-sugars. The first extract was never very 
 active, owing probably to traces of acid developed during the 
 growth of the organism in the original culture-liquid. 
 
 The extract prepared in either of these ways was then 
 allowed to digest with trehalose obtained from trehala, a kind 
 of waxy excretion, which a certain Coleopterous larva pours out 
 to form its cocoon. In a typical experiment, 10 c.c. of the 
 extract were added to 10 c.c. of a solution containing about 
 2 per cent, of the sugar. The rotatory power of the mixture 
 was then ascertained to be 3° 36' of a polarimeter when 
 examined in a tube 200 mm. long. Hydrolysis at once 
 commenced and after 18 hours' digestion at 12° — 15° 0. the 
 deviation was only 2° 20'. It continued to diminish steadily 
 till the sixth day, after which no change took place. The final 
 deviation was 1° of the polarimeter. The reducing-sugar 
 was then titrated and found to be present in the proportion of 
 •98 grm. per 100 c.c. On the assumption that all the trehalose 
 used was hydrolysed to glucose, the calculated results would 
 be a deviation of the polarised ray equal to 1'013° of the 
 polarimeter, and an amount of reducing sugar equal to "962 grm. 
 per 100 c.c, numbers which agree fairly closely with those ob- 
 tained during the experiment. 
 
 The same results were yielded by the trehalose which was 
 extracted from various fungi as already mentioned. 
 
 Bourquelot states that the enzyme which effects the 
 hydrolysis of trehalose is different from either invertase or 
 glucase, though it co-exists with them in the mycelium of the 
 fungi which he describes. He has given it the name trehalase, 
 and describes it as working most advantageously in a faintly 
 acid medium, the best degree of acidity being about -003 
 per cent, of sulphuric acid. Larger quantities than this are 
 
IX] SUGAR-SPLITTING ENZYMES. TREHALASE. 139 
 
 deleterious, the enzyme being almost without action in the 
 presence of "2 per cent, of the acid. This behaviour is 
 very much like that of invertase under corresponding con- 
 ditions. 
 
 As the extract of Aspergillus, and the precipitate obtained 
 from it by the action of alcohol, act upon cane-sugar and upon 
 maltose as well as upon trehalose it is necessary to carefully 
 examine its behaviour in these three cases to determine 
 whether the so-called trehalase is really not identical with 
 either invertase or glucase. The former of these enzymes 
 however can be prepared from several sources besides Asper- 
 gillus, and when perfectly isolated it has no power of hydro- 
 lysing trehalose. It is apparent therefore that the decom- 
 position observed in the experiments described was not due 
 to the invertase contained in the fungus. 
 
 There is more difficulty in deciding as to the identity of 
 trehalase with glucase, as the latter enzyme cannot be readily 
 obtained without admixture with others. Careful experiments 
 upon the effect of heating the extract of Aspergillus to dif- 
 ferent temperatures and then digesting it with trehalose 
 on the one hand and with maltose on the other, have led 
 Bourquelot to pronounce unhesitatingly in favour of there 
 being two separate enzymes hydrolysing these two sugars 
 respectively. 
 
 He heated a quantity of the extract in a double water-bath, 
 using a series of 18 test tubes each containing 24 c.c. The 
 temperature of the bath was allowed to rise very gradually and 
 the first of the series was withdrawn when it reached 44° C. 
 No. 2 was taken out at 46° C. and the others in regular order 
 at a constant difference of 2° 0. The temperature was raised 
 to 78° 0. when the last was withdrawn. The tubes were then 
 all cooled and 10 c.c. of the contents of each were mixed with 
 10 c.c. of a 2 per cent, solution of trehalose. Another 10 c.c. 
 from each was mixed with 10 c.c. of a -25 per cent, solution of 
 maltose. The rotatory power of the trehalose mixtures was 
 determined by a polarimeter and found to be 3° 40' ; that of 
 the maltose mixtures was 3° 16' of the same scale. They were 
 all allowed to stand for 36 hours at the temperature of the 
 
140 SUGAR-SPLITTING ENZYMES. TBEHALASE. [CH. 
 
 laboratory and the rotation again observed. The results of 
 
 16 duphcate tubes are recorded in the following table: — 
 
 Temperature of Botation of the Eotation of the 
 
 heating. Trehalose mixture. Maltose mixture. 
 
 50° rio' r32' 
 
 52° r 10' 1° 32' 
 
 54° 1° 20' r 32' 
 
 56° r24' r32' 
 
 58° 1°38' 1°32' 
 
 60° 2° 18' 1° 32' 
 
 62° 2° 30' 1° 32' 
 
 64° 3° 40' r32' 
 
 66° 3° 40' r 50' 
 
 68° 3° 40' 2° 05' 
 
 70° 3° 40' 2° 12' 
 
 72° 3°'4D 2° 24' 
 
 74° 3° 40 2° 54' 
 
 76° 3° 40 3° 16' 
 
 78° 3° 40 3° 16 
 
 The influence of temperature consequently enabled Bour- 
 quelot to discriminate between the two enzymes. Trehalase 
 begins to be affected at 54° C, while glucase is not weakened 
 below 66° C. The activity of trehalase is destroyed at 64° C, 
 while glucase survives up to 74° C. 
 
 In a subsequent paper Bourquelot has shown that trehalase 
 exists also in Polyporus sulphureus, the extract of which is 
 capable of converting the trehalose of trehala into a reducing- 
 sugar during a digestion extending over 48 hours. Working 
 with H6rissey he has detected it also in germinating barley, 
 where it exists side by side with several other enzymes. In 
 conjunction with Gley, Bourquelot has shown that intestinal 
 juice is capable of hydrolysing trehalose, and that pancreatic 
 extract and the serum of blood do not exercise such an action. 
 Trehalase therefore appears to be present in animal as well as 
 vegetable tissues. Its occurrence in the former is supported 
 by the researches of Fischer and Niebel, who have ascertained 
 that trehalose is hydrolysed slowly by an extract of the 
 duodenum of some animals, while a similar extract prepared 
 from others has no effect upon this sugar. The serum of 
 certain fishes, especially that of the carp, has the same hydro- 
 lysing power. 
 
IX] SUGAR-SPLITTING ENZYMES. EAFFINASE. 141 
 
 Raffinase (Melibiase). 
 
 Another enzyme belonging to this group decomposes the 
 sugar known as raffinose. This carbohydrate was discovered by 
 Loiseau in 1876. It occurs in the root of the beet, the seed of 
 the cotton-plant {Oossypium) and in barley and wheat during 
 germination. Tollens has shown it to be identical with the 
 so-called melitose of Eucalyptus manna. 
 
 Raffinose is a hexatriose having the formula CisHsaOia; on 
 hydrolysis by dilute mineral acids it splits up into glucose, 
 fructose, and galactose. According to Scheibler, by using very 
 dilute acids at a low temperature, the hydrolysis can be shown 
 to take place in two stages : — 
 
 Raffinose Fructose Melibiose 
 
 and 
 
 C12H22O11 -f- HjO = CsHiaOe -I- CeHiaOe 
 Melibiose Glucose Galactose. 
 
 Its hydrolysis has been attributed to invertase by the older 
 writers, and recently by O'SuUivan, but Pautz and Vogel, and 
 Fischer and Niebel, point out that the small intestines of the 
 dog and of the horse, which contain invertase or invertin, 
 cannot hydrolyse raffinose. The extract of yeast is able to 
 effect this change as well as the inversion of cane-sugar. It is 
 probable that the hydrolysis is due to the presence of a special 
 enzyme which is contained in the yeast but not in the animal 
 membrane. 
 
 Raffinose has a specific rotatory power of (a)B = + 103'12°, 
 and is not capable of reducing cupric oxide when boiled with it 
 in alkaline solution. 
 
 Bourquelot found that its hydrolysis can be effected by an 
 extract of Aspergillus niger. In his experiments he mixed 
 25 c.c. of a 2 per cent, solution of the sugar with 25 c.c. of the 
 extract of Aspergillus, and digested it for 7 days at 12—15° C, 
 taking precautions against the introduction of micro-organisms. 
 The specific rotatory power of the solution was then only about 
 (a)j, = -I- 50°, while 10 c.c. of Fehling's solution were completely 
 reduced on boiling with 7-4 c.c. of the digestion. 
 
142 SUGAR-SPLITTING ENZYMES. BAFFIN ASE. [CH. 
 
 The enzyme (which may be termed raffinase) has been shown 
 by Bourquelot to be present also in baker's yeast and in low 
 fermentation beer-yeasts. He digested 40 c.c. of the extract of 
 each of these fungi with 40 c.c. of 2 per cent, solution of 
 raffinose, keeping the vessels for an hour at 45° C. and then 
 allowing them to stand at the ordinary laboratory temperature 
 for 5 days, taking the same precautions as before against the 
 introduction of micro-organisms. The rotation of the polarised 
 ray in a 200 mm. tube was at first 2° 4' of the scale of the 
 polarimeter. After 20 hours it had declined to 1° ; after 48 
 hours to 0° 58'. The baker's yeast was a little the more active. 
 The reduction of Fehling's solution was about the same as in 
 his experiments with the extract of Aspergillus. 
 
 The occurrence of this enzyme has also been observed by 
 Fischer and Lindner, who were able to extract it from low 
 fermentation yeasts of the Froberg and Saaz types, but not from 
 the high fermentation forms. They dried the yeasts for 3 days 
 in air at 20° — 25° C after draining them for some time on 
 porous earthenware. When dry the yeasts were extracted by 
 digesting them in water at 33° 0. for 20 hours. The extract 
 was found capable of hydrolysing melibiose. 
 
 Barr has extracted the enzyme from low fermentation 
 Froberg yeast but he attributes to it only the final stage in 
 the hydrolysis, the conversion of melibiose into glucose and 
 galactose, and he has named it melibiose in consequence. Barr 
 holds that the first hydrolysis of rafiinose or melitriose into 
 fructose and melibiose is efifected by invertase. He differs 
 from Fischer and Lindner in saying the melibiose is insoluble 
 in water. This however appears very unlikely from a con- 
 sideration of the properties of other enzymes. 
 
 Melizitase. 
 
 A sugar to which the name melizitose has been given was 
 discovered in 1859 by Berthelot in Bran9on manna, and was 
 ascertained in 1877 by Villiers to exist in considerable quantity 
 in the manna yielded by Alhagi maurorum, a leguminous plant 
 
IX] SUGAR-SPLITTING ENZYMES. MEIJZITASE. 143 
 
 of shrubby habit which grows in Persia and Bokhara. The 
 manna is an exudation from the leaves and branches of the 
 plant, and appears in hot weather in the form of drops which 
 soon harden on exposure to the air and can be collected by 
 merely shaking the branches. Like raffinose, melizitose is a 
 hexatriose, having the formula dsHsaOis. It melts at 148° C, 
 is without the power of reducing Fehling's solution, and has 
 a specific rotatory power of (a)^ = + 88-15°. When hydrolysed 
 with dilute mineral acids it splits up into three molecules of 
 glucose, showing in that way a resemblance to maltose. 
 
 Bourquelot found that a limited hy4rolysis of this sugar can 
 be effected by an extract of Aspergillus niger. He mixed 
 15 c.c. of this extract with 15 c.c. of a solution containing about 
 2'5 per cent, of melizitose and digested it for four days at the 
 temperature of the laboratory, heating it every few hours to 
 50° C. and keeping it at that point for a few minutes. Bour- 
 quelot says that this treatment was effectual in preventing the 
 access of micro-organisms. At the commencement of the 
 experiment the observed rotation of the polarised ray in a 
 200 mm. tube was 2° 9' of the scale of the polarimeter. After 
 three days it had gone down to 1° 32', and at the end of the 
 experiment it was 1° 28'. The liquid then reduced Fehling's 
 solution. 
 
 The hydrolysis of the sugar was not complete. The specific 
 rotatory power fell from (a)D = -!- 88-15° to (a)j, = H- 61-2°. The 
 melizitose was converted into glucose and a hexabiose known 
 as touranose, the decomposition being represented by the 
 equation 
 
 OjgHaaOis + H2O = CjHiaOs + CijHsiiOu 
 
 Melizitose Glucose Touranose. 
 
 Touranose in turn can be further hydrolysed into two 
 molecules of glucose, being isomeric with maltose. The enzyme 
 of Aspergillus, which may be called melizitose, seems to be 
 incapable of effecting this latter change, which is easily brought 
 about by dilute mineral acids. 
 
144 SUGAE-SPLITTING ENZYMES. LACTASE. [CH. 
 
 Lactase. 
 
 The nutritive value of a milk diet partly depends upon the 
 sugar which it contains. This, which is known as lactose, or 
 milk-sugar, is a member of the group of polysaccharides, and 
 upon hydrolysis under the influence of mineral acids a mole- 
 cule of it splits up into a molecule of glucose and another of 
 galactose. It has only recently been ascertained that there 
 exists in the animal body a soluble enzyme which is capable of 
 carrying out a similar hydrolysis, and which may consequently 
 be named lactase. Our knowledge of it is chiefly due to 
 the researches of Rohmann and Lappe, who discovered it 
 in the mucous membrane of the small intestine of calves and 
 dogs. Prior to the appearance of these researches the trans- 
 formations of lactose in the alimentary canal were ascribed 
 to bacterial action and were thought to lead invariably to 
 the formation of lactic acid. Rohmann and Lappe prepared 
 extracts of the mucous membrane of the small intestine with 
 antiseptic precautions and allowed them to act on solutions of 
 lactose. After digestion lasting for several hours glucose was 
 found to be present in the liquid by means of the phenyl- 
 hydrazine-acetate reaction. The osazone produced gave all the 
 reactions of glucosazone. Rohmann and Lappe precipitated 
 the enzyme from the extracts of the intestine by the usual 
 treatment with alcohol, and found that a solution of the 
 precipitate possessed the power of hydrolysing milk-sugar. 
 
 The work of Rohmann and Lappe is supported by some 
 researches of Pautz and Vogel, who ascertained that the 
 mucous membrane of the jejunum of dogs and of new-born 
 children is capable of slowly hydrolysing lactose. Pregl has 
 found that lactase is not present in the intestinal juice of the 
 lamb obtained by the Thiry-Vella method. Fischer and Niebel 
 also have noticed that lactose is hydrolysed by extracts of 
 portions of the small intestine, particularly of young animals. 
 They have shown further that lactase is not present in blood 
 serum. 
 
 Weinland also has found it in the intestine of sucking 
 animals, and in that of the pig, dog, and horse. He states 
 
IX] SUGAR-SPLITTING ENZYMES. LACTASE. 145 
 
 that after several months' feeding on a milk diet, the enzyme 
 makes its appearance in the intestines of the rabbit and the 
 hen. He has also found it in the pancreas of both young and 
 adult dogs. 
 
 The extract of the Kephir organism has been said by 
 Beyerinck to be capable of hydrolysing lactose. He claims to 
 have separated a soluble enzyme from the organism, and to 
 have shown that it possesses the same power. His work has 
 been disputed by other observers, who say that his enzyme 
 splits up cane-sugar and raffinose, but not milk-sugar. Fischer 
 confirms Beyerinck as to its action on the latter, and says that 
 it hydrolyses cane-sugar also. Besides being yielded by Kephir 
 it can, according to Fischer, be extracted from certain yeasts 
 when they are dried at the ordinary temperature, ground up 
 with powdered glass and suspended in water. It diifers by its 
 greater stability from the glucase already described as occurring 
 in some other yeasts. 
 
 Dienert also states that he has extracted lactase from 
 certain yeasts that are capable of fermenting lactose. 
 
 Fischer says that the emulsin of the bitter almond is 
 capable of hydrolysing lactose, a view which is supported by 
 Herissey. A careful examination of their experiments suggests 
 however that their emulsin contained a small amount of lactase. 
 Reference will be made to this more fully in a subsequent 
 chapter. If this view is correct, lactase exists in the seeds 
 of the bitter almond. 
 
 G. F. 10 
 
146 
 
 CHAPTER X. 
 
 GLUCOSIDE-SPLITTING ENZYMES. 
 
 Many bodies of very complex character exist in plants, 
 which have one property in common. When they undergo 
 decomposition under the influence of either mineral acids or 
 soluble enzymes, they yield a sugar, generally glucose, as one of 
 the products of the action. The other bodies which are 
 produced simultaneously are very varied, but one or more 
 of them usually belong to the aromatic series of organic 
 compounds. They are on the whole not very unlike the 
 polysaccharides which have already been discussed, but while 
 the latter are formed of molecules of sugars united to each 
 other, in the former we find the sugar grouped with or united 
 to other radicles. From the fact that the sugar is almost 
 always glucose, these bodies have been termed glucosides. The 
 best known of them are the amygdalin of the almond and other 
 Rosaceous plants, the sinigrin of the Cruciferae, the tannin 
 which is so widely distributed in the vegetable kingdom, the 
 salicin of the willow, and the coniferin of the fir-trees. Many 
 others however exist in other plants. 
 
 The decomposition which they undergo is generally of a 
 hydrolytic character and frequently leads to the total disruption 
 of their molecule. Sometimes it is less complete and consists 
 only in the separation of part of their sugar, a less complex 
 glucoside remaining. The latter decomposition is less frequent 
 than the former and is only known in connection with some 
 members of the group, particularly amygdalin. Mineral acids 
 effect complete hydrolysis as they do in the case of the 
 
CH. X] GLtrCOSIDE-SPLITTING ENZYMES. EMULSIN. 147 
 
 polysaccharides. The more limited decomposition is also 
 known in the latter group, one member of which, melizitose, 
 we have already seen, can be split up by one of the enzymes of 
 Aspergillus, yielding glucose and touranose, the latter on final 
 hydrolysis by acids yielding two more molecules of glucose. 
 
 While it would be too much to say that there is a special 
 ferment for the decomposition of each glucoside it is yet certain 
 that a great number of such enzymes exist. We are acquainted 
 with emulsin, which hydrolyses amygdalin, myrosin, which de- 
 composes sinigrin, erythrozym, which splits up the glucoside of 
 the madder, rhamnase, which acts upon xantho-rhamnin, found 
 in the seeds of the Persian berry, and gaultherase, which de- 
 composes a glucoside of methyl-salicylic ether, occurring in 
 Monotropa hypopythis, a parasite upon the roots of many trees. 
 Others also are known to exist in various plants, though 
 no definite examination of them has at present been made. 
 
 Emulsin (Synaptase). 
 
 The glucoside amygdalin was first obtained in 1830 by 
 Eobiquet and Boutron, who prepared it in crystalline form from 
 the seeds of the bitter almond {Amygdalus communis). They 
 showed this body to be the antecedent of the so-called essence 
 of bitter almonds. In 1837 Liebig and Woehler found that the 
 transformation leading to the appearance of the latter was 
 brought about under the influence of a certain albuminoid 
 matter also existing in the kernel of the almond, and to this 
 principle they gave the name emulsin. They ascertained that 
 during the action of the latter on amygdalin, sugar and 
 prussic acid were formed. Robiquet in the following year 
 suggested that the action belonged to the same category as 
 the action of diastase on starch and gave the name synaptase 
 to the enzyme. The older name however has continued to be 
 applied to it. 
 
 The decomposition of amygdalin which emulsin effects is 
 one of hydrolysis, and can be expressed by the equation 
 
 C2oH,,NO„-|-2H,0 = C,H,0 + HON + 2(CeHaA). 
 Amygdalin Benzoic Prussio Glucose 
 
 aldehyde acid 
 
 10—2 
 
148 GLUCOSIDE-SPLITTING ENZYMES. EStULSlN. [CH. 
 
 Besides amygdalin it is able to effect the decomposition of 
 several other glucosides, among which are scdidn, helidn, phlo- 
 rizin and arbuiin. The several reactions may be expressed by 
 the following equations, the reaction being one of hydrolysis in 
 each case : — 
 
 Salicin Saligenin Glucose 
 
 Helicin Salicylic Glucose 
 
 aldehyde 
 
 C^H^Oio + H,0 = C,5H,,05 + CgH,A- 
 Fhlorizm Phloretin Glucose 
 
 C„HjA + H,0 = CeHA + CeH^O^ 
 Arbutin Hydro- Glucose 
 
 quinone 
 
 Emnlsin has been found by Fischer to be capable of eflFectrng 
 also the decomposition of certain of the artificial glucosides 
 which he prepared as described in the last chapter. 
 
 The occurrence of emulsin has been observed in several of 
 the higher plants and in certain fdngL Of the former, the 
 most striking are the Almond and the Cherry-laurel, both of 
 which belong to the Natural Order Rosaceae. Its distribution 
 in these plants is different ; in the former it is chiefly foimd in 
 the seeds ; in the latter it occurs in the young stems and leaves. 
 In 1865 Thome was led to the opinion that the enzyme exists 
 in the seeds of the bitter Almond only, and is localised there 
 in the fibre- vascular bundles of the cotyledons. Portes in 1877 
 concluded emulsin to be confined to the axis of the embryo and 
 amygdalin to be present in the cotyledona In 1887, Johansen 
 foimd emulsin in the seeds of both the sweet and the bitter 
 vaiieties of the Almond, in the fibro-vascular bundles and the 
 ceUs abutting on them, particularly in those of the cotyledons. 
 He found amygdahn in the parenchyma of the cotyledons of 
 the bitter variety only. Its distribution in these plants and in a 
 few others closely allied to them has been the subject of a care- 
 ful study in recent years by Guignard, whose methods have been 
 partly chemical and partly based upon certain reactions^'leading 
 
X] GLUCOSIDE-SPLTTTING ENZYMES. EMULSIN. 149 
 
 to the development of definite coloration in the cells con- 
 taining the enzyme. The adoption of the latter method of 
 research is attended by great difficulties, and conclusions based 
 solely on such reactions must be received with caution. We 
 may especially allude to the fallacious character of the blue 
 reaction with tincture of guaiacum said by many workers to be 
 characteristic of enzymes, but now known to be given by many 
 other substances usually or frequently present in vegetable cells. 
 Guignard has been careful not to rely exclusively on 
 colour-reactions for the identification of the enzyme but to 
 supplement these by chemical tests. His first experiments 
 were made with the leaf of the Cherry-laurel. When he 
 examined under the microscope some of the tissue of this 
 organ, which he found capable of hydrolysing amygdalin, he 
 observed that certain cells surrounding the vascular-bundles 
 were marked by the very finely-granular character of their pro- 
 toplasm and their freedom from starch and chlorophyll, though 
 they contained a certain amount of tannin, and were richer 
 in proteids than the other cells of the leaf. He treated sections 
 of this tissue with Millon's reagent, which is a mixture of the 
 nitrates of mercury with 4 cm. of nitric acid, and found that all 
 the cells of the leaf darkened in colour, becoming almost black, 
 but the tint was much deepest in the cells in question. On 
 warming the section the black tint gradually disappeared, and 
 was replaced by an orange-red in the cells suspected to contain 
 the enzyme and by a faint pink in the ordinary parenchyma. 
 In an allied species, Cerasus lusitanica, which contains tannin 
 but no emulsin, this orange-red coloration was not developed 
 in the corresponding cells by the same treatment. The re- 
 action was not therefore due to the tannin, but to some proteid 
 constituent with which the enzyme is associated or possibly to 
 the enzyme itself. Probably the reacting body is a proteid 
 rather than thp enzyme, for proteids give a very similar reaction 
 when heated with Millon's reagent, the coloration which they 
 assume being a dull brick-red. Confirmatory tests for proteids 
 bore out this opinion. Copper-sulphate and caustic potash 
 coloured these cells a violet-pink, as they do proteids in solution, 
 while cells containing only tannin did not react to these 
 
150 GI.UCOSIDE-SPLITTING ENZYMES. EMULSIN. [CH. 
 
 reagents more than ordinary parenchyma cells with their 
 lining of protoplasm, which stained a pale pink with no 
 admixture of violet. 
 
 The chemical experiments by which Guignard confirmed his 
 conclusion that these cells contain emulsin consisted of a 
 digestion of the tissues with a solution of amygdalin. The 
 cells in question were observed to occur in definite positions in 
 the leaf, and with care Guignard found it possible to separate 
 them from the adjacent tissues. The difficulties of such 
 separation were mainly mechanical but delicate manipulation 
 enabled him to overcome them. In the leaves and young 
 branches of the laurel the cells occur in the endodermis, which 
 is a sheath surrounding the collection of fibro-vascular bundles, 
 but belonging to the cortex, of which it is the innermost layer. 
 When this sheath was carefully dissected out under a micro- 
 scope and the piece of tissue placed in a solution of amygdalin 
 in a watch-glass, and kept at 50'^ C, the decomposition of the 
 glucoside was speedily effected, and was recognized by the 
 odour of the benzoic aldehyde and prussic acid which we have 
 seen to result from its hydrolysis. Sections made through the 
 leaf produced the same decomposition when they were cut so 
 as to include part of this endodermal sheath, but not otherwise. 
 
 By these methods Guignard determined that the emulsin 
 of the Cherry-laurel exists in the endodermis; that of the 
 Almond was found in the axis of the embryo in the many- 
 layered pericycle which lies immediately under the endodermis 
 and closely surrounds the fibrovascular bundles; in the coty- 
 ledons it is in the endodermis as well as in the pericycle. The 
 enzyme exists in both the sweet and bitter Almond, though 
 amygdalin is only present in the latter. 
 
 Probably the glucoside and the enzyme do not naturally 
 exist in the same cells, as the decomposition of the former in 
 the seed of the Almond only takes place during germination. 
 The distribution of the amygdalin is not however definitely 
 known. It seems probable that the fluid sap containing it may 
 travel along the cellular tissue of the axis of the plant, and as 
 the ferment which decomposes it is in the immediate neigh- 
 bourhood of the bast, which is a great conducting tissue for 
 
X] GLUCOSIDE-SPLITTING ENZYMES. EMULSIN. 151 
 
 elaborated products, it is not unlikely that the decomposition 
 of the glucoside may take place during its transit along the 
 axis in consequence of its diffusion into the cells containing 
 the emulsin, so that the latter is charged with the duty of 
 preparing from the amygdalin certain nutritive products, es- 
 pecially sugar, which may thence easily make their way to the 
 conducting tissues, and so travel to the actual seats of con- 
 structive metabolism. On the other hand it may be that the 
 amygdalin descends by the conducting tissue of the bast and 
 undergoes decomposition there as it passes downwards, the 
 emulsin diffusing into the bast to meet it, though this seems 
 less likely, as Guignard did not lind emulsin at any time apart 
 from the cells which he describes as the seats of its formation. 
 
 The probability that the enzyme and the glucoside are 
 formed in different cells has been challenged by no less an 
 authority than Pfeffer, who suggests that they both exist in the 
 same cells, and that the only degree of separation is that the 
 ferment is in the protoplasm and the glucoside dissolved in the 
 cell-sap. This view however does not harmonise with Guig- 
 nard's experiments. 
 
 The plants mentioned so far all belong to the Rosaceae. 
 Emulsin is not however confined to members of this Natural 
 Order and their near allies. It has been found in plants belong- 
 ing to the Gymnosperms and the Monocotyledons as well as 
 other Dicotyledons. 
 
 Emulsin has been shown during recent years to have a 
 somewhat wide distribution among the Fungi. It was observed 
 almost simultaneously in 1893 by G6rard in Penicilium glau- 
 cum and by Bourquelot in Aspergillus niger. Bourquelot 
 cultivated the latter fungus in Raulin's nutritive fluid till it 
 was about to put out its fructification. The liquid was then 
 decanted off, and the fungus washed several times with dis- 
 tilled water and macerated with water for several days. The 
 filtrate from the maceration contained a mixture of enzymes, 
 of which emulsin was one. When a little of it was added to a 
 solution of amygdalin and kept warm, the characteristic odour 
 of the essence of bitter almonds could be recognised in the 
 course of an hour. 
 
152 GLUCOSIDE-SPLimNG ENZYMES. EMULSIN. [CH. 
 
 Bouiquelot investigated its action on several other gluco- 
 sides and found it capable of hydrolysing some, but not alL 
 He has published the following series of experiments. 
 
 10 c.c. of an aqueous solution of salidn containing "2 grm. 
 were mixed with 10 c.c. of the Aspergillus extract and kept for 
 40 hours at a temperature of 23° C. At the eud of that time 
 55 per cent, of the salicin was decomposed. 
 
 To 10 C.C. of the Aspergillus extract "2 grm. of coni/erin 
 was added. This glucoside being scarcely soluble in water the 
 liquid remained turbid, the coniferin being only in a state of 
 suspension. The digestion was carried on for 30 hours at 23° C. 
 and then for a further three hours at 45° C. During this 
 exposure the liquid became less and less turbid till it was 
 nearly clear. A little later it became milky, and a new 
 precipitate was formed which settled down in the tube, and 
 increased in quantity till the end of the experiment. 
 
 At the end of the digestion the liquid contained '093 grm. 
 of glucose, which was nearly as much as the 2 grm. of coniferin 
 was capable of yielding on complete hydrolysis. The final 
 precipitate was the coniferilic alcohol resulting from the de- 
 composition, which Kke the coniferin itself is almost insoluble 
 ia water. 
 
 H6rissey has prepared the enzyme from the resting seeds of 
 the Almond, by the following method. The seeds, divested of 
 their outer coatings were powdered in a mortar as finely as 
 possible and extracted for 24 hours with twice their weight of 
 water containing a little chloroform. They were then strained 
 off and a little glacial acetic acid was added to the liquid. This 
 precipitated the bulk of the proteid matter contained in the 
 extract. After filtration a clear limpid liquid was obtained. 
 On the addition of about four volumes of alcohol the enzyme 
 was precipitated This was collected on a filter, washed with 
 a mixture of alcohol and ether and finally dried in vacuo, over 
 sulphuric acid. The powder thus obtained was soluble in water, 
 and the solution retained the properties of emulsin. It was 
 however not pure but was mixed with a carbohydrate re- 
 sembling araban. 
 
 A more active preparation was obtained from cultuies of 
 
X] GLUCOSIDE-SPLITTING ENZYMES. EMULSIN, 153 
 
 Aspergillus. The fungus was cultivated from spores in Raulin's 
 solution till its fructification was mature, generally for about 
 three days. The culture fluid was then syphoned off and replaced 
 by distilled water. After a few hours this was again replaced 
 by a further quantity, and the process was repeated till the 
 water contained no trace of the salts of the original liquid. It 
 was then macerated in fresh distilled water for about three 
 days at the ordinary temperature, and the liquid filtered off. 
 So prepared the solution was perfectly clear and limpid, and 
 contained only "2 grm. of residue per litre. It was much more 
 active than the preparation made from the almonds. 
 
 Besides amygdalin, salicin, and coniferin, Bourquelot and 
 Hdrissey found the emulsin of Aspergillus to be capable of 
 hydrolysing many other glucosides, including phloridzin and 
 populin. They obtained negative results however with solanin, 
 hesperidin, convallarin, digitalin, jalapin, and atractylate of 
 potassium, 
 
 Herissey has found that the emulsin of the almond has no 
 action on either phloridzin or populin. Either there are two 
 varieties of emulsin or there is another enzyme in Aspergillus, 
 which decomposes the other two glucosides. 
 
 Beijerinck states that the emulsin of the almond hydrolyses 
 indican. 
 
 Gerard's results with Penicilium were very similar to those 
 of Bourquelot. He prepared his enzyme by macerating the 
 mould in distilled water, and concentrating the extract to a 
 small bulk in vacuo. The emulsin was then precipitated from 
 the concentrated extract by the addition of alcohol. He found 
 it capable of hydrolysing amygdalin and salicin. 
 
 Bourquelot has ascertained that emulsin is present in many 
 other fungi than the species mentioned. It is not confined to 
 saprophytic forms, but exists in a large number of parasites, 
 prominent among which may be mentioned the genus Poly- 
 porus. In all he investigated 43 fungi of parasitic habit, and 
 found 34 to contain emulsin. Most of the latter infest living 
 trees, especially attacking the old wood. It is well known that 
 the cortex, the cambium, and even the woody parts of trees 
 contain glucosides. The distribution of the enzyme in the 
 
154 GLUCOSIDE-SPLITTING ENZYMES. EMULSIN. [CH. 
 
 fungi seems to bear some relationship to the peculiarities of 
 their parasitism. 
 
 Fischer has stated that emulsin is capable of decomposing 
 lactose, yielding glucose and galactose. Hdrissey made the 
 same observation. It appears however from a consideration, of 
 the conditions of the preparation of the enzyme that it is 
 probable that the almond contains also a little lactase. It is 
 a matter of very common occurrence that several enzymes are 
 present in various seeds, and unless careful means of separation 
 are adopted it is rash to assume that the various decompositions 
 set up by an extract of them are all due to a single ferment. 
 The probability that the almond contains lactase is increased 
 by an observation made by H^rissey, that the much more active 
 extract yielded by Aspergillus was altogether without action on 
 lactose. 
 
 A similar association of two enzymes in the same tissue was 
 noted by Schunck who found emulsin and erythrozyme together 
 in the root of the Madder plant. 
 
 Recently H^rissey has discovered emulsin in several Lichens, 
 among which may be mentioned Usnea barbata, Physcia cili- 
 aris, Parmelia caperata and two species of Ramalina. He 
 bruised the lichen, and placed about half a gramme in contact 
 with a solution of amygdalin for two or three days at a tem- 
 perature of 35° C. At the end of that time both hydrocyanic 
 acid and sugar were present. 
 
 It is doubtful whether emulsin exists in the animal orga- 
 nism. KoUiker and Miiller have stated that the pancreatic 
 juice is capable of effecting the decomposition of amygdalin, 
 and Moriggia and Ossi, and Gerard, have separately shown that 
 the secretion of the small intestine of certain herbivora has the 
 same power, but the specific enzyme has not been isolated. 
 
 Emulsin acts most energetically at temperatures between 
 40° and 50° C. ; above the latter point its power gradually 
 declines, but it is not entirely destroyed till heated to near 
 80° C. It works best in neutral solutions, but its activity is not 
 materially impeded by the presence of small quantities of either 
 acids or alkalis. It is precipitated by a solution of tannin. 
 
X] GLUCOSIDE-SPLITTING ENZYMES. MYROSIN. 155 
 
 Myrosin. 
 
 . This body, which is the characteristic enzyme of the Cruci- 
 ferae, was first discovered by Bussy. Not only is it widely 
 distributed throughout the Cruciferae but it occurs also in 
 several closely allied Natural Orders. It acts especially upon 
 the glucoside sinigrin or myronate of potassvwm, effecting its 
 decomposition into sulpho-cyanate of allyl, or essential oil of 
 Mustard, gliocose and hydrogen-potassium sulphate being simul- 
 taneously formed, according to the equation 
 
 CioHisNKS^Oio = QH^CNS + CeH,,0« + KHSO4 
 
 Sinigrin Sulpho- Glucose Hydrogen 
 
 cyanate potassium 
 
 of Allyl sulphate 
 
 It is remarkable that the decomposition of the glucoside is 
 apparently not associated with the incorporation of a molecule 
 of water, so that it does not appear to be a process of hydro- 
 lysis, as in all the other cases so far examined. Van Rijn has 
 stated however that sinigrin contains a molecule of water and 
 that its formula may preferably be given as CioHisNSaKOg-hHjO. 
 
 When the seed of the black mustard, Sinapis (Brassica) 
 nigra, is bruised and treated with water, the odour of the 
 sulpho-cyanate of allyl is easily recognisable. Both the myrosin 
 and the glucoside are contained in the seed, but in separate 
 cells, and the reaction is the result of their being brought 
 together by the solvent. 
 
 Sinigrin can be prepared from the seeds of the black 
 mustard by the following method, which is a modification 
 of that used by Bussy. A kilogramme of the powdered seeds 
 is extracted with 1"5 litre of alcohol of 82 per cent, strength ; 
 and boiled till the alcohol is reduced to 1250 c.c. The residue 
 is pressed while hot, and again boiled with a further quantity 
 of alcohol. After decantation of the spirit it is then pressed 
 and dried at 100° C, and digested for 12 hours with three 
 times its volume of cold water. The residue is again separated 
 by decantation and digested with two volumes of water. The 
 two aqueous solutions are evaporated to a syrupy consistency, 
 
156 GLUCOSIDE-SPLITTING ENZYMES. MYROSIN. . [CH. 
 
 after the addition of a little carbonate of barium. This syrupy 
 residue is then exhausted by boiling alcohol, of 85 per cent, 
 concentration, which dissolves the sinigrin. After filtration the 
 spirit is distilled off, and the glucoside crystallises out. 
 
 The localisation of myrosin has been the object of a very 
 elaborate research by Guignard, vs^ho has investigated its occur- 
 rence in a very large number of plants belonging to the 
 Natural Orders, Crudferae, Capparidaceae, Resedaceae, Tro- 
 pceolaceae, Limnanthaceae and Papayaceae, and has carefully 
 scrutinised the various regions and organs of tjrpical members 
 of all these groups. In 1886, Heinricher showed that in many 
 of the plants of the Cruciferae, special cells, very variously 
 distributed, could be recognised by the peculiar nature of their 
 contents. They gave very strongly-marked proteid reactions 
 and hence he considered them to be reservoirs of albuminoid 
 material. Guignard has found similar cells widely distributed 
 in plants belonging to all the Natural Orders mentioned and 
 by similar tests to those he employed in the cases of the 
 cherry-laurel and almond, he has identified them as the cells 
 which contain mjrrosin. They are recognisable by their finely 
 granular contents and by their being free from starch, chloro- 
 phyll, fatty matter, and aleurone grains, though they are situated 
 in various regions among other cells which contain one or more 
 of these constituents. When the tissue in which they lie is 
 treated with Millon's reagent and warmed, these cells become 
 orange-red in colour, while the parenchyma in which they are 
 embedded only takes on a pale pink tinge. They give a violet- 
 red coloration with cupric sulphate and caustic potash. These 
 cells contain, associated with their protoplasm, a quantity of 
 amorphous proteid matter, which is coagulated by alcohol, and 
 then separates from the peripheral protoplasm in the form of 
 coarsely-granulated masses, which on subsequent treatment 
 with Millon's reagent are coloured a more vivid red than 
 the protoplasm. The cells can be distinguished among the 
 parenchyma in which they lie by staining with methyl-green 
 and other anilin dyes. Usually they are slightly larger than 
 the surrounding cells and are longer and less regular in shape. 
 
 The power of decomposing sinigrin possessed by pieces of 
 
X] GLUCOSIDE-SPLITTING ENZYMKS. MYROSIN. 157 
 
 different tissues carefully dissected out was found by Guignard 
 to vary with the number of such cells which the tissue con- 
 tained. The myrosin seems to be associated in them with 
 the proteid matter, just as in the case of emulsin as described 
 above. The demonstration of the presence of myrosin in these 
 cells was most easily effected in the Wall-flower, in which they 
 form a readily separable layer of the pericycle. When this 
 was isolated with great care and warmed with a 2 per 
 cent, solution of sinigrin, the characteristic odour of the 
 sulpho-cyanide of allyl was perceptible almost immediately. 
 Guignard found throughout his experiments that any tissue 
 containing these cells could effect the decomposition, but that 
 if they were not present the tissue could not act upon the 
 glucoside. 
 
 The results of Guignard's researches into the localisation 
 of these cells in the plants of the various Natural Orders 
 mentioned may be briefly summarised as follows: — 
 
 Roots. Chiefly in the cortex, but sparsely in the wood. In 
 fleshy roots the tissue representing the wood is mainly paren- 
 chymatous, and contains them. In woody roots they are found 
 in the secondary bast as well as in the cortex and to a less 
 extent in the medullary rays. In the Capparidaceae the wood 
 contains none, but some are found in the pith. In the 
 Kesedaceae they do not occur further inwards than the bast. 
 In the Papayaceae the root is not very rich in myrosin. 
 
 Stems. There is a good deal of variety in the distribution 
 in different species. Speaking generally, the pericycle and the 
 tissues derived from it are richest in the enzyme, while the 
 secondary bast comes next in importance. When the special 
 cells occur in the region of the wood they are generally in the 
 medullary rays. The pith also contains some of them. Guig- 
 nard recognises nine types of distribution in the stems of the 
 Cruciferae. 
 
 (1) The pericycle alone. Lepidium sativa etc. 
 
 (2) The pericycle and the primary and secondary basr. 
 
 Erysimum cheiranthoides. 
 
 (3) The cortex chiefly, but the pericycle to a less extent. 
 
 Moricandia hesperidiflora. 
 
158 GLUCOSIDE-SPLITTING ENZYMES. MYROSIN. [CH. 
 
 (4) The cortex, and the bast under a thin and scleren- 
 
 chymatous pericycle. Iberis amara etc. 
 
 (5) The cortex, pericycle and pith. Nasturtium officinale 
 
 etc. 
 
 (6) The cortex, pericycle, and secondary bast. Bunias 
 
 orientalis etc. 
 
 (7) The cortex, pericycle, secondary bast and pith. Ra- 
 
 phanus sativus etc. 
 
 (8) The cortex and pericycle chiefly; also the primary 
 
 and secondary bast and pith. Brassica nigra etc. 
 
 (9) The cortex, pericycle, primary and secondary bast, 
 
 woody parenchyma and pith. Gochlearia armora- 
 cea etc. 
 
 The distribution in the other families referred to is similar, 
 with the exception of the Papayaceae, in which group the 
 stems contain but little mjrrosin. In the Limnanthaceae the 
 special cells lie mainly in the lacunar cortex, but a few are to 
 be found in the bast ; in the Tropceolaceae they generally form 
 groups or nodules of cells in the hypodermal layer of the 
 cortex, a few being in the bast. 
 
 Leaves. Species that contain many of the myrosin-secreting 
 cells in the axis of the plant generally exhibit them in the 
 leaves also, and the relative proportion is often greatest there. 
 They occur throughout the mesophyll but are usually most 
 numerous towards the lower surface. In some leaves they are 
 localised in the mesophyll and the pericycle ; in others chiefly 
 in the pericycle and in the bast of the veins. In a few cases 
 they occur in the endodermis of the bundles. In the Cappari- 
 daceae they are found two or three together, the groups 
 extending through the parenchyma. In the Limnanthaceae 
 they are principally in the epidermis of the lower surface. 
 They are very long in comparison with their breadth, and 
 appear almost tubular ; sometimes two or more lie side by side. 
 In the Papayaceae different species vary as to the abundance 
 of myrosin they contain. A good deal is present in the leaves 
 of Carica condinamarcensis and Vasconcella querci/olia, the 
 cells being chiefly found in the lamina. 
 
 Flowers. In the Capparidaceae the flower contains large 
 
X] GLUCOSIDE-SPLITTING ENZYMES. MTROSIN. 159 
 
 numbers of the secreting cells both in the sepals and petals. 
 They are very numerous in the wall of the ovary. In this 
 order they generally occur in little groups, each of which is 
 developed from a single cell which becomes conspicuous while 
 the tissue is still very young. In Tropceolum they are very 
 prominent in the tissue of the spur. In the flowers of the 
 Cruciferae they are met with chiefly in the carpels, where they 
 lie in the neighbourhood of the flbro-vascular bundles. 
 
 Seeds. In the Cruciferae the distribution of the secreting 
 cells varies ; either the integuments alone, or both embryo and 
 integuments may contain them. Guignard distinguishes flve 
 types exhibiting the following regions of localisation : — 
 
 (1) The cotyledonary parenchyma and the cortex of the 
 
 axis of the embryo. 
 
 (2) The tissue abutting on the back of the cotyledonary 
 
 fibro-vascular bundles, or the pericycle if that layer 
 is differentiated ; also the cortex of the axis. 
 
 (3) Both regions described under (1) and (2). 
 
 (4) Sometimes they are absent from the radicle and the 
 
 cotyledons, 
 
 (5) The integuments only. 
 
 In the Limnanthaceae these cells can be detected in the 
 parenchyma of the cotyledons when the seed is very young 
 and before reserve-materials are stored in any of the cells. 
 Later, on germination of the seed, they can be recognised in 
 the lower epidermis as soon as the cotyledons become green. 
 The other orders except Papayaceae show a similar distribution. 
 In these the myrosin is almost confined to the outer integu- 
 ment, which forms a thin pellicle swelling considerably when 
 in contact with water. It is doubtful whether any of the 
 enzyme is secreted in the inner layer of the seed-coat. 
 
 Jadia has found myrosin in nearly all parts of Moringa, a 
 plant closely allied to the Capparidaceae though not a member 
 of that Natural Order. 
 
 There is no myrosin in the latex of the Papayaceae. 
 
 Besides these Natural Orders, Guignard has discovered 
 myrosin in several species of Manihot from Brazil. It is not 
 localised there in the laticiferous system as in emulsin. 
 
IGO GLUCOSIDE-SPLITTING ENZYMES. MYROSIX. [CH. 
 
 Though the action of rayrosin has been examined chiefly 
 with regard to the decomposition of sinigrin, it is equally 
 efficacious in splitting up sinalbin, the glucoside present in the 
 White Mustard, Brassica alba, and there seems no doubt that 
 it can decompose all the glucosides which are so prominent in 
 the Natural Orders mentioned. These are much alike in the 
 products of their decomposition, the aromatic molecule varying 
 in diflferent cases. 
 
 Guignard has carried out a research on some of the chemical 
 and physical properties of myrosin, in which he has found it to 
 possess many points of agreement with other enzymes. His 
 material was drawn from two sources, the outer integument 
 of the seed of Carica papaya, and the extract of the ground 
 seed of the White Mustard. The enzyme behaved in an 
 almost identical manner in the two cases. 
 
 The outer coat of the seed of Carica can be easily separated 
 from the rest of the structure. It swells up considerably in 
 water and assumes an almost mucilaginous consistency. The 
 inner coat is hard and sclerenchymatous, so that the outer 
 envelope can be easily separated from it. The cells of the 
 outer coat contain hardly anything but myrosin, and a fairly 
 pure preparation of the enzyme can be obtained by swelUng 
 up this layer, separating it from the inner coat, drying it and 
 grinding it to powder. A very minute quantity of this powder 
 is sufficient to set up decomposition of the glucoside. 
 
 Guignard prepared it from the White Mustard by grinding 
 a number of the seeds and soaking them in water at 40° C. for 
 several hours. The filtrate from the paste so formed was then 
 heated to 70° C to coagulate the larger part of the proteids 
 which were subsequently separated by a further filtration. The 
 myrosin was thrown down from this filtrate by the addition of 
 two volumes of alcohol of 90 per cent, concentration, filtered 
 off and dried over a water-bath at 30° C, being finally washed 
 with ether. 
 
 In his experiments on the properties of myrosin, Guignard 
 used both these preparations side by side throughout. 
 
 The enzyme was found to show certain peculiarities with 
 regard to its power of resisting destruction by high temperatures. 
 
X] GLUCOSIDE-SPLITTING ENZYMES. MYROSIN. 161 
 
 A series of tubes was prepared, each containing 5 c.c. of 
 the extract of the seeds of Carica or Mustard, mixed with 
 5 c.c. of water, and they were heated on a water-bath very 
 gradually. When a temperature of 50° C. was reached, the 
 first tube was withdrawn, and another was removed at each 
 increment of 1° C. The heating was continued till the tem- 
 perature of 85° C. was reached. The tubes were then allowed 
 to cool and '02 grm. of sinigrin was added to each, and they 
 were digested at the temperature of the laboratory for 18 to 
 24 hours. The retention of the power of decomposing the 
 glucoside was ascertained by observing whether or no there 
 was a production of the odour of the sulpho-cyanate of allyl in 
 each case. It was found that no deterioration of the power 
 of the myrosin to liberate this body had taken place in those 
 tubes which had not been heated to 81° C. From this point 
 upwards the temperature partially destroyed the enzyme and 
 the destruction was total at 85° C. The curve representing 
 the action at different temperatures was seen to show accord- 
 ingly a very rapid fall between 81° and 85° C, a much steeper 
 slope than that exhibited by the corresponding curve of any 
 other enzyme so far examined. The maximum temperature 
 or point at which destruction takes place is not very different 
 from that given by Kjeldahl for diastase, which is 86° C, but 
 it is considerably higher than that for invertase, which is 70° C. 
 
 Salicylic acid when present in the proportion of less than 
 1 per cent, does not interfere with the action of myrosin; 
 1*5 per cent, weakens it very considerably, and the enzyme is 
 quite inoperative in the presence of 2 per cent. Diastase is 
 affected in a very similar way by salicylic acid. 
 
 The presence of 1 per cent, of tannin nearly stops the action 
 of both myrosin and emulsin. If the digestion is carried on 
 at a high temperature, such as 80° C. the inhibitory effect of 
 tannin is much greater, '05 per cent, being sufficient to suspend 
 altogether the decomposition of the glucoside. 
 
 Chloral has much less effect on the action of either myrosin 
 or emulsin, which is rather surprising, as this reagent combines 
 very energetically with proteids. Guignard prepared a series 
 of tubes each containing 5 c.c. of extract of the testa of the 
 
 G. F. 11 
 
162 GLUCOSIDE-SPLITTING ENZYMES. ERYTHROZYME. [CH. 
 
 seeds of Carica, 20 c.c. of water, and 02 grm. of sinigrin, and 
 to the several tubes of the series he added gradually increasing 
 proportions of chloral, and digested them at 40° C. He found 
 that a tube containing 1 per cent, of chloral evolved a strong 
 odour of allyl sulpho-cyanate in 15 minutes ; in the presence 
 of 2 per cent, much less was produced ; with 3 per cent, the 
 odour was only just perceptible in that time ; with 4 per cent, 
 there was no odour till after 30 minutes ; and with 5 per cent, 
 the action could not be detected till the expiration of an 
 hour. Subsequent digestion for 12 hours did not increase the 
 intensity of the odour. When a tube was heated to 80° C. 
 for 2 minutes with 1 per cent, of chloral, the enzyme was de- 
 stroyed. That temperature alone was without effect upon it. 
 
 Alum and borax, which have antiseptic properties, exercised 
 but little influence upon myrosin, quantities of less than 6 to 8 
 per cent, being innocuous. 
 
 Eri/throzyme. 
 
 A third enzyme which is capable of splitting up a glucoside 
 was discovered by Schunck in 1852 in the root of the Madder 
 plant {Rvbia tinctoria). Unlike the others so far described, it 
 has a very limited distribution and acts upon only a single 
 glucoside. The latter, which Schunck named rubian, was in- 
 vestigated by him two years earlier. He found that by soaking 
 the cortical tissue of the root in water he could extract from it 
 a body which on the addition of either protochloride of tin, or 
 acetate of lead, yielded a beautiful purple precipitate. After 
 separating this from the liquid by filtration, on suspending it 
 in water and passing a stream of sulphuretted hydrogen through 
 it, the sulphide of tin which was formed carried down with it part 
 of the colouring matter, leaving another part in solution. The 
 mixture of these two had been previously described by Kuhl- 
 mann as a single colouring matter under the name of Xanthine. 
 
 The precipitated sulphide of tin with its adherent colouring 
 matter when collected on a filter and well washed with cold 
 water, gave up to boiling alcohol the colouring matter alone, 
 forming a yellow solution. This on evaporation deposited the 
 
X] GLUCOSIDE-SPLITTING ENZYMES. ERYTHROZYMB. 163 
 
 pigment, which Schunck named rubian. When it was hydro- 
 lysed by dilute mineral acids, the beautiful purple colour 
 returned and was proved to be due to the formation of alizarin, 
 glucose and other bodies of complex composition being also 
 present in the solution. 
 
 The enzyme, like the glucoside, was prepared from a watery 
 extract of the madder root. A quantity of the latter was ground 
 and placed upon a layer of fine canvas and extracted with a 
 large volume of distilled water, 4 quarts being used for each 
 pound of ground root. During the process the temperature of 
 the liquid was maintained at 38° 0. The watery extract was 
 added to an equal bulk of alcohol, when a brown flocculent 
 precipitate separated out. This was separated by decantation 
 and washed on a filter with alcohol till the latter failed to extract 
 any more colouring matter. A reddish-brown mass then re- 
 mained on the filter. 
 
 This crude material contained the enzyme, mixed however 
 with various pectic bodies and probably some pectase, a peculiar 
 enzyme which will be described in a subsequent chapter. 
 
 When a quantity of it was added to a solution of rubian and 
 left at the ordinary laboratory temperature for a few hours, the 
 liquid became a jelly of a light brown colour, tasteless, and 
 insoluble in cold water. After standing for a time, any water 
 which was passed through it remained uncoloured. Schunck took 
 this point to be that at which the action of the enzyme ceased. 
 The liquid remained neutral in reaction, and there was no evo- 
 lution of any gas. The jelly in all probability resulted from 
 the action of the pectase on certain constituents of the extract, 
 as we shall see later. In addition to the antecedent of the 
 jellythe extract contained certain substances formed from the 
 rubian. By appropriate treatment Schunck separated from it 
 alizarin, the purple colouring matter which is formed from 
 rubian by acids, verantin and rubiretin, two bodies of resinous 
 nature, rubiafin, rubiagin, and rubiadipin, the last named being 
 of a fatty character. The water with which the jelly had been 
 washed contained glucose and certain pectic bodies. 
 
 The decomposition was apparently of a very complex cha- 
 racter, for under various modifications of the treatment, the 
 
 11—2 
 
164 GLUCOSIDE-SPLITTING ENZYMES. ERYTHROZYME. [CII. 
 
 proportions of the various resulting products were found to 
 vary considerably. Under the conditions described the sugar 
 and the rubiafin and rubiagin were in greatest amount, and 
 the alizarin in smallest, the quantity of rubiretin and verantin 
 being intermediate between the others. When a trace of 
 sulphuric acid was added to a solution in which the action 
 was proceeding, the bulk of the product consisted of rubi- 
 retin and verantin, a little alizarin and rubiagin also being 
 formed. When the liquid was made alkaline instead of acid, 
 carbonate of soda being used in small quantities, there was 
 formed a large amount of rubiafin, more than the average 
 quantity of alizarin, a moderate amount of rubiretin and 
 verantin, and no rubiafin. Excess of caustic alkali produced 
 the same result as dilute sulphuric acid. The more the 
 action was retarded by any cause, the more rubiretin and 
 verantin and the less alizarin resulted. 
 
 From his numerous experiments under varying conditions 
 Schunck came to the conclusion that the decomposition involved 
 three portions of rubian. The first lost water and gave rise to 
 alizarin. The second also lost water, but produced rubiretin 
 and verantin in equal proportions. The third only was truly 
 hydrolysed, taking up water and producing sugar and rubiafin, 
 or with more water, rubiagin. The rubiadipin he found to be 
 present in very small quantity, and he held both its origin and 
 nature to be very uncertain. Schunck attributed all these 
 actions to the influence of the enzyme, which he named 
 erythrozyme. 
 
 This body is insoluble in water, but remains in suspension in 
 it, and can be thrown down irom such a suspension by the ad- 
 dition of acetate of lead or bichloride of mercury. Its activity 
 is destroyed by heating it to 100° C. in the presence of water. 
 
 Erythrozyme is apparently related to emulsin, for the latter 
 enzyme has also a limited power of action on rubian. Schunck 
 used a preparation of emulsin from bitter almonds, which he 
 obtained by precipitating the watery extract of the seeds by 
 alcohol, after getting rid of the oil which they contain. He 
 found that emulsin formed alizarin, verantin, and rubiretin firom 
 the i-ubian, the alizarin being in greater quantity than when 
 
X] GLUCOSIDE-SPLITTING ENZYMES. RHAMNASE. 165 
 
 erythrozyme was employed. He has made no statement as to 
 the occun-ence of either rubiafin, rubiagin, or glucose in his 
 emulsin digestions. 
 
 Schunck considered that though the two enzymes were 
 allied, emulsin had a feebler action than erythrozyme. 
 
 Rhaninase. 
 
 A fourth enzyme also belonging to this group has a still more 
 limited distribution than either of those so far described. It 
 occurs in the seeds of Rhamnus infectorius, the so-called Persian 
 berry, a species whose fruits yield a brilliant yellow dye. This 
 enzyme, which may be called rhamnase, has been investigated 
 by Marshall Ward and Dunlop, and more recently by C. and 
 G. Tanret. The fruits contain a glucoside which is known as 
 Xanthorhamnin and which has the formula C48He8029. When 
 decomposed it yields rhamnetin or rhamnin and two sugars, 
 rhamnose and galactose. If the pulp of the fruits, or an 
 extract of the pericarp is digested with an extract of the seeds 
 and kept for a short time at a temperature of 35° C. a copious 
 yellow precipitate falls, which consists of the rhamnin, the 
 sugars remaining in solution. Boiling the extract of the seeds 
 destroys its power of producing the precipitate. Very careful 
 histological investigations have proved that the enzyme is 
 confined to the raphe of the seed, which is composed of 
 parenchymatous cells, containing a brilliant oily-looking, colour- 
 less substance. The cells exhibit two or three large vacuoles 
 in which a few brilliant graniiles can be observed. When Ward 
 and Dunlop prepared a solution of xanthorhamnin from the 
 pericarp of the fruit and floated a small portion of the raphe 
 of the seed upon its surface, the action of the rhamnase became 
 visible in the course of a few minutes. In ten minutes the 
 floating piece of tissue was covered with a golden-yellow 
 precipitate of the colouring matter ; in twenty minutes clouds 
 of the same precipitate were sinking through the solution, and 
 in less than an hour the bottom of the test tube which con- 
 tained it was covered by a layer nearly two millimetres deep. 
 
 Rhamnase has only been observed in the species alluded to. 
 
166 GLUCOSIDE-SPLITTING ENZYMES. GAX7LTHERASE. [CH. 
 
 It can be precipitated by the addition of alcohol to the cold 
 water extract of the fi-uit. Its optimum temperature is 70° C. 
 and it is destroyed on heating its solution to 85° C. 
 
 Gaultherase (Betulase). 
 
 The fifth enzyme of this group which calls for notice has 
 been the subject of investigations by Schneegans and by Bour- 
 quelot. It was detected by the former of these observers in 
 the bark of Betula lenta and he gave it the name of hetulase. 
 Bouxquelot found it independently in several plants, among 
 which may be mentioned especially Monotropa hypopythis, 
 Gaultheria procumbens, Spiraea Ulmaria, S. Filipendula, and 
 several species of Poly gala. It hydrolyses gaultherin with the 
 formation of methylsalicylic acid (oil of winter-green) and 
 glucose, the following equation representing the reaction: — 
 
 CuH^Os + H„0 = CAOHCOOCH, + CeH, ,0, 
 gaultherin methylsalicylic acid glucose 
 
 Bourquelot has named it gaultherase, from the name of the 
 glucoside. 
 
 Gaultherin was first discovered in 1844 by Procter in the 
 bark of Betula lenta ; he was not successful in preparing it in a 
 pure condition, but he ascertained that on decomposition it 
 yielded methylsaHcyUc acid, and he stated that the same bai-k 
 contained a ferment which was capable of splitting it up. Fifty 
 years later Schneegans and Gerock prepared the glucoside from 
 the same source but by a method which enabled them to get it 
 in a crystalline form and to determine its formula. 
 
 In the same year Bourquelot found that the oU of winter- 
 green could be prepared from several species of Pol^gala (P. 
 vulgaris, P. calcarea, and P. depressa) and from Monotropa 
 hypopythis, a parasite which grows upon the roots of several 
 trees, especially some species of Pinus. Monotropa is the 
 tj-pical genus of the Natural Order Monotropaceae, which is 
 sometimes considered only a sub-order of Ericaceae. 
 
 On bruising the roots of the species of Polygala or the stems 
 of Monotropa the characteristic odour of the oil of winter-green 
 
X] GLUCOSIDE-SPLITTING ENZYMES. GAULTHEEASE. 16T 
 
 is very soon perceptible. Bourquelot states that it arises in 
 consequence of the decomposition of the glucoside under the 
 influence of an enzyme. 
 
 Bourquelot prepared both the glucoside and the enzyme 
 from the stems of Monotropa and ascertained that though both 
 are present, they are not found in the same cells, and that con- 
 sequently the methylsalicylic acid noticed soon after bruising 
 the tissues does not exist in the plant during its intact condition 
 but is formed as a result of the injury. 
 
 He prepared the enzyme by the following treatment: — 
 recently gathered Monotropa plants were triturated in a mortar 
 with washed sand ; the mixture was digested for half-an-hour 
 with 95 per cent, alcohol and the latter removed by filtration. 
 The contents of the filter were then washed with alcohol and 
 subsequently with ether, and dried in contact with the air. 
 The powder so produced contained the enzyme. 
 
 It is of course apparent that the latter is not in any degree 
 isolated by this method. The powder must contain all the 
 constituents of the plant except such as are soluble in alcohol 
 and ether. As the glucoside however is soluble in the former 
 medium, the method can be relied on to separate the two bodies 
 which take part in the hydrolysis. 
 
 Bourquelot found that a little of the powder when added to 
 an aqueous solution of gaultherin very speedily produced the 
 odour of methylsalicylic acid. 
 
 Schneegans states that the activity of the enzyme is in- 
 creased by the presence of small quantities of alkalis or mineral 
 acids. 
 
 Gaultherase is present in considerable quantity in the leaves 
 and berries of Oaultheria procumhens. The characteristic pro- 
 duct of its action, methylsalicylic acid, can also be easily 
 extracted from the same parts of the plant by distilling them 
 with water. 
 
 Bourquelot has prepared the enzyme also from the roots of 
 Polygala calcarea and P. vulgaris, the roots of Spirwa Ulmaria, 
 S. Filipendula, and S. salicifolia, and from the petals and leaves 
 of several varieties of Azalea. 
 
 Bourquelot has confirmed the early work of Procter and the 
 
168 GLUcosiDE-spLirrrNG enzymes, taxxase. [ch. 
 
 researches of Schneegans, showing that the same enzyme exists 
 
 in the bark of Betula lenta. It does not appear to be quite 
 
 cei-tain that the glucosides of Monotropa, Polygala and the 
 
 other plants are identical with the gaultherin prepared from 
 
 Betula by Schneegans and Gerock but it seems probable, as 
 
 in all cases the same enzyme effects its decomposition and 
 
 the oil of -winter-green appears as one of the products of the 
 
 hydrolysis. 
 
 Tannase. 
 
 The fermentation of tanniu has been examined by Strecker 
 
 and by Van Tieghem. The former observer showed it to be a 
 
 glucoside and to undergo hydrolysis according to the equation 
 
 C2,H2,0„ + 4,H,0 = SCrReO, + CeH, A 
 T annin Gallic aoid Glucose 
 
 Van Tieghem showed that certain moulds can decompose 
 tannin, gaUic acid being one of the products. 
 
 Recently Fembach and Pottevin, working independently and 
 in ignorance of each other's researches, have ascertained the 
 presence of an enzyme in one of these moulds, Aspergillus 
 niger, and have proved the hydrolysis to be due to its action. 
 Both prepared the enzyme by cultivating a quantity of the 
 fongus in Rauhn's solution, in which tannin in the proportion 
 of about 3 per cent, replaced the sugar of the normal fluid. 
 
 Fembsich prepared the enzyme from the crop of fungus by 
 the method adopted by Lintner for the preparation of diastase, 
 which has already been described, obtaining it as a giey powder, 
 soluble in water. Mixing some of this with a 10 per cent, solu- 
 tion of tannin and keeping it at 50° C. all the tannin disappeared 
 and on cooling the vessel a precipitate of needle-shaped crystals 
 of gallic acid rapidly followed. 
 
 Pottevin made an extract of the mycelium in chloroform 
 water and sterilised it by filtration through a Chamberland filter. 
 He then prepared two series of sterilised tubes, in each of which 
 he put 10 c.c. of a 30 per cent, solution bf tannin which had 
 been similarly filtered. To each he added 10 c.c. of the stei-ihsed 
 extract, and then boiled one series for several minutes. He then 
 sealed them all with the blow-pipe and kept them at 35° C. 
 After a few days the unboiled tubes contained a deposit of gallic 
 acid while the boiled controls remained unaltered. 
 
X] GLUCOSIDE-SPLITTING ENZYMES. LOTASE. 169 
 
 Pottevin has found the decomposition to be attended "with a 
 formation of glucose, the quantity depending on the purity of 
 the tannin used. It varied from 15 to 98 per cent, of the gallic 
 acid simultaneously formed. 
 
 Fembach also found he could filter his extracts through 
 a Chamberland filter without destroying their activity. 
 
 Pottevin says that tannase can be precipitated by alcohol ; acts 
 in neutral or faintly acid media, has an optimum temperature of 
 67° C. and can completely decompose the tannin in a solution. 
 
 Tannase attacks not only tannin but the compound of 
 tannin and gelatin, as well as other tannates; also the sali- 
 cylates of methyl and phenyl. 
 
 Pottevin has prepared the enzyme not only fi-om Aspergillus 
 but also from the sumac {Rhus). 
 
 Lotase. 
 
 Another recently discovered enzyme of this group, like 
 rhamnase, has only been observed at present in a single plant. 
 The latter is Lotus Arahicus, a member of the Leguminosse, 
 which abounds in Northern Africa, Egjrpt, and the Nile Valley. 
 The plant is known to the natives of those parts by the name of 
 Khuther. It is one of the few poisonous members of the Natural 
 Order. It has for some time been known that at certain 
 stages of its growth, and particularly just before its time of 
 flowering, its vegetative parts, especially the young leaves and 
 stems, are very deleterious to horses and cattle feeding upon 
 them, fatal results being not infrequent. During the year 
 1900 an extended investigation of the plant was conducted in 
 the scientific department of the Imperial Institute by Messrs 
 Dunstan and Henry, which led not only to the identification 
 of a particular glucoside, but also to the recognition of a new 
 enzyme, provoking the decomposition of the latter with the 
 production of hydrocyanic (prussic) acid. The name lotase has 
 been given to the enzyme by its discoverers. 
 
 When the leaves are bruised with water they are found to 
 give off prussic acid with considerable readiness. 
 
 The plant contains in certain of its cells the glucoside 
 referred to, which is a yellow crystalline body having the 
 empirical composition OsaHuNOjo. 
 
170 GLUCOSIDE-SPLITTING ENZYMES. [CH. 
 
 On hydrolysis by dilute acids after separation from the 
 plant, this body yields hydrocyanic acid as one of the products, 
 together with glucose and a new yellow colouring matter, which 
 has received the name lotoflavin. It appears to be isomeric 
 with the pigments which are characteristic of Reseda luteola, 
 and Rkus cotinus. 
 
 Dunstan and Henry have named the new glucoside lotusin. 
 They consider the action of the enzyme like that of dilute acids 
 to be one of hydrolysis and they represent it by the following 
 equation : 
 
 C^H,,NO„ + 2H,0 = C„H„Oa + HON + OeH,A 
 
 Lotusin Lotoflavin Prussic Glucose 
 
 acid 
 
 In the plant the glucoside and the enzyme appear to be 
 contained in different and distinct cells of the tissue of the 
 leaves, so that they do not come into contact with each other 
 till the latter are injured. Lotase is rapidly destroyed by 
 contact with alcohol. 
 
 Besides these enzymes, several others are known to occur in 
 different plants, but they have not been so completely investi- 
 gated. One of them is described by Schiitzenberger as beiag 
 capable of hydrolysing phillyrin, a glucoside existing in the bark 
 of PMllyrea latifolia, and populin, which is found in that of the 
 Aspen. These glucosides are not attacked by yeast nor by the 
 emulsin of the almond but appear to be hydrolised by the 
 lactic Bacterium. 
 
 The enzyme does not appear to have been isolated. 
 
 Jorissen and Hairs have described some experiments with 
 a species of Linum which point to the presence of an enzyme 
 differing somewhat from emulsiu but yet able to hydrolyse 
 amygdaUn. Linum contaios a glucoside to which the name 
 linamarin has been given. On hydrolysis it yields hydrocyanic 
 acid, a fermentable sugar, and a body related to the ketones. 
 The hydrolysis cannot be effected by the emulsin of the almond, 
 but is brought about by an extract of the seed of Linum. The 
 latter also hydrolyses amygdalin, as already mentioned. 
 
 Br^audat has discovered another glucoside-splitting ferment 
 in the leaves of Isatis alpina, which decomposes indican, giving 
 
X] GLTJCOSIDE-SPLITTING ENZYMES. 171 
 
 rise to indigo-white, and a peculiar sugar known as indiglucine. 
 It resembles emulsin in being able to split up amygdalin, but 
 the author does not say whether the two are identical. It does 
 not seem improbable that this is the case as Beijerinck has 
 since shown that emulsin can hydrolyse indican. 
 
 It has recently been claimed by Sigmund that the enzymes 
 of this group have also the power of splitting up fats into 
 glycerin and free fatty acids. He says that he caused myrosin 
 and emulsin to act upon olive oil in closed glass vessels at a 
 temperature of 38° — 40° C. and that free fatty acid was gradually 
 and continuously developed in the mixture, its presence being 
 demonstrated both by litmus and by phenolphthalein. 
 
 His mode of preparing the enzymes is however open to 
 criticism. He bruised seeds of the mustard in the one case, and 
 of the almond in the other, and allowed them to macerate in 
 a quantity of water for twelve or fourteen hours. He then 
 decanted the supernatant fluid and added an excess of alcohol, 
 throwing down a precipitate, which he removed by filtration, 
 washed and dried at about 40° C. This method is not likely 
 to isolate either myrosin or emulsin ; if any other enzyme, e.g. a 
 fat-splitting one, was present in the seeds as well as either of the 
 former, it would certainly have been present in his dried residue. 
 Though hitherto no one has isolated a fat-splitting enzyme from 
 these seeds there seems to be reason for suspecting its presence, 
 as both mustard-seeds and almonds contain oil. Sigmund further 
 states that certain fat-splitting enzymes which he detected in 
 various seeds were able to split up amygdalin and salicin. The 
 same criticism may be applied to this statement. The mode 
 of extraction was similar to that described above, and it is at 
 least possible that his residues contained two enzymes, rather 
 than one as he supposed. 
 
 How far these various glucosides are of value in the nutri- 
 tion of the plant is uncertain. No doubt the sugars which 
 they yield on hydrolysis can be utilised in its metabolism, but 
 whether the other residues can be applied to nutritive purposes 
 has not been determined. Some researches recently published 
 by Treub seem to show that in the case of one plant at least, 
 Pangium edule, some compounds of hydrocyanic acid are made 
 use of Further investigation is necessary on these points. 
 
172 
 
 CHAPTER XI. 
 
 PROTEOLYTIC ENZYMES. PROTEOLYSIS. 
 
 The next group of enzymes which must come under con- 
 sideration comprises those which effect the decomposition of 
 proteids. They occur in hoth the animal and the vegetable 
 organism and play a very important part ia the metabolic 
 processes. Like the diastasic ferments, they may either exert 
 their action in the interior of the cell, as in the case of most 
 vegetable organisms, or they may be extruded in solution ia 
 special secretions, as in the alimentary canal of animals. 
 
 In examining their action we are at once confronted with 
 greater difficulties than in the case of the diastasic enzymes. 
 The constitution of starch has been at any rate approximately 
 determined and the sequence of changes which it undergoes on 
 zymolysis can be expressed in the form of chemical equations, 
 which certainly appear probable if they cannot be regarded as 
 finally established. But iu the case of proteids we meet with 
 similar difficulties to those presented in the case of cellulose. 
 We have no conception at present of their chemical constitution 
 and cannot represent them by any formula. As ia the case of 
 cellulose again we are not dealiag with a single substance, but 
 with a large group the members of which show considerable 
 differences in behaviour and are no doubt differently related to 
 each other. 
 
 We find that certain proteids, such as albumins and glo- 
 bulins, can be split up in various ways by different reagents, 
 and that as a result of such splitting other proteids are formed, 
 which we have reason to think have a simpler composition thau 
 
CH. Xl] PROTEOLYTIC ENZYMES. PROTEOLYSIS. 173 
 
 either albumin or globulin. We find further that on very pro- 
 found decomposition certain of these can give rise to crystalline 
 bodies which are not proteid, but which belong to the group 
 of substances known by chemists as amido-acids. All these 
 bodies occur naturally both in the animal and the vegetable 
 organism. The possibility of a gradual decomposition of a 
 primary proteid such as an albumin appears thus to be in- 
 dicated, resulting in the appearance of a certain number of 
 derived or secondary proteids, which can be broken down finally 
 into crystalline chemical compounds, the constitution and rela- 
 tionships of which have been fully ascertained. 
 
 But this representation of the decomposition appears very 
 crude when compared with the hydrolysis of starch, in which we 
 have been able to see group after group of atoms successively 
 split off and the subsequent fate of each group fairly satisfactorily 
 traced. 
 
 The decomposition of the proteid molecule has been effected 
 by the action of dilute mineral acids at moderate and at high 
 temperatures, and different products have been obtained in each 
 case. 
 
 When an albumin is subjected to a prolonged digestion with 
 •25 per cent, of hydrochloric acid at 40° C. the liquid after a 
 while is found to contain at least four products of decomposi- 
 •tion, known as antialbumate, antialbumid, hemialbumose and a 
 variety of peptone. Antialbumate can be separated from the 
 rest by careful neutralisation of the liquid with a weak alkali. 
 Gillespie says it falls just before complete neutralisation. It 
 is soluble only in faintly acid and alkaline solutions and differs 
 from albumin and globulin by not being coagulated when 
 its solutions are boiled. Antialbumid appears to result from 
 the further action of the acid on the antialbumate ; it is almost 
 or quite insoluble in acid liquids, so that it separates out as a 
 granular-looking residue as the action of the acid proceeds. It 
 is soluble in a somewhat stronger solution of an alkali, such as 
 sodic hydrate of 1 per cent, concentration. Hemialbumose 
 does not appear to be derived from either of the other two. It 
 is soluble in acid and in neutral solutions, so that it is not thrown 
 down on neutralisation. It is precipitated on the addition 
 
174 PBOTEOLTTIC ENZYMES. PROTEOLYSIS. [CH. 
 
 of a small quantity of nitric acid, or of acetic acid in the presence 
 of potassic ferrocyanide, and the precipitate dissolves on warming 
 but reappears on subsequent cooling of the liquid. The peptone, 
 sometimes called hemipeptone, appears to result from the further 
 decomposition of this body. It is soluble in acid, alkabne, or 
 neutral solutions, and is not precipitated on addition of nitric 
 acid or of acetic acid and potassic ferrocyanide, nor is its solution 
 coagulated on boiling. Further reactions of all these bodies 
 will be given subsequently. 
 
 The decomposition eflfected by dilute hydrochloric acid at 
 40° C. is represented by the following scheme by Kuhne, to 
 whose researches much of our knowledge on this point is 
 due : — 
 
 Albumin 
 
 I 
 
 Antialbnmate Hemialbnmose 
 
 Antmlbuimd Hemipeptone Hemipeptone 
 
 A more complete decomposition is brought about by 3 — 5 
 per cent, of sulphuric acid when the digestion is conducted at a 
 temperature of 100° C. The first splitting into two groups 
 appears to be the same, but either no antialbumate is formed 
 or it is at once converted into antialbumid. On the side of the. 
 hemi-groups, both hemialbumose and hemipeptone are formed, 
 and the latter is stiU further decomposed, with the formation of 
 various amido-acids and other bodies, among which leucin and 
 tyrosin are conspicuous. The decomposition is represented by 
 Kiihne as follows : — 
 
 Albumin 
 
 ! 
 
 Antialbmnid Hemialbnmose 
 
 Hemipeptone Hemipeptone 
 
 Amido-acids (lencin &c.) Amido-acids (leacin &e.) 
 
 The idea which Kiihne has propounded that the proteid 
 molecule is a double one and may be separated into an anti- 
 and a hemi-moiety, each of the latter then undergoing further 
 
Xl] PROTEOLYTIC ENZYMES. PROTEOLYSIS. 175 
 
 and different decompositions, is supported by the observations 
 of Schiitzenberger on the action of boiling mineral acids on 
 proteids. As a result of his experiments he comes to the con- 
 clusion that only half the proteid molecule is easily decomposed 
 by the acid, the other remaining in a somewhat modified 
 condition, but yet comparatively unaltered. This moiety 
 Schiitzenberger called hemiprotein ; it is the same body as 
 Ktlhne's antialbumid. 
 
 There has been a good deal of controversy about the nature 
 of the decompositions effected by the proteolytic enzymes. As 
 we shall see later there are two groups of these bodies, the 
 types of which are respectively the pepsin of the gastric secretion 
 of mammals and the trypsin of the pancreas. Each possesses 
 well marked peculiarities; while they resemble each other in 
 the general features of their action, they differ in the extent 
 to which they carry the decomposition of the primary pro- 
 teid. 
 
 Neglecting for the present the earlier researches upon these 
 enzymes, the first investigations which call for discussion are 
 those of Meissner and his pupils, carried out during the years 
 1859 — 62. While Meissner recognised the power of an extract 
 or infusion of the pancreas to split up proteids, he carried out 
 his work chiefly with pepsin in the presence of a small per- 
 centage of hydrochloric acid. He digested various primary 
 proteids with these reagents for varying times at the tempera- 
 ture of the body, about 38° C, and examined the various 
 products of the several decompositions. On neutralising the 
 mixture a precipitate settled out just before the neutral point 
 was reached, which possessed properties resembling those of the 
 body already described as having been found later by Kiihne 
 and named by him antialbumate as already mentioned. Meiss- 
 ner called this body parapeptone. A fter its removal by filtration, 
 the addition of a trace of acid sometimes yielded a small amount 
 of another precipitate, to which he gaye the name metapeptone. 
 When casein was the proteid under examination, a small quantity 
 of a different proteid was formed, which he called dyspeptone. 
 The liquid of the digestion, after being freed from these bodies, 
 contained three other proteids which he considered to be 
 
176 PROTEOLYTIC ENZYMES. PROTEOLYSIS. [CH. 
 
 varieties of peptone and which he accordingly named a, h, and 
 c peptones. 
 
 The reactions of these bodies are described as under : — 
 
 (1) Parapeptone. Soluble in dilute acids or alkalis; 
 
 solution not coagulated on boiling. Precipitated 
 on neutralisation. Incapable of being converted 
 into peptone by pepsin, but digestible by an extract 
 of the pancreas. 
 
 (2) Metapeptone. Insoluble in very dilute acids (-05 to 1 
 
 per cent.), soluble in '2 per cent. 
 
 (5) Dyspeptone. Insoluble in dilute acids ; soluble in dilute 
 
 alkalis. 
 
 (4) a-peptone. Soluble in acid, alkaline or neutral liquids, 
 not coagulated on boiling; precipitated by strong 
 nitric acid, and by potassic ferrocyanide in presence 
 of dilute acetic acid. 
 
 (-5) 6-peptone. Much like (4) but not precipitated by 
 strong nitric acid. Precipitated by potassic ferro- 
 cyanide in presence of an excess of strong acetic acid. 
 
 (6) c-peptone. Like (4) and (5) but not precipitated by 
 
 either strong nitric acid or potassic ferrocyanide in 
 the presence of any proportion of acetic acid. 
 
 Meissner's contemporary opponents failed to confirm his 
 results except with regard to the occurrence of his parapeptone 
 and c-peptone. Meissner claimed that all the bodies he described 
 were due to the specific action of the pepsin upon the primary 
 proteids. The view which came to be generally accepted, largely 
 owing to the influence of Briicke, was that the two bodies 
 mentioned were the sole products of a peptic digestion; that 
 the former, parapeptone, resulted rather from the action of the 
 acid used ; and that the pepsin was capable of transforming it 
 into peptone at a subsequent stage of the digestion. 
 
 Later work however has shown that Meissner's results come 
 nearer the truth than his opponents would admit. 
 
 The subject was again taken up several years later by 
 Klihne and his school, who investigated with great care and 
 thoroughness the decomposition of a primary proteid by both 
 
Xl] PROTEOLYTIC ENZTMKS. PROTEOLYSIS. 177 
 
 pepsin and trypsin, the former being as we have seen essentially 
 an acid digestion, the latter in the animal body being equally 
 certainly an alkaline one. Kiihne found that in the early 
 stages of both a proteid was produced which was precipitated 
 on neutralisation of the digesting fluid, falling just before the 
 neutral point was reached. He found further that the final 
 products differed when trypsin was employed instead of pepsin, 
 the amido-acids leucin and tyrosin appearing only in the former 
 case. However long the digestion was continued the conversion 
 of the proteid into amido-acids was never complete, the final 
 products being a mixture of these with peptone and with hemi- 
 albumose, the same substances as he obtained under the influence 
 of acids. The final product of a peptic digestion was similar 
 except that no amido-acids were produced. 
 
 Kuhne was led to the view that the decomposition of proteid 
 is essentially a process of hydrolysis, comparable with that of the 
 splitting up of cane-sugar under the influence of invertase, and 
 as the latter decomposition yields two different but similar 
 sugars, so he suggested that the hydrolysis of proteids leads to 
 the formation of two forms of peptone which show certain 
 differences from each other. This idea was supported by the 
 constant presence of some peptone side by side with the amido- 
 acids resulting from a long-continued tryptic digestion. Kuhne 
 held that the amido-acids were derived from the decomposition 
 of one moiety of the peptone formed, and that the other moiety 
 was incapable of such a splitting up and hence was present un- 
 changed however long the digestion lasted. 
 
 Taking a quantity of the peptone formed during a peptic 
 digestion and submitting it to the action of trypsin Kiihne 
 found his views confirmed by the impossibility of converting the 
 whole of it into amido-acids. 
 
 He ascertained that the hemialbumose which was present 
 in both digestions was capable of being transformed by pepsin, 
 partially at least, into a peptone which could be further 
 converted by trypsin into leucin and tyrosin, while the neutra- 
 lisation product of the earlier stages of the digestion yielded a 
 peptone which was not capable of such further decomposition. 
 
 Thus he was led to formulate the decomposition of proteid by 
 G. p. 12 
 
178 PROTEOLYTIC ENZYMES. PROTEOLYSIS. [CH. 
 
 the two enzymes severally as under ; the successive stages being 
 in each case hydroly tic : — 
 
 Pepsin. 
 Albumin 
 
 itumose Hemialbmnose 
 
 Antialbumose 
 
 Antipeptone Antipeptone Hemipeptone Hemipeptone 
 
 Trypsin. 
 Albumin 
 
 Antialbnmose Hemialbamose 
 
 ! ! 
 
 -I 
 
 Antipeptone Antipeptone Hemipeptone Hemipeptone 
 
 I I 
 
 Amido-acids Amido-aclds Amido-acids Amido-acids 
 
 The result of either peptic or tryptic digestion is thus to 
 split the primary proteid into two residues, each of which gives 
 rise on further decomposition to a group of compounds. The 
 first or anti-residue is peculiarly resistant to decomposition and 
 never gives rise under natural conditions to a simpler body than 
 a peptone; the other or hemi-residue behaves differently with 
 pepsin and with trypsin, the former only converting it into 
 peptone, the latter decomposing the peptone into leucin, tyrosin, 
 and other bodies. 
 
 The reactions of these products of the cleavage of a primary 
 proteid may be briefly described as under : 
 
 Antialbumose. Soluble in faintly acid or alkaline fluids but 
 insoluble in neutral ones. Not coagulated on boiling. 
 Capable of being converted into peptone by either pepsin 
 or trypsin, the peptone being antipeptone. 
 Hemialbumose. Soluble in neutral, acid, or alkaline fluids. 
 Not coagulated on boiling. Precipitated by a small 
 quantity of nitric acid, by potassic ferrocyanide in 
 presence of a trace of acetic acid, or by a few drops of a 
 saturated solution of sodic chloride in presence of strong 
 
Xl] PROTEOLYTIC ENZYMES. PROTEOLYSIS. 179 
 
 acetic acid, the precipitate in each case disappearing on 
 warming the liquid and reappearing as it cools. 
 Antipeptone. Soluble in neutral, alkaline, or acid solutions 
 which are not coagulated on boiling. Not precipitated 
 by mineral acids nor by acetic acid and potassic ferro- 
 cyanide. Incapable of decomposition into amido-acids by 
 trypsin. 
 Hemipeptone. Differing from antipeptone only in being 
 capable of conversion into leucin, tyrosin &c. by trypsin. 
 Both peptones can be separated from the albumoses by 
 saturation of a boiling solution with ammonium sulphate, 
 which precipitates all the latter. 
 
 A mixture of these two peptones, such as is present in a 
 peptic digestion, is spoken of as amphopeptone. 
 
 This scheme of hydrolysis though in its general lines accepted 
 to the present time is not quite complete. Further research 
 has shown that it needs modification in some important par- 
 ticulars, the albumoses mentioned not being such definite bodies 
 as Kiihne supposed. If antialbumose is prepared and subjected 
 to the influence of either pepsin or trypsin a peculiar insoluble 
 residue alwaj'^s appears during the digestion, which gives the 
 same reactions as the antialbumid resulting from the action of 
 acids. The greater part of it can be converted with some diffi- 
 culty into antipeptone by trypsin but not by pepsin. The hemi- 
 albumose has also been shown to be a mixture of several 
 bodies which are formed successively rather than simultaneously. 
 The decomposition products of the hemipeptone are more 
 varied than Kuhne supposed. His researches however mark 
 a very distinct advance in our knowledge of proteolysis, though 
 the changes are more gradual than his theory suggests, as it 
 was first propounded. 
 
 It will be interesting here to compare the bodies recognised 
 by Kiihne with those described 15 years earlier by Meissner. 
 When we consider their reactions we see that the latter 's para- 
 peptone resembles antialbumose in every respect except that 
 Meissner describes his body as incapable of peptonisation by 
 pepsin. Dyspeptone is probably identical with antialbumid, 
 though Meissner's body was most likely mixed with nucleins. 
 Hemialbumose agrees very closely with Meissner's a-peptone, 
 
 12—2 
 
180 PROTEOLYTIC ENZYMES. PROTEOLYSIS. [CH. 
 
 though the reactions of the latter have not been very fully 
 described. The c-peptone of Meissner was what is now called 
 amphopeptone, a mixture of the two peptones of Kiihne's 
 
 scheme. 
 
 Further researches on the hemialbumose carried out by 
 Klihne and Chittenden have shown that it is not a definite body, 
 but a mixture of four closely allied but distinct albumoses. The 
 name albumose is being abandoned in favour of proteose for the 
 members of the group, the latter terra indicating that they are 
 the results of decomposition of primary proteid matter in general 
 and not of albumin especially. The proportions of the ditferent 
 constituents in any given amount of hemialbumose vary accord- 
 ing to the conditions of preparation of the latter. The four 
 characteristic proteoses are the following: — 
 
 (1) Proto-proteose. Soluble in hot or cold water and dilute 
 
 neutral saline solutions ; precipitated by saturation 
 of the solution with sodium chloride or magnesium 
 sulphate. 
 
 (2) Hetero-proteose. Insoluble in water and therefore 
 
 precipitable by separating the salt from its solu- 
 tions by dialysis. Soluble in solutions of sodium 
 chloride and precipitated from these on addition of 
 the salt to the point of saturation. 
 
 (3) Deutero-proteose. Soluble in water ; not precipitated 
 
 by saturation with sodium chloride unless an acid 
 is added at the same time. Precipitated by satura- 
 tion of its solution with ammonium sulphate. This 
 body approaches the peptones most nearly. 
 
 (4) Dys-proteose. This is very much like hetero-proteose, 
 
 from which it differs only in being insoluble in salt 
 solutions. It is probably produced from hetero- 
 proteose by a species of coagulation. 
 All the proteoses can be separated from peptone by saturat- 
 ing a solution containing them with ammonium sulphate, or, 
 according to Bomer, with zinc sulphate. The saturation must 
 be very complete and the liquid must be made successively neutral, 
 acid, and alkaline. Saturation throws down the proteoses. 
 
 A modification of Kiihne's scheme of hydrolysis has been 
 
XI] PROTEOLYTIC ENZYMES. PROTEOLYSIS. 181 
 
 advanced by Neumeister. According to him the proteoses do 
 not all come from the hemi-residue of the early hydrolysis, but 
 some of them are derived from the anti-group. On this theory 
 albumin may be considered to be composed of hemi-albumin 
 and anti-albumin. The first stage of the decomposition is 
 the splitting of hemi-albumin into proto-proteose and hetero- 
 proteose, while the anti-albumin gives rise to acid-albumin 
 and hetero-proteose. The next step is the conversion of the 
 proto-proteose and both hetero-proteoses into deutero-proteose, 
 and the final stage is the production of anti- and hemi-peptones 
 from the members of the separate groups. 
 
 Neumeister says that in the hydrolysis brought about by 
 trypsin proto- and hetero-proteose are not formed, but that 
 several deutero-proteoses appear as the first stage of the action. 
 
 During the past decade very careful and detailed investiga- 
 tions of the transformation of proteids under the influence of 
 pepsin and hydrochloric acid have been carried out by Chittenden 
 and his pupils, which point to the process of hydrolysis being 
 even more gradual than this. Some experiments upon the 
 digestion of the crystallized globulin or vitellin prepared from 
 hemp seeds show that if the digestion is carried out for different 
 periods, varieties of proto-vitellose can be detected, which are 
 exactly alike in their chemical reactions, but which differ in 
 their percentage composition and perhaps in their specific 
 rotatory power. The deutero-vitellose obtained under the 
 varied conditions also showed differences in its percentage com- 
 position. In the early stages of all the digestions Chittenden 
 noted the formation of a body resembling Kiihne's anti- 
 albumose and having the reactions of an acid-albumin; this 
 gradually disappeared, leaving an indigestible residue of anti- 
 albumid. On filtering this off the liquid contained only 
 proteoses and peptones. He states emphatically that he finds 
 no evidence of the production of peptone directly from the 
 acid-albumin but only from the proteoses. 
 
 Pepsin-proteolysis thus leads to the formation of a series of 
 products by hydration and cleavage, of which four are well 
 defined, viz. proto-proteose, hetero-proteose, deutero-proteose, and 
 amphopeptone. The proteoses may each represent a group of 
 
182 PROTEOLYTIC ENZYMES. PROTEOLYSIS. [CH. 
 
 closely allied bodies, so closely related as not to be distinguish- 
 able by chemical tests, and the exact composition of each group 
 may depend upon the length and intensity of the proteolytic 
 process. 
 
 These proteoses represent definite stages in the progress of 
 the hydrolysis ; the proto- and hetero-bodies yielding deutero- 
 proteose and the latter becoming converted into peptone. This 
 conversion is not however complete, the amount of proteoses 
 and peptones existing together at the conclusion of a digestion 
 varying considerably. In a prolonged digestion of vitellin, of 
 10 days' duration, there was found to be present 23 per cent, 
 of proteoses and 77 per cent, of peptone. In another experiment 
 carried out on egg-albumin, after 17 days' action the proteoses 
 amounted to 44 per cent, and the peptone to 54 per cent. 
 The difficulty of the final conversion is not dependent on 
 an inhibitory influence on the action of the enzyme caused by 
 the presence of the products of the transformation as in the 
 case of the hydrolysis of starch into maltose, for the same 
 results were found in the case of digestions carried out in 
 dialysing tubes, which permitted the escape of the products 
 after their formation. 
 
 A comparison of proteolysis with the hydrolysis of starch 
 shows a similarity in another respect. The final product is one 
 which is capable of passing by dialysis through a moist mem- 
 brane such as vegetable parchment. Sugar is completely 
 dialysable, starch not at all. The primary proteids such as 
 albumin and globulin are also incapable of dialysis — peptone on 
 the other hand' can pass through a membrane with considerable 
 facility. The proteoses, seen to be intermediate between the two, 
 show also an intermediate power of dialysis. Hetero-proteose 
 is very feeble in this respect, deutero-proteose comes next, and 
 proto-proteose next, the two not showing much difference, and 
 all being much inferior to peptone. Ktlhne represents the 
 relative powers of dialysis as 5-22 for hetero-proteose, 241 for 
 deutero-proteose, 28-3 for proto-proteose and 51 for peptone. 
 
 According to Gillespie the comparative rates of dialysis of 
 proto-albumose, deutero-albumose, and peptone prepared from 
 egg-albumin are 2o, 5-4 and 238. 
 
Xl] PROTEOLYTIC ENZYMES. PROTEOLYSIS. 183 
 
 The probability that the process of decomposition of proteids 
 by both acids and enzymes is one of hydrolysis is not only sup- 
 ported by analogy with the action of other enzymes and by the 
 general course of the action so far as it can be traced without 
 actual knowledge of the molecular constitution of proteid matter, 
 but also by comparison of the percentage compositions of the 
 various products of the decomposition prepared as pure as 
 possible with our present methods. The proportion of carbon 
 becomes gradually less as we pass from albumin towards pep- 
 tone, which corresponds to such an increase in the amount of 
 hydrogen as would result from the incorporation of water into 
 the molecule. Too much stress must not however be laid upon 
 this, for our criteria of purity in the case of any proteid are not 
 exact, and with such a large molecule as proteids must possess 
 there is room for considerable error in such a computation. 
 
 The decomposition of proteid has still more recently been 
 accomplished by submitting it to the prolonged action of water 
 heated under pressure to temperatures considerably above 
 100" C. Under such conditions Neumeister has effected the 
 splitting up of fibrin by water at 160° G, with the formation of 
 other proteids, which he calls respectively atmid-albumin and 
 atmid-albumose, there being at the same time a formation of 
 peptone, and the evolution of a quantity of sulphuretted 
 hydrogen, ammonium sulphide also being distinguishable in 
 the solution. Both the new proteid bodies show resemblances 
 to others of the groups resulting from the action of enzymes 
 and both can be transformed into deutero-albumose by treat- 
 ment with sulphuric acid. Chittenden and Meara similarly 
 found that egg-albumin could be decomposed by heating it 
 with water in sealed tubes to 150° — 160° for several hours. 
 Their resulting products were chiefly "atmid" bodies corre- 
 sponding to those which Neumeister obtained from fibrin, but 
 they distinguish two forms of atmid-albumose. They also 
 detected the formation of peptone, leucin and tyrosin, while 
 on opening the tubes the evolution of sulphuretted hydrogen 
 and other disagreeable gases could be noticed. 
 
 The action of water at such pressures and temperatures as 
 were employed is eminently hydiolytic. The similarity of the 
 
184 PROTEOLYTIC ENZYMES. PROTEOLYSIS. [f'H. 
 
 products obtained to those yielded on zymolysis greatly supports 
 the view that the latter also is a hydrolytic process. 
 
 This view is further supported by the fact that it is 
 possible by the action of dehydrating agents to convert peptone 
 into a bod\' resembling acid-albumin, which we have seen to be an 
 intermediate product formed during the conversion of albumin 
 or globulin into peptone. If 10 parts of dry peptone are mixed 
 with twice their weight of acetic anhydride and the mixture 
 heated for a long time to 80° C, and finally the excess of acetic 
 anhydride distilled off and the residue dialysed, it is found to 
 be changed into a proteid that is not diffusible, is soluble in 
 dilute alkali, is precipitated by acetic acid and potassic ferro- 
 cyanide, and by many metallic salts, as ordinary proteids are. 
 According to Hofmeister a similar eflFect may be produced by 
 heating peptone for a long time to 140" C. The resulting 
 brown mass contains a part soluble in water and another 
 not so, which react after the manner of a globulin and a 
 derived albumin respectively. 
 
 Other views of the relationship between ordinary proteids 
 and peptone have been advanced, some observers believing that 
 peptones are polymers and othera that they are isomers of primary 
 proteids. Adamkiewicz suggests that they differ in the re- 
 moval of salts and a rearrangement of the molecule. Schtitzen- 
 berger suggested that peptone is a mixture, which, by treat- 
 ment with phosphotungstic acid, can be separated into two 
 parts, one containing a little more oxygen than the other, and 
 both being ureide bodies. Fibrin on the other hand he held 
 to be a kind of compound ether, which is saponified by the en- 
 zyme, and on taking up water splits into the two bodies found. 
 The transformation is thus one of hydration, being the result of 
 the decomposition of an ether by saponification. 
 
 Kiihne's cleavage theory of the hydrolysis of proteids is not 
 now universally accepted. Objections to it are based on the 
 fact that hemipeptone has never been isolated but remains 
 a theoretical substance. The quantities of the various proteoses 
 are not so regular as the theory suggests they should be. The 
 nature of antipeptone is also disputed ; Siegfided and Balke 
 deny its proteid character, while Kutscher states that he has 
 
Xl] PROTEOLYTIC ENZYMES. PROTEOLYSIS. 185 
 
 found it to be a mixture of hexone bases such as argenin and 
 hystidine, with traces of tyrosin, leucin, glutamic acid &c. If 
 this is confirmed we must consider that both hemi- and anti- 
 groups can be converted into crystalline compounds of nitrogen, 
 or that the hydrolysis of proteid is not a cleavage into two 
 moieties but a series of successive decompositions. 
 
186 
 
 CHAPTER XII. 
 
 PROTEOLYTIC ENZYMES (continued). 
 
 We have seen in the preceding chapter that the enzymes 
 which can effect the decomposition of primary proteids, such as 
 albumin, fibrin, etc. fall into two categories, represented by the 
 pepsin of the gastric secretion and the trypsin of the pancreas. 
 They are alike in their power of splitting up the proteid 
 molecule, probably inducing hydrolysis or decomposition after 
 incorporation of one or more molecules of water. The first 
 products of the cleavage of the primary proteid are probably 
 the same in both cases, identical or at any rate corresponding 
 secondary products being formed. The two ferments differ how- 
 ever in the extent to which the decomposition is carried out 
 by tliem, the peptic enzyme never taking it beyond a point 
 at which the final product is still undoubtedly a proteid, i.e. 
 peptone ; the tryptic ferment splits this up still further, with 
 the formation of amido bodies such as leucin, tyrosin, asparagin, 
 and others. That these bodies are products of the decomposition 
 of proteid is shown further by the fact that in vegetable meta- 
 bolism, they, or some of them, have been shown to be antecedents 
 of proteid or to take part in its construction, disappearing from 
 the cells as proteid is formed. The tryptic enzymes are accord- 
 ingly to be regarded as much the more energetic. They are 
 found to be of much wider distribution both in animals and 
 plants, so much so that when only one proteolytic enzyme is 
 present in an organism it is almost invariably a tryptic one. 
 In the metabolic processes concerned in nutrition it seems 
 necessary, or at any rate advantageous, that the proteid 
 
CH. XIl] PROTEOLYTIC ENZYMES. 187 
 
 molecule should be broken up so far as to produce very much 
 simpler bodies than itself, such indeed as may be compared 
 in complexity with such carbohydrates as the sugars. 
 
 Pepsin. 
 
 This enzyme can be most conveniently obtained from the 
 stomach of the dog or pig by removing the mucous membrane 
 from the underlying muscular coats, and after finely mincing it 
 allowing it to macerate in a solution of hydrochloric acid of 
 about '2 per cent, concentration. During the maceration most 
 of the membrane undergoes digestion. After a time the liquid 
 can be filtered from the undigested debris, when it will be found 
 to be strongly proteolytic. Or the membrane may be extracted 
 with glycerine, which has the advantage of not dissolving the 
 proteids to anything like the same extent as the hydrochloric 
 acid solution. As the maceration in glycerine can be conducted 
 in the absence of the acid, digestion of the membrane takes 
 place to only a very slight extent, so that the glycerine extract 
 is much purer than the acid one. Neither method however 
 yields the pepsin free from proteids. Pekelharing says that 
 such an artificial gastric juice can be purified by dialysis, though 
 there is a considerable loss in the process. 
 
 The pepsin of commerce is a very crude product ; it is gene- 
 rally prepared according to a method first used by Schefier, 
 which consists of saturating with a neutral salt an acid extract 
 of the mucous membrane of the stomach of the pig. As we 
 have seen, this treatment separates from the liquid the proteoses 
 of digestion ; the pepsin adheres to them and can so be with- 
 drawn in large part from the extract. The sticky precipitate 
 is removed and dried at a low temperature, forming a pale 
 yellow or buff-coloured powder. 
 
 The enzyme can however be prepared from the stomach in 
 a relatively pure condition by taking advantage of a property 
 it possesses of adhering to any inert precipitate formed in the 
 liquid in which it is dissolved. The method which first yielded 
 it in anything like a pure state was adopted by Briicke in 1861. 
 He extracted the minced gastric mucous membrane with a 
 
188 PROTEOLYTIC ENZYMES. PEPSIN. [CH. 
 
 considerable quantity of water containing 5 per cent, of phosphoric 
 acid, and after straining off the debris filtered his extract and 
 added lime water almost to the point of neutralisation. A 
 heavy precipitate of calcic phosphate was thus obtained in the 
 extract and the pepsin was almost entirely carried down ^vith 
 it, mixed however with a certain amount of proteid matter. 
 The precipitate was next dissolved carefully in dilute hydro- 
 chloric acid, avoiding great excess, and again thrown down by 
 addition of lime water. A certain portion of the contaminating 
 proteid escaped precipitation, so that the pepsin clinging to 
 the lime salt was much purer than before. It was again 
 dissolved in dilute hydrochloric acid, and a solution of chole- 
 sterin in a mixture of four parts of alcohol and one part of 
 ether was poured through a thistle funnel to the bottom of the 
 vessel containing the pepsin. Cholesterin is insoluble in water, 
 and therefore as the light liquid containing it rose towards 
 the surface, it separated out as a very fine precipitate. The 
 separation was facilitated by shaking the flask in which the 
 operation was conducted. The pepsin adhered as before to the 
 inert precipitate, which was removed by filtration and washed 
 with very dilute acetic acid and subsequently suspended in a 
 little water. The moist mass was then agitated with ether, 
 which dissolved the cholesterin and left the pepsin in solution 
 in the water. The latter was removed by means of a separating 
 funnel. It was necessary to repeat the extraction by ether 
 several times to ensure the removal of the whole of the chole- 
 sterin. The separation of the ether from the watery extract was 
 completed by exposure to the air and the extract filtered and 
 further purified by dialysis. 
 
 A modification of this process has been adopted by Maly, 
 who instead of using the method of separating the pepsin 
 by cholesterin, dialysed the hydrochloric acid solution till it 
 was free from chlorides and pliosphates. By this means he got 
 rid of nearly all the admixed substances. 
 
 The methods adopted by von Wittich, Wurtz, and Loew, 
 described in connection with the preparation of diastase, are 
 also applicable to the extraction of pepsin. Loew's lead method 
 was formerly used in the preparation of the pepsin of commerce 
 
^^^] PROTEOLYTIC ENZYMES. PEPSIN. 189 
 
 but was abandoned because it was found very difficult to remove 
 the last traces of lead, and a great proportion of the pepsin was 
 destroyed in the process of manufacture on the large scale. 
 
 Pekelharing adopted the plan of dialysing artificial gastric 
 juice into distilled water till the acid and salts had disappeared. 
 The pepsin was thus precipitated upon the dialyser. Collecting 
 it and dissolving it again in dilute hydrochloric acid a purer 
 product was obtained, much insoluble proteid being left behind. 
 Further purification was secured by repeating the operation 
 several times. Ultimately Pekelharing obtained a product 
 sufficiently pure to undergo analysis. He found that it gave 
 proteid reactions and contained phosphorus, the latter com- 
 prising less than 1 per cent, of the whole. 
 
 Other methods of preparation differing slightly from these 
 have been described by Ktihne, Petit, and Sundberg. 
 
 The chief seat of the formation of pepsin is the stomach of 
 the Vertebrata, in the mucous membrane of which numerous 
 closely-packed glands occur which secrete it in abundance. The 
 whole of the mucous membrane is furnished with these glands, 
 but its different regions do not supply equally active gastric 
 juice. Most of the pepsin is yielded by the glands which are 
 situated at the cardiac end. As the pylorus is approached 
 by way of the greater curvature the glands produce pepsin in 
 rapidly decreasing amount, and those in the pyloric region itself 
 secrete it in very small quantity. 
 
 Pepsin is associated in the gastric secretion with another 
 enzyme which curdles milk. 
 
 In some of the lower vertebrates the formation of pepsin is 
 not confined to the glands of the stomach but is produced 
 also by similar structures at the lower end of the oesophagus. 
 A curious distribution of the enzyme has been described by 
 Miss Alcock as occurring in the larva of Petromyzon. In this 
 animal the alimentary canal consists of thrae regions, a pharynx, 
 an anterior intestine, and an intestine proper. The most active 
 preparation of the ferment that is yielded by the walls of the 
 alimentary canal is derived from the pharynx, while those of the 
 intestine contain little, if any, when cleansed from the secretion 
 of the so-called liver, which is poured into that portion of the 
 
190 PEOTEOLYTIC ENZYMES. PEPSIN. [CH. 
 
 tube. The glandular structures of the wall correspond in their 
 distribution to this variation of enzymic power. The chief seat 
 of formation of the ferment is however the so-called liver, 
 whose secretion is discharged during life into the intestine. 
 Miss Alcock found that the skin of the larva also secretes the 
 enzyme, the superficial layer of cells being the active ones. 
 
 The statement is made both by this author and by Kruken- 
 berg that the enzyme of the liver is pepsin. The evidence on 
 which the statement is based is meagre, and room is left for 
 doubt whether it is not trypsin, as is the case with the so-called 
 liver of many invertebrates. Levy says, however, that this 
 organ in Helix pomatia secretes pepsin, and Bourquelot claims 
 to have proved the presence of pepsin in the liver of various 
 Cephalopoda, in which however it is associated with trypsin. 
 
 Krukenberg found a proteolytic enzyme in the eggs of the 
 fowl, which is active in the presence of "1 per cent, of hydro- 
 chloric acid. He states also that the mesenteric filaments of 
 the Actinozoa can digest fibrin. Haxtog has stated recently 
 that pepsin exists in the spawn of the fi"og. He confirms 
 Krukenberg as to its occurrence in hens' eggs. Dixon and 
 Hartog found it in Pelomyxa, an Amoeba. 
 
 It is very doubtful whether a peptic ferment exists in the 
 vegetable organism, most of the enzymes which have been in- 
 vestigated proving to be tryptic. The most notable case which 
 is stni uncertain is that of the secretion of Drosera, one of the 
 insectivorous plants. Darwin, who first pointed out the exist- 
 ence of the enzyme, classes it strictly with the enzyme of the 
 mammalian gastric juice, but as he did not examine in detail 
 the products of its action, the matter cannot be regarded as 
 definitely settled. A description of the enzyme of Drosera 
 will be found in a subsequent section. 
 
 Besides occurring in the gastric secretion, pepsin has been 
 found in the blood, Tnuscles, and urine of the higher animals. 
 Many physiologists consider that this is not normal pepsin, and 
 that its occurrence there indicates the way in which so much of 
 the enzyme as has been made use of is removed fi:om the body. 
 Bechamp on the other hand suggests that the pepsia in the 
 blood, which can be extracted from fibrin, is formed by the 
 
XIl] PROTEOLYTIC ENZYMES. PEPSIN. ].91 
 
 leucocytes, and secreted by them into the blood-stream. This 
 view has also been upheld by Leber, who claims for the leucocytes 
 the property of softening and dissolving proteid matter in the 
 tissues by the secretion of an enzyme. This view can however 
 hardly be considered to be proved. 
 
 PepsiQ exhibits a peculiarity in the conditions of its working 
 by virtue of which it stands almost if not entirely alone among 
 enzymes. It can only carry out its specific decomposition 
 when it is in a state of combination with a weak acid. 
 The pepsin of the stomach of the Vertebrata is always found 
 combined with hydrochloric acid, the latter being found in 
 gastric juice in the proportion of '2 — "5 per cent. So close is 
 the union between the two that many physiologists prefer to 
 speak of the proteolytic fluid as "pepsin-hydrochloric acid." 
 Other mineral acids, especially nitric or phosphoric acid, can 
 replace the hydrochloric. Organic acids can play the same 
 part. Wroblewski has published the results of some very 
 careful researches on this point, using pepsin from dogs, fi:'om 
 pigs, and from the human subject. He finds that though acid 
 is essential for the action of all, the different pepsins behave 
 differently in the presence of different acids and are moreover 
 different from each other. He prepared his pepsin by Witte's 
 glycerin method from the mucous membrane of the stomachs 
 in each case. Using pig's pepsin he found it to be most active 
 in the presence of oxalic acid of such strength that one volume 
 of normal alkali solution neutralised 20 volumes of the acid. 
 Then in order came hydrochloric, nitric, phosphoric, tartaric, 
 lactic, citric, malic, paralactic, sulphuric and acetic acids. With 
 human pepsin lactic acid was slightly more efficacious than 
 phosphoric. 
 
 The action of pepsin is influenced by several physical condi- 
 tions, of which the most important is temperature. Klug states 
 that it can act at 0° C, but is then very feeble ; as the tempera- 
 ture rises the activity increases to an optimum which Klug 
 puts at from 50° to 60° C. ; it then falls off' as the digestion is 
 warmed still further, and at 80° 0. it ceases and the enzyme is 
 destroyed. Other writers have put the optimum point much 
 lower, at about 38° — 40° C, the normal body temperature. 
 
192 PKOTEOLTTIC ENZYMES. PEPSIN. [CH. 
 
 Langley says that pepsin is rapidly destroyed by warming to 
 55°— 57°C. 
 
 There appears to be some difference as to the optimum point 
 between the pepsia of warm-blooded and that of cold-blooded 
 animals. Hoppe-Seyler found the pepsin of the pike digested 
 fibrin faster at 15° C. than at 40° C. Many observers have 
 found however that this is not the case with the pepsin of the 
 frog. 
 
 Dilution has a conspicuous effect upon the activity of pepsia ; 
 the best results are obtained when the digestive fluid contains 
 fi-om '5 to "01 per cent, of the enzyme, which is a fairly wide 
 range. With greater or less concentration the activity lessens, 
 though proteolysis will take place with only "005 per cent, 
 present. The pepsia of the dog is more active than that of 
 the pig or the bullock. 
 
 Various neutral salts have been found to have a markedly 
 retarding effect upon the action of the enzyme, especially 
 ammonium sulphate and sodium chloride, the latter if present 
 in greater amount than "5 per cent. Alkahne salts are also 
 prejudicial in a very high degree, sodium carbonate in such 
 small proportion as '005 per cent, causing an appreciable de- 
 struction of pepsin in one to two hours at the body temperature. 
 The destruction varies according to the strength of the alkali 
 or alkaline salt, the time during which it is allowed to act, and 
 the amount of proteids present, the latter berag preservative 
 up to a certain amount. The protection is however far fi-om 
 complete. Langley and Edkins found that in the presence of 
 2'5 per cent, of peptone, seven-eighths of the pepsin ia aa 
 extract of cat's gastric mucous membrane was destroyed at 
 17° C. by '5 per cent, of sodium carbonate in 60 seconds. 
 Carbonic acid gas is also deleterious, but much less so than 
 an alkali. Peptone is very efficacious as a protection against 
 this gas. 
 
 A great difference may be observed between the effect of 
 alkabne and that of neutral salts. While the former destroy 
 the enzyme entirely, the latter only inhibit its action. If they 
 are removed by dialysis or other means, the pepsin is found to 
 be still capable of canning out proteolysis. 
 
XIl] PEOTEOLYTIC ENZYMES. PEPSIN. 193 
 
 Loew states that an inhibitory effect is exerted by the 
 presence of 1 per cent, of quinine. 
 
 Chittenden and Amerman have found that the removal of 
 the products of the action of pepsin during a digestion has little 
 or no influence upon the course of proteolysis. 
 
 The action of pepsin has been chiefly studied upon fibrin 
 or coagulated egg-albumin. The nature of the decomposition 
 which it sets up in these cases has already been discussed. 
 Babcock and Kussell state that when it is made to act on 
 casein the products of the digestion consist almost entirely of 
 proteoses, scarcely a trace of peptone being found. Chittenden 
 and Hartwell and later Chittenden and Mendel have studied 
 the proteolysis of the crystalline globulin or vitelliu which can 
 be prepared from the seeds of the Hemp, and which may be 
 taken as a representative vegetable proteid. The course of the 
 action was found to be much the same as with fibrin or 
 egg-albumiu, syntonin or a form of acid-albumin being formed 
 at the commencement of the digestion, together with a small 
 amount of an insoluble residue correspondiag to antialbumid. 
 Later proto- and deutero-vitelloses and peptone could be 
 detected as in the other cases, but only a very small quantity 
 of hetero-vitellose was formed. By varying the length of time 
 during which the digestion was carried on, Chittenden and 
 Mendel identified two proto-vitelloses. 
 
 Harlay has recently observed among the products of de- 
 composition produced by pepsin a peculiar chromogen, which, 
 when acted on by tyrosinase, an oxidising enzyme present 
 in the juice of Russula delica, gives a red colouration which 
 slowly becomes green. When this chromogen is digested with 
 trypsin it gives rise to tyrosin and tryptophane, a peculiar 
 substance discovered in tryptic digestions by Neumeister, which 
 turns red on treatment with chloriae water. The peptic 
 chromogen is not decomposed by pepsin. 
 
 Besides the proteids, gastric juice by virtue of its pepsin can 
 effect the decomposition of gelatin. Tiedemann and Gmelin 
 were the first to observe that this body, under the action 
 of the secretion of the stomach becomes liquid, and loses 
 the power of gelatinisation. Metzler, Schweder, Etzinger and 
 
 G. F. 13 
 
194 PKOTEOLYTIC ENZYMES. PEPSIN. [CH. 
 
 other observers have obtained the same result with a solution 
 of pepsin. 
 
 The process of the decomposition of gelatin has been studied 
 in detail by Chittenden and Solley, and has been ascertained to 
 be almost exactly similar to the course of proteolysis by the 
 same enzyme. The chief difference between the two is the 
 absence of any neutralisation precipitate corresponding to 
 acid-albumin. The bulk of the products arising during the 
 course of the digestion consists of proto-gelatose and deutero- 
 gelatose, gelatin-peptone also being formed, but only in small 
 amount. These various bodies show properties very much Uke 
 the corresponding ones produced during digestion of albimiin. 
 
 Dastre and Floresco have arrived at similar conclusions to 
 those of Chittenden and Solley. They also found similar 
 changes to those induced by pepsin to follow the action of 
 water at high temperatures under pressure. 
 
 Pepsin also acts upon the so-caUed nucleo-alhumins, or 
 micleo-proteids, which appear to be compounds of proteids 
 with a substance called nuclein, the latter being the principal 
 material foimd in the nuclei of cells. The enzyme appears 
 to attack chiefly the albumin of the compound peptones and 
 albumoses being formed, together with a phosphorised residue 
 which is probably slightly altered nuclein. Pepsin does not act 
 upon the substance of the nuclei. 
 
 The action of pepsin upon the caseinogen of milk is 
 interesting from this point of view. The latter has generally 
 been considered to be a proteid, much resembling the derived 
 albiunins. Lubavin has found that when digested with pepsin 
 a phosphorus-containing body closely resembling if not identical 
 with nuclein is separated out as an insoluble residue. This fact 
 lends support to Hammarsten's view that caseinogen is rather a 
 nucleo-albumin than a true proteid. 
 
 Pepsin also digests elastin, but is apparently without action 
 on mucin. 
 
XIl] PKOTEOLTTIC ENZYMES. TRYPSIN. 195 
 
 Trypsin. 
 
 This is the characteristic enzyme of the pancreatic secretion 
 of the higher animals and the chief agent in the proteolysis 
 which takes place in plants. It differs from pepsin in two 
 important respects. It effects as we have seen a more com- 
 plete disruption of the primary proteid, breaking some of it 
 down so completely that crystalUne amide bodies result from 
 the decomposition. Further it is not dependent like pepsin 
 upon the presence of an inorganic ally such as hydrochloric 
 acid. The trypsin of the pancreas acts most vigorously in the 
 presence of a small amount of an alkaline salt such as carbonate 
 of soda, but there is no definite relation between the alkali and 
 the enzyme. Digestion by pancreatic juice can go on indeed 
 in a neutral or even a faintly acid medium; Ewald says that 
 the trypsin of the ox can digest fibrin in the presence of '3 per 
 cent, of hydrochloric acid. Mays obtained the same result. 
 The vegetable trypsins agaia are most active in a faintly acid 
 medium. 
 
 In the pancreatic secretion trypsin is associated with several 
 other ferments, one of which, pancreatic diastase, has already 
 been discussed. Among the other enzymes which it contains 
 are a form of rennet, which curdles milk, and pialyn or lipase, 
 which decomposes fats. The two latter will be considered 
 subsequently. Pancreatic juice as ordinarily obtained contains 
 also a considerable quantity of proteids. 
 
 Similar admixture with other enzymes is frequently found 
 to mark the trypsin of vegetable origin. 
 
 The preparation of trypsin from the pancreas or its secretion 
 is attended with difficulty, though a proteolytic extract of the 
 organ can be obtained by digesting the miaced tissue with a 
 dilute acid or with glycerin, the operation being conducted^ 
 at 35° — 40° C. Such an extract contains however the other 
 enzymes described as well as the trypsin. 
 
 The separation of the three especially digestive enzymes, 
 trypsin, diastase, and pialyn or lipase, was first effected by 
 Danilewski in 1862. He took a freshly extracted pancreas and 
 ground it up in a mortar with sand and cold water. Eemoving 
 
 13—2 
 
196 PEOTEOLTTIC EXZTMES. TRYPSIX. [CH. 
 
 the debris he saturated the liquid with magaesic oxide, which 
 caused the precipitation of the pialyn, associated with certaia 
 other constituents of the extract. The filtrate from this pre- 
 cipitate contained the diastase and the trypsin, and these were 
 separated by shaking it with an alcoholic and ethereal solution 
 of collodion. The precipitate of the collodion carried down the 
 trypsin, leaving the diastase in solution. The two can be 
 separated in another way, by acidulating the extract with 
 phosphoric acid and then neutralising by lime water. The 
 precipitate so caused carries down the diastase, leaving the 
 trypsin dissolved. 
 
 Kiihne prepared trypsin in what is generally regarded as an 
 approximately pure condition by the following process. The 
 minced tissue of a fresh pancreas was macerated in alcohol and 
 ether and two extracts were prepared from it ; the first was acid, 
 the acid used being salicylic acid in the proportion of '1 per 
 cent. After maceration in this liquid for 4 hours at 40° 0. the 
 extract was separated from the tissue by pressure through a hnen 
 strainer. The residue was next extracted by a '25 per cent, solu- 
 tion of sodium carbonate, the digestion as before being conducted 
 at the body temperature; after 12 hours' maceration the liquid was 
 again separated by pressure. The two extracts, which contained 
 the enzymes together with a good deal of proteid matter, were- 
 then mixed, and sodium carbonate added to the whole tiU it 
 contained about "5 per cent, of the salt. The mixture was then 
 allowed to digest in the presence of some antiseptic till the 
 trypsin it contained had converted the proteids into peptones. 
 A somewhat prolonged digestion was necessary, sometimes ex- 
 tending over a week. The liquid was then allowed to stand iu 
 the cold for 24 hours and filtered. It was next made faintly 
 acid with acetic acid and saturated with neutral ammonium 
 sulphate. As this reagent precipitates albumoses, it was very 
 necessary that the preHminary digestion should be prolonged 
 till these were converted into peptones. In the absence of 
 albumoses, the ammonium sulphate precipitated the trypsin in 
 a fairly pure condition and the latter was collected on a filter, 
 washed and dissolved in a little alkali. 
 
 The method seems open to criticism to a certain extent- 
 
XIl] PROTEOLYTIC ENZYMES. TRYPSIN. 197 
 
 It has been found by many observers that the total conversion 
 of the albumoses into peptones is a very difficult process and 
 probably never takes place, a certain portion of albumose always 
 remaining in a digestion. When Kiihne examined the compo- 
 sition of his product he found it always gave up to boiling 
 water a certain amount of coagulated proteid and nearly 80 per 
 cent, of albumose. He attributed this to decomposition of the 
 enzyme itself, but it appears certainly possible that it may 
 have arisen from incomplete hydrolysis of the albumoses of the 
 original extracts. 
 
 While trypsin is the most potent proteolytic agent in 
 vertebrate animals it has also a very wide distribution among 
 the lower forms. The course of proteolysis has however been 
 examined only very incompletely among these and in many it 
 is not certain whether it is peptic or trj^tic. Passing by these 
 for the present and dealing only with those whose secretions or 
 tissues have been investigated we notice that Fredericq has 
 prepared an enzyme from certain sponges which converts pro- 
 teids into peptones and partially into leucin and tyrosin. He 
 has met with the same body in certain Echinodermata. In the 
 pyloric cseca of TIraster, which are situated in the rays of the 
 animal, there is found a digestive fluid which is strikingly like 
 pancreatic juice. It decomposes fats, saccharifies starch and 
 dissolves coagulated albumin. Fredericq extracted the enzyme 
 by the method of macerating the tissue in glycerin after 
 dehydration by alcohol. So prepared, it digested muscle-fibre 
 with the formation of leucin and tyrosin. He says it is most 
 active in alkaline media, less so in neutral fluids and possesses 
 hardly any power in acid extracts. In the Gcelenterata markedly 
 glandular cells are found in the endoderm, and the body-fluid 
 possesses digestive properties. 
 
 In Worms the intestine is partly surrounded by a yellowish 
 glandular tissue which is said to be pancreatic in function. 
 Fredericq extracted fi*om the dehydrated tissue of the whole 
 body of the worm an enzyme which digested fibrin in both acid 
 and alkaline solutions of various strengths. The most advan- 
 tageous one was '6 — 1'2 per cent, of hydrochloric acid. Though 
 the extract when neutral was less potent, it was yet capable of 
 
198 PROTEOLYTIC EXZYMES. TRYPSIN. [CH. 
 
 carrying out a slow digestion. Later Fredericq dissected out 
 the intestine of a large earth- worm and found ia the cavity a 
 slightly alkaline fluid which was capable of digesting fibrin. 
 This fluid was the secretion of the glandular cells which have 
 been mentioned as covering the intestine (page 43). Griffiths 
 has found iadol in the products of a proteid digestion effected 
 by the enzyme prepared fi-om the worm. The evidence thus 
 points to a tryptic enzyme in the gland. 
 
 In Insecta there is also evidence of the presence of trypsin ; 
 Griffiths has found the pyloric caeca of Blatta to be pancreatic 
 in function. In the stomach of both larva and imago of certain 
 Lepidoptera are glandular follicles which secrete a juice corre- 
 sponding in proteolytic activity to that of a pancreas of the 
 Yertebrata. Abelous and Heim detected trypsin in the eggs of 
 certain Crustacea, in which diastase was also present, as already 
 mentioned (page 44). 
 
 The so-called liver of the Crustacea and the Molhisca appears 
 to be in part at all events a digestive gland of the type of 
 the pancreas. Bourquelot has studied it very carefully in. the 
 Cephalopoda and concludes that it secretes both pepsin and 
 trypsin. Fredericq who has examined it in many families says 
 that in them all the fluid it secretes is capable of digesting 
 starch, splitting up fats, and dissolving fibrin with formation of 
 leucia and tyrosin. The enzyme can be prepared fi-om the 
 tissue by dehydrating it with alcohol and subsequently ex- 
 tracting it with glycerin, according to the methods of von 
 Wittich or Krawkow which have been already described. In 
 the Tunicata the tubules which ramify over the wall of the 
 intestine are said to constitute a pancreatic gland. 
 
 The distribution of trypsin in the vegetable kingdom will 
 be treated of subsequently. 
 
 In the conditions of its action trypsin much resembles 
 pepsin; it is similarly affected by temperature, and by even 
 sUght excess of acids or alkaUs. Pancreatic trypsin is destroyed 
 by •! per cent, of hydrochloric acid and totally inhibited by 
 ■05 per cent, of lactic acid. Neutral salts in excess affect it 
 much as they do pepsin. 
 
 The removal of the products of the digestion as they are 
 
XIl] PEOTEOLYTIC ENZYMES. TRYPSIN. 199 
 
 formed has a considerable influence upon the course of pro- 
 teolysis. Lea has carried out a very careful research upon this 
 point, using trypsin prepared by Kiihne's method, and carrying 
 out two operations side by side, the one in a glass flask and the 
 other in a dialysing tube surrounded by a solution containing 
 •25 per cent, of sodium carbonate and a little thymol. He has 
 found that the typical disintegration of fibrin in its earlier 
 stages is much more rapid in the dialyser than in the flask, and 
 that the digestion is never so complete in the latter case, more 
 antialbumid being left at the conclusion of the experiment. 
 On the other hand the amount of leucin and tyrosin formed in 
 a flask-digestion is always greater than in a dialyser-digestion, 
 other conditions being the same in both cases. 
 
 The course of the decomposition of primary proteids 
 effected by trypsin has already been described. A difference 
 between this enzyme and pepsin in the case of the proteolysis 
 of fibrin may be noticed here. When fibrin is digested by 
 pepsin it swells up under the influence of the hydrochloric acid 
 with which the enzyme is associated and is subsequently dis- 
 solved, the fluid remaining clear until nearly the end of the 
 digestion, when the resulting antialbumid is lefb as a granular 
 residue. With trypsin the course of action is different. The 
 fibrin does not become swollen or translucent, but is gradually 
 corroded rather than dissolved, so that the digestive liquid is 
 always turbid and full of a finely granular debris. 
 
 It has already been mentioned that the decomposition 
 products arising from the tryptic digestion of hemipeptone 
 are more varied than Kuhne supposed. The amido-acids of the 
 fatty series include not only leucin (amido-caproic acid) but 
 amido-valerianic acid, asparagin, aspartic acid and glutamic 
 acid. Hedin also obtained two basic substances from a pan- 
 creatic digestion, lysin and Vysatinin. These were originally 
 prepared firom proteid by Drechsel, but he did not prove them 
 to be the result of the action of trypsin. Hedin has since shown 
 lysatinin to be a mixture of lysin and argenin, the latter of 
 which can be made to give rise to urea. The amido-acids of 
 the aromatic series are represented by tyrosin. 
 
 The tryptic digestion of fibrin has been shown by Hirschler 
 
200 PROTEOLYTIC ENZYMES. TRYPSIN. [CH. 
 
 and by Stadelmann to be attended with the liberation of a 
 small amount of ammonia. 
 
 Nencki identified among the products of tryptic digestion a 
 certain quantity of xanthin, guanin, hypoxanthin and adenine- 
 kypoxanthin. Kossell denies that these are due to the action of 
 trypsin on proteids and attributes their presence to the nucleins 
 of leucocytes entangled in the fibrin which was undergoing 
 digestion in Nencki's experiments. 
 
 Among the usual products of decomposition set up by 
 trypsin a substance is found which assumes a red colour on the 
 addition of chlorine water and a violet one when bromine water 
 is added to it. This substance was originally observed ia an 
 extract of the pancreas by Tiedemann and GmeHn. It was 
 ascertained by Neumeister to be formed simultaneously with 
 tyrosin, so constantly indeed that its presence in a digestion 
 liquid might be taken to be an indication of the formation 
 of tyrosin. He gave it the name tryptophane. Stadelmann 
 has suggested the name of proteinochromogen for this body and 
 proteinochrome for the substance produced from it by bromine 
 water. 
 
 According to Nencki the chromogen consists of two sub- 
 stances, from the bromine compound of one of which he 
 prepared skatol, pyrrolin, and indol by fusing it with caustic 
 potash. 
 
 Kossell states that among the products of the action of 
 trypsin on proteids certain bodies occur which he calls pro- 
 tamines ; by further digestion with the same enzyme a number 
 of basic bodies are formed, to which he has given the name of 
 hexones. They include argenin, histidin, and others. 
 
 Harlay attributes the formation of tryptophane to the 
 action of trypsin upon the chromogen which he says is formed 
 during peptic digestion. This substance undergoes decom- 
 position into tryptophane and tyrosin, which explains their 
 constantly simultaneous appearance, already mentioned as 
 observed by Neumeister. 
 
 Trypsin is capable of digesting mitcin, which resists the 
 action of pepsin; it cannot attack the collagen of connective 
 tissue or tendons, though it can digest the gelatin into which 
 
XIl] PROTEOLYTIC ENZYMES. TRYPSIN. 201 
 
 this material can be converted by prolonged boiling with water. 
 The products of its action on gelatin according to Nencki are 
 gelatin-peptone, leucia, glycin (amido-acetic acid) and ammonia. 
 Elastin is more or less rapidly dissolved by it, yielding, according 
 to Walchli, glycin, leucin, valerianic acid and ammonia. 
 
 A curious peculiarity attending the action of pancreatic 
 juice upon milk was first noticed by Eoberts and subsequently 
 confirmed by other observers. Milk is an emulsion of fat 
 (cream) ia a liquid containing certain proteid, carbohydrate, 
 and mineral constituents. The chief proteid is the one generally 
 called casein, which in. its reactions approaches the group of 
 derived albumins. It is so much like them indeed that it was 
 formerly considered to be a form of alkali-albumin. This body 
 differs from a primary albumin or a globulin by not undergoing 
 the process of heat-coagulation as the temperature of its solvent 
 is raised. Roberts has shown that when milk is digested for a 
 short time with pancreatic juice and then boiled, the casein 
 separates out in the form of a scum or coagulum. He suggests 
 that this change is due to the action of the trypsiu and iudi- 
 cates a very early stage in the course of proteolysis. Longer 
 digestion with pancreatic juice causes the disappearance of this 
 property and then the ordinary decomposition products of the 
 casein, proteoses, etc. can be detected. Roberts has named this 
 new body, derived fi-om casein and coagulating on boiling 
 "metacasein." His facts have been corroborated, but a dif- 
 ferent interpretation of them is now suggested. Later writers 
 consider that the action is not connected with proteolysis by 
 the trjrpsin, but that the metacasein is formed by the rennet 
 enzyme under particular conditions. This point will be further 
 discussed subsequently. 
 
 Litracellular proteolytic digestion is not of such general 
 occurrence in the animal kingdom as diastasic. It is however 
 far fi:om unknown, but whether it is brought about by peptic 
 or by tryptic enzymes, and how far the direct action of the pro- 
 toplasm is involved, is uncertain in most cases. Metschnikoff 
 has described digestion as occurring ia the cells of the mesoderm 
 of Synapta and Phyllirhoe, and has claimed that the leucocytes 
 
202 PEOTEOLYTIC ENZYMES. [pB. 
 
 of the higher Vertebrata possess digestive power. Leidy has 
 described the ingestion of an Amoeba by a larger one of another 
 species, and other observers have recorded similar circumstances. 
 There is a general agreement among them that when such 
 ingestion has taken place the absorbed matter often lies for 
 a time in a vacuole in the animal's substance, surroimded by 
 what is apparently a digestive liquid. 
 
 Miss Greenwood has described the processes of digestion in 
 several of the lowHest forms. In both ATnceba and Actino- 
 sphcBrium she has watched the ingestion of solid food particles 
 and the subsequent changes which such particles undergo. 
 The ingested matter is surrounded by fluid, sometimes in not 
 inconsiderable quantity, so that it lies in a vacuole in the 
 animal's substance. If the absorbed matter is innutritions the 
 vacuole of ingestion soon disappears ; if nutritious it undergoes 
 change which is not effected by direct contact with the living 
 substance, but by something passing out of the protoplasm 
 into the vacuole, which thus becomes a digestive cavity, 
 and receives a secretion from the living substance. This 
 secretion is probably not acid, but it exerts a digestive action 
 on the imprisoned food material, which after a time disappears. 
 If the ingested matter is an Alga surrounded by a. cellulose 
 membrane, the latter is not dissolved but its proteid contents 
 are digested. This fact clearly shows that in these cases the 
 digestive action is exerted by a secretion which can diffuse 
 through the membrane, and not by contact with the protoplasm 
 itself. The fact that innutritions substances do not provoke 
 this formation of a digestive vacuole points to the absorption of 
 food-material being a stimulus setting up the secretion of a 
 digestive juice. 
 
 In Actinosphcerium soon after the ingestion of food-material 
 there is a marked appearance of granularity in the region of 
 the living substance surrounding the prey. These granules are 
 not formed round indigestible matter that may be absorbed. 
 Miss Greenwood states that this granular gathering is significant 
 of secretory activity. 
 
 It is worthy of notice that various observers have indicated 
 that the vacuoles round ingested matters frequently contain an 
 
XIl] PEOTEOLYTIC ENZYMES. 203 
 
 acid liquid. Among others, Engelmann states that litmus 
 particles turn red when ingested by Faramcemum, Stylonichia, 
 and some species of Amosba. 
 
 In Carchesium, one of the Vorticellidce, the digestive changes 
 can be seen within even greater clearness. Miss Greenwood 
 describes the appearances which can be made out when the 
 animal is fed with coagulated white of egg in a fine state of 
 division. A number of particles suspended in fluid are ingested 
 together and a vacuole containing them can be seen in the 
 animal's sarcode. They pass from the mouth towards the more 
 deeply-seated substance of the animal, becoming aggregated 
 together and losing the fluid which surrounds them. They are 
 then stored for a time, which sometimes lasts for more than an 
 hour, when vacuoles again form around them, containing freshly- 
 secreted fluid which has a solvent action on proteids. The 
 included particles are gradually dissolved, the vacuole con- 
 taining them moving slowly towards that region of the animal 
 fi-om which the finally undigested portions are discharged. 
 
 The process of secretion of the proteolytic enzymes in the 
 cells of the mammalian glands follows the same course as that of 
 the formation of diastase in the saKvary glands. The general 
 structure of the pancreas and of the glands of the stomach is 
 similar to that already described in the case of the latter 
 organs. When the glands are at rest there is a conspicuous 
 formation of peculiar granules in them, which may accumulate 
 so as to make the whole of the cell opaque, or which may be 
 stored more or less on the side which abuts upon the lumen 
 of the gland. In the pancreas we have thus two well-marked 
 zones, the outer being small but transparent, while the inner is 
 opaque. When secretion takes place the granularity gradually 
 becomes less evident, the granules retreating from the outer 
 parts of the cells towards the lumen of the gland and the whole 
 outlines of the cells being well-defined. At the conclusion of 
 the process the cells are nearly iree from granularity. The 
 granules are then gradually reformed till the resting condition 
 is again attained, the cells at the same time becoming some- 
 what larger. Comparison of cells taken from the gland during 
 different conditions of secretion lead to the view that the act of 
 
204 PROTEOLYTIC EXZYMES. [CH. 
 
 secretion consists of three well-marked stages. (1) The growth 
 of the cell at the expense of material brought to it by the 
 lymph. (2) The construction of the granules from the proto- 
 plasm of the cell. (3) The extrusion of these with a certain 
 amount of fluid in which they are more or less completely dis- 
 solved. In some glands these stages are apparently more or 
 less consecutive. In others they appear to go on simultaneously 
 but at relatively different rates. In such cells the clear and 
 granular zones cannot be clearly recognised, though the amount 
 of granularity varies from time to time. 
 
 The granules do not appear to be the actual enzymes but 
 to be composed of substances which are readily and rapidly 
 converted into them. If a fresh pancreas, taken from the body 
 immediately after death, is extracted with water or glycerin, the 
 extract is almost without action on proteids. If instead of 
 being extracted at once it is kept warm for 24i hours, or if it 
 is warmed for a shorter time in dilute acetic acid, and then 
 extracted, the extract has well-marked proteolytic powers. The 
 inert extract prepared from the fresh pancreas can also be made 
 active, by slightly acidulating it with acetic acid and keeping it 
 for some time at 40° C. The cells contain the enzyme in the 
 condition of a zymogen, which is readily converted into the 
 actual ferment by the acid used. The zymogen of the pancreas 
 can also be converted into the enzyme by passing a current of 
 oxygen for about 15 minutes through the neutral extract. 
 That the granules really contain this zymogen appears evident 
 from the fact that the amount of enzyme which can be formed 
 is proportional to the granulaaity. 
 
 There is little doubt that these changes are controlled by 
 nervous action as in the case of the salivary glands, but the 
 paths of the impulses are not so clearly understood. Stimula- 
 tion of the medulla oblongata increases the secretion of the 
 pancreas, but it is not dependent altogether upon nervous supply 
 from the central nervous system. When all the nerves going 
 to the gland are cut, the secretion is not materially affected. 
 In some animals there is probably a reflex nervous mechanism 
 influencing it, for secretion commences immediately food is 
 taken into the stomach. 
 
XIl] PROTEOLYTIC ENZYMES. 205 
 
 The secretion of both pepsin and trypsin is affected if not 
 altogether provoked by the absorption of nutritive products. 
 Schiff found that when an insoluble proteid such as fibrin or 
 coagulated albumin was introduced into the stomach of a fasting 
 animal no pepsin was secreted and the proteid remained un- 
 digested. If with the latter certain soluble matters, to which he 
 gave the name oi peptogens, were introduced, pepsin began to be 
 secreted immediately on their absorption. Among these pep- 
 togens Schiff mentions solutions of dextrin, extract of meat, 
 gelatin and peptone. He found that they were just as effective 
 when introduced into the blood by injection, or when absorbed 
 in any other way. The action they set up is clearly due, 
 therefore, not to their mere presence in the stomach, and they 
 must act chemically and not mechanically upon the gland cells. 
 They may be of value as setting up the necessary nutritive 
 changes in the gland cells, acting directly upon the protoplasm ; 
 or they may work indirectly by stimulating some nervous 
 mechanism in the gland or in the stomach wall, so causing 
 secretion. Some physiologists hold the view that there are 
 in the pancreas as in the salivary glands two sets of nerve 
 fibres, the one influencing the formation of the enzymes, and 
 the other their discharge during the activity of the gland. 
 This view rests not so much upon experimental proof as upon 
 analogy with the salivary gland, in which there is evidence of 
 such nerve supply. 
 
 There is a marked difference between the two enzymes with 
 regard to the way in which they are affected by acids, alkalis, 
 and neutral salts. Pepsin can only effect proteolysis in the 
 presence of a dilute acid ; in gastric juice it is closely associated 
 with hydrochloric acid in quantity varying from "2 to '6 per 
 cent. Pancreatic trypsin on the other hand is destroyed by a 
 relatively small quantity of free acid. Trypsin is aided in its 
 activity by most salts in small amount, particularly alkaline 
 salts; of these sodium carbonate is the most favourable, the 
 best results being obtained when from '9 to 1'2 per cent, of the 
 salt is present. This body is most injurious to pepsin, '5 per 
 cent, destroying almost the whole of the enzyme in a solution in 
 as short a time as 15 seconds. The presence of soluble proteids 
 
206 PROTEOLYTIC ENZYMES. [CH. 
 
 lessens the rate of destruction, but not very materially, even 
 25 per cent, of peptone not preventing it. The pepsin of the 
 frog is less easily destroyed than that of a mammal. 
 
 The presence of an excess of neutral salts in a solution of 
 either enzyme is deleterious, but pepsin is more readily 
 affected than trypsin. The action of the latter, according to 
 Edkins, is assisted by the presence of 1 — 2 per cent, of sodium 
 chloride, but very greatly retarded by 8 per cent. 
 
 A still more marked difference between pepsin and trypsin 
 lies in their relative powers of effecting the decomposition of 
 proteid. Pepsin can only carry the hydrolysis up to the stage 
 at which peptone is formed, while trypsin can split up one 
 form of peptone so far that the proteid is completely de- 
 composed. The initial stages of the proteolysis do not differ 
 greatly in the two cases, and the chief cause of such difference 
 as there is seems to be closely related to the fact that the one 
 acts in an acid, and the other in an alkaline medium. 
 
 The process of peptonisation by the aid of pepsin is a slow 
 and incomplete process, the relative amount of proteoses and 
 peptones found when such a digestion approaches its completion 
 being roughly 65 and 30. Chittenden and Amerman believe 
 that complete peptonization is not a property of gastric diges- 
 tion either in the natural or artificial process. The action of 
 pepsin is rather a preliminary stage in proteolytic digestion, a 
 preparation for the more vigorous transformations brought 
 about in the intestine under the action of trypsin. This view 
 is not a new one, having been advanced originally by Claud 
 Bernard. It is supported by the consideration that among the 
 lower animals pepsin is of rare occurrence, the only proteolytic 
 ferment secreted by them being a tryptic one. Trypsin acts 
 more energetically than pepsin, converting primary proteids 
 rapidly into proteoses, and these more or less completely into 
 true peptone. The only rapid transformation effected by pepsin 
 is the first change into primary proteoses, the subsequent action 
 being slow and incomplete. 
 
XIl] PROTEOLYTIC ENZYMES. GALACTASE. 207 
 
 Galactose. 
 
 An enzyme which belongs to the tryptic group has heen 
 discovered by Babcock and Russell in the course of some 
 researches they carried out on the ripening of cheese. They 
 have given it the name of galactase. 
 
 They found in the course of experiments on the changes 
 which take place in milk that is kept for a long time under 
 antiseptic conditions that there was a gradual increase in the 
 nitrogenous constituents which are soluble in water, which are 
 contiauously formed at the expense of the caseinogen. In a milk 
 12 days old the nitrogen existing in this condition was 30 per 
 cent, of the total nitrogen ; in one 240 days old it had become 
 63 per cent. In another sample, 12 hours after milking it was 
 less than 10 per cent., but after three weeks standing it had 
 risen to 30 per cent. During all this time bacteria were found 
 to be entirely absent. 
 
 These changes in the nitrogenous constituents of the milk 
 did not take place in samples that were boiled at the outset. 
 
 Similar results were obtained with a cheese which was kept 
 under a bell-jar in the presence of the vapour of ether and 
 chloroform. 
 
 The experiments clearly pointed to the presence of a proteo- 
 lytic enzyme, and this after considerable trouble they succeeded 
 in extracting from fresh milk. Taking advantage of the property 
 of enzymes to attach themselves to finely divided material in 
 suspension, they passed the milk through a centrifugal separator 
 and collected the slime which remained adhering to the instru- 
 ment. They mixed the slime with an equal weight of 40 per 
 cent, alcohol and added an antiseptic. After 24 hours it was 
 filtered and the filtrate concentrated by evaporation at 25° C. to 
 one-tenth its original volume, more antiseptic being added. 
 The solution was faintly acid, and was therefore neutrahsed 
 by adding a little sodic carbonate, when a precipitate of syn- 
 tonin and a little calcic phosphate fell. The :fitrate from this 
 precipitate was found capable of curdling milk and subsequently 
 dissolving the curd. It contained the proteolytic ferment 
 
208 PROTEOLYTIC EXZYMES. GALACTASE. [CH. XII 
 
 together \nth a certain quantity of another enzyme, rennet, 
 which will be discussed in a subsequent chapter. 
 
 More complete examination of the proteolytic enzyme showed 
 it to differ in some important respects from trypsin, with which 
 its discoverers were at first inclined to identify it. The general 
 course of digestion of caseiuogen by its activity resembles that 
 brought about by trypsin ia that it gives rise to amido-acids 
 as well as to albumoses and peptones. It differs however in 
 cartying the decomposition of the proteid further, a certain 
 quantity of ammonia being always found. Trypsin from 
 pancreatic juice was found not to be capable of doing this, 
 though it is stated by Hirschler and by Stadelmann, the 
 formation of ammonia takes place during the tryptic diges- 
 tion of fibrin. 
 
 The enzyme has been named galactose by its discoverers. 
 Its principal features seem to be the following: — 
 
 It ia capable of hydrolising proteids, particularly caseinogen,, 
 caiTj-ing the decomposition to the stage of the liberation of 
 ammonia. 
 
 It also liquefies gelatin. 
 
 It is active in neutral, feintly alkaline and Mntly acid 
 solutions, but very sUght excess of acidity rapidly inhibits it. 
 
 It decomposes peroxide of hydrogen with liberation of 
 oxygen. 
 
 Its optimum point is 37° — 42° C. ; its activity at 52° C. is 
 about equal to that it exhibits at 28° C. ; its proteolytic power 
 is destroyed at 76° C, and its action on gelatin at 65° C. 
 
 It is inhibited by mercuric chloride, fonnaUn, phenol and 
 carbon-disulphide, but not by chloroform, ether, benzol or toluol. 
 
209 
 
 CHAPTER XIII. 
 
 PROTEOLYTIC ENZYMES (continued). VEGETABLE TRYPSINS. 
 
 It is uncertain whether pepsin is represented in the 
 vegetable kingdom. All the proteolytic enzymes which have 
 been fully investigated have been found capable of carrying 
 the hydrolysis beyond the stage of peptone. The work of the 
 earlier observers did not include a careful examination of the 
 products of the decomposition, and hence for the present it 
 remains uncertain whether or no some of the ferments belong 
 to the peptic category. 
 
 The chief enzymes of the proteolytic class that have been 
 examined with any minuteness are papain, obtained from the 
 juice of Garica papaya ; hromelin, from the fruit of the Pine- 
 apple; the trypsin of germinating seeds; and the enzyme of 
 the secretion of Nepenthes, one of the so-called pitcher-plants. 
 Several others are known to occur in different plants, but our 
 information respecting them is not very complete. 
 
 Bromelin. 
 
 Though this enzyme was not the earliest to be investigated, 
 it is the one about which we have at present the fullest 
 information, and as it seems typical of most of them it is 
 advisable that its consideration should be undertaken first. 
 It occurs in great quantity in the juice of the Pine-apple 
 (Ananassa sativa) and is becoming of commercial importance. 
 Attention was first called to it by Marcano in 1891, and it has 
 been examined very fully since then by Chittenden and his 
 G. F. 14 
 
210 PROTEOLYTIC ENZYMES. BROMELIN. [CH. 
 
 pupils in America, who have given it the name of bromelin, 
 from the Natural Order to which the pine-apple belongs. 
 
 The juice of the fruit is extremely abundant, nearly a litre 
 being yielded by one of average size. It is distinctly acid, 
 the acidity being equal to that of a solution of hydrochloric 
 acid of about '45 per cent, strength. The proteolytic enzyme is 
 associated in the juice with a rennet or milk-curdling ferment, 
 just as pepsin is in the gastric secretion of animals. 
 
 The juice contains two proteids, one of which appears to be 
 a protoproteose and the other either a heteroproteose or a 
 globulin. The former is associated chiefly with the bromelin, 
 and coagulates when the juice is heated to 75° C If the 
 latter is neutralized before warming the coagulating point is 
 raised to 82° C. The second proteid is associated chiefly with 
 the rennet and is only coagulated when the juice is boiled. 
 
 Bromelin can be extracted from pine-apple juice by 
 saturating the latter with crystals of either sodium chloride, 
 magnesium sulphate, or ammonium sulphate. The first of 
 these salts gives the best result, the digestive activities of the 
 three preparations having been ascertained by submitting 
 10 grams of moist coagulated egg-albumin suspended in 100 c.c. 
 of water to the action of "05 gm. of each for 6 hours at 40° C. 
 The sodium chloride preparation under these conditions di- 
 gested 276 per cent, of the proteid, the magnesium sulphate 
 one 14"1 per cent., and the ammonium sulphate one 12'3 per 
 cent. only. The ferment loses some of its activity during the 
 process of extraction, the neutralised juice always having 
 greater proteolytic powers than solutions of the different salt 
 precipitates made up to the original bulk. 
 
 The action of the enzyme shows slight variations when it 
 acts in neutral and acid solution respectively, and its course is 
 not quite the same when acting on different proteids. As the 
 sodium chloride precipitate is the most potent, it was used by 
 Chittenden and his pupils in most of their experiments. 
 
 When fibrin was used as the proteid to be digested the 
 course of the action was shown in the following experiment : — 
 300 grams of moist fibrin were warmed at 40° C. with 
 2500 c.c. of 025 per cent, hydrochloric acid and '25 gram of 
 
Xm] PROTEOLYTIC ENZYMES. BEOMELIN. 211 
 
 bromelin for 5 hours : it had then been all dissolved except a 
 small flocculent residue. The filtrate from this residue when 
 neutralised deposited a small amount of precipitate. After 
 filtration the neutralised fluid was boiled for a short time, and 
 a heavy coagulum was formed, which dissolved in dilute acids 
 and alkalis. A further precipitation occurred on adding 
 alcohol to the concentrated filtrate from the heat-coagulated 
 proteid, and from the alcoholic filtrate leucin and tyrosin 
 separated out in large quantities under appropriate treatment. 
 The original undissolved residue was found to consist of an 
 antialbumid mixed with a proteose which resisted further 
 action of the enzyme. The neutralisation precipitate was 
 probably not acid-albumin but a form of hetero-proteose, as it 
 became converted into dysproteose on standing. The heat- 
 coagulated proteid was a heteroproteose which was not precipi- 
 tated by neutralisation. In different experiments the quantity 
 of the neutralisation precipitate and that thrown down on 
 boiling varied inversely, pointing therefore to their both being 
 the same proteid, which did not always behave in the same 
 way with regard to the reaction of the liquid. The precipitate 
 given by the alcohol was a mixture of proteoses and peptone. 
 When dissolved in water the former were separated from the 
 latter by saturation of the solution with ammonium sulphate, 
 and were found to consist of a mixture of proto- hetero- and 
 deutero-proteose. The peptone was almost if not entirely 
 antipeptone. The course of action was thus found to be almost 
 identical with that of pancreatic trypsin, but there was no 
 hemipeptone. The amount of crystalline amides indicated that 
 the latter had been entirely decomposed. 
 
 When the digestion was carried out in neutral solution, in 
 the presence of 1 per cent, sodium chloride, the course of action 
 was similar, but the heat-precipitate was in greater quantity 
 and consisted chiefly of dysproteose, which yielded deutero- 
 proteose on further digestion. The greater part of the peptone 
 was antipeptone, but some hemipeptone appeared to be present 
 as well. The proteoses mixed with the peptone contained no 
 heteroproteose, and the leucin and tyrosin were in smaller 
 
 quantities than in the acid digestion. 
 
 X4* — ^ 
 
212 PBOTEOLTTIC ENZYMES. BROMELIN. L^H- 
 
 Fibrin was not digested so easily in neutral as in acid 
 solution of the enzyme, and the presence of such a salt as 
 sodium chloride was almost essential to the activity of the 
 bromelin in the former case. 
 
 When coagulated egg-albumin was substituted for fibrin 
 several differences were manifested. The coagulated albumin 
 from 3 dozen eggs was digested with 2 litres of distilled water 
 and -8 gram of bromelin for 40 hours at 40° C. under antiseptic 
 precautions. There was then still some insoluble residue, con- 
 sisting of antialbumid with a little undigested albumin. The 
 liquid had become slightly acid but neutralisation gave no 
 precipitate, nor was there any coagulnm formed on boiling. 
 Alcohol threw down a mixture of proteoses and peptone, and 
 the filtrate from this yielded leucin and tyrosin. There was 
 very little proto- or hetero-proteose, these having probably been 
 further hydrolysed during the somewhat prolonged period of 
 digestion; abundant deutero-proteose was present. 
 
 The action of bromelin on myosin was also examined. It 
 digested this proteid best in "025 per cent, hydrochloric acid 
 solution. The course of digestion showed a difference in one 
 respect from what was observed in the other cases. Shortly 
 after it had begun the solution became thick and semi- 
 gelatinous from the separation of what appeared to be acid- 
 albumin. This disappeared however as the action proceeded. 
 In other respects myosin behaved like the other proteids, 
 yielding proteoses, peptone, and amides. 
 
 Careful quantitative analyses of the bodies formed during 
 the digestions showed a gradual decrease in the nitrogen they 
 contained, suggesting successive separations of nitrogen-con- 
 taining radicals. Thus while myosin contains 16'86 per cent, 
 of nitrogen, the neutralisation precipitate only contained 15'8, 
 and the deutero-albumose 13'9 per cent. Somewhat singularly 
 the peptone prepared from albumin contained 14'5 per cent, 
 and that from myosin 15 "7 per cent, of nitrogen. 
 
 The effect of various quantities of different acids on the 
 action of bromelin on fibrin is expressed in the following 
 table. 
 
XIIl] 
 
 PROTEOLYTIC ENZYMES. BROMELIN. 
 
 213 
 
 Acid 
 used 
 
 None 
 
 HCl 
 
 Acetic 
 
 Citric 
 
 Tartaric 
 
 Oxalic 
 
 With NaCl preparation 
 
 Enzyme almost inactive 
 
 With -Oia'/o active : activity 
 increases with increase of 
 acid up to -025 '/o ; then is 
 not altered up to '05 %; 
 then decreases, and ceases 
 with -I'/o 
 
 Almost inactive up to •25''/() ; 
 then is active, and remains 
 so up to 1 °/o 
 
 Activity begins with '03 % ; 
 remains good up to '12 "/o i 
 then declines, is inactive 
 with l«/o 
 
 Slight action with -06 "/j; 
 maximum with "IS— l^/j 
 
 Active only with -12- 
 inactive with 1 "/j 
 
 ■25% 
 
 With MgSOi 
 preparation 
 
 As with NaCl 
 {^reparation 
 
 Increases up to 
 •05%; above 
 this concen- 
 tration activ- 
 ity declines 
 
 With (NHjjjSO^ 
 preparation 
 
 As with NaCl pre- 
 paration 
 
 As with MgSO^ 
 preparation 
 
 As with NaCl pre- 
 paration 
 
 Maximun> activity 
 with -5—1 -5 70 
 
 Maximum activity 
 with -25— -5 % 
 
 With egg-albumin coagulated by boiling, bromelin was most 
 active in neutral solutions ; slight amounts of hydrochloric 
 acid (-012— •025 "/o) did not inhibit it, nor did "025 "/o of sodium 
 carbonate ; organic acids interfered with the action ; '1 % of 
 citric acid reduced it in the proportion of 31 : 125. 
 
 With raw egg-albumin different acids showed further varia- 
 tions. Hydrochloric acid in a series of experiments gave the 
 following results when lOc.c. of albumin solution, '05 gm. 
 bromelin, and 90c.c. water, were digested for 16 hours at 40° C. 
 in the presence of thymol : 
 
 Neutral (standard) HCl •012«/„ HGl -025% HCl -1% 
 
 digested"l6-67o digested 17-3 »/„ digested 21-9 "/„ digested6-27„ 
 
 Sodium carbonate was prejudicial to a greater extent ; in 
 the presence of -05 % only -8 % of the albumin was digested. 
 
 In another series of experiments, carried out with the same 
 relative proportion of bromelin and proteid, the effect of other 
 acids was examined, with the results shown in the following table: 
 
 
 Neutral 
 (standard) 
 
 Tartaric 
 0-25 % 
 
 Tartaric 
 0-5% 
 
 Oxalic Citric Citric 
 0-1% 0-1 «/„ 0-2 «/„ 
 
 Percentage of 
 proteid digested . 
 
 35-5 
 
 23-3 
 
 16-6 
 
 19-0 18'6 18-7. 
 
214 PBOTEOLYTIC ENZYMES. PAPAIN. [CH. 
 
 The effect of neutral salts on the action of bromelin varied 
 again with the preparation of the enzyme used, with the 
 particular proteid digested, and with the reaction of the 
 digesting fluid. With neutral preparations digesting fibrin, the 
 NaCl preparation was assisted by the presence of 1 — 3»/o of 
 neutral salts, the other two by proportions ranging from 
 1 — 5°/o. but larger quantities than these were prejudicial to 
 the action. Neutral salts did not affect acid digestions unless 
 they were present in large amounts. 
 
 They did not facilitate neutral digestions of coagulated 
 egg-albumin. 
 
 The neutrahsed pine-apple juice works most powerfiilly at 
 a temperature ranging from 50° — 60° C, and still shows con- 
 siderable action even when heated to 70° C. 
 
 With the precipitated enzyme the effect of temperature 
 upon the working is shown in the following table, the conditions 
 of the digestion being 10 grams of coagulated egg-albumin, 
 •05 gram of the precipitate, and 100 c.c. of water, digested at 
 the temperatures quoted for 5 hours : 
 
 Percentage of 
 Temperature proteid digested 
 
 40° C. 7-8 
 
 45° C. 9-6 
 
 60° C. 11-4 
 
 55° C. 11-9 
 
 60° C. 12-5 
 
 65° C. 9-4 
 
 70° C. 8-3. 
 
 The optimum point here is about the same as for the 
 neutralised juice. 
 
 Papain. 
 
 The fruit of the Papaw tree has long had traditionally the 
 property of rendering meat tender when cooked with it. 
 During recent years it has been ascertained that the juice of 
 the fruit contains a proteolytic enzyme of considerable power. 
 It was first investigated by Wtirtz in 1879 ; he discovered that 
 
XIIl] PROTEOLYTIC ENZYMES. PAPAIN. 215 
 
 the enzyme is not confined to the juice of |;he fruit, but is 
 present also in the sap obtainable from the stem and leaves. 
 It can be prepared in a very crude condition by expressing the 
 juice from the tissues of the plant, adding alcohol till a pre- 
 cipitate falls, collecting the latter by filtration, washing it with 
 absolute alcohol, and drying it at a low temperature. The sap 
 extracted from the plant is neutral in reaction and contains 
 proteid matter in solution. Wiirtz considered that the enzyme 
 was itself a proteid, and gave as its reactions that it is not 
 precipitated on boiling, gives no precipitate with corrosive 
 sublimate, gives a precipitate with nitric or hydrochloric acid, 
 which is soluble in excess of the precipitant ; also gives a pre- 
 cipitate with acetic acid and potassic-ferrocyanide. Wurtz says 
 further that in a neutral solution it dissolves animal proteids, with 
 formation of peptones and leucin. At the time when he wrote, 
 the differences between peptone and intermediate products, 
 particularly albumoses, were not understood, and many bodies 
 of the latter class were incorrectly considered to be peptones. 
 
 A more complete examination of the papaw juice and of the 
 enzymes it contains was made by Martin in 1883 and 1884. 
 He found that the proteids in the neutral juice consisted of an 
 albumin, a globulin, and two forms of albumose, which he 
 named a- and ;8-phytalbumose. Comparison of these bodies 
 with those more recently investigated points to the former 
 being a form of proto-proteose and the latter related to the 
 hetero-proteoses. As we have seen, comparatively little was 
 known of the character and behaviour of the proteoses, or 
 albumoses until they received careful study in the laboratory 
 of Professor Chittenden, of Yale University. A comparison 
 of Martin's two bodies with the several classes of proteoses 
 described by Chittenden leaves little doubt that they may 
 be considered forms of proto-proteose and hetero-proteose re- 
 spectively. Martin found that the papaw juice contains two 
 enzymes, one capable of curdling milk (rennet), and the other 
 the proteolytic one, now known as papain. The latter is 
 associated closely with the a-phytalbumose, or proto-proteose. 
 
 Martin agrees with Wurtz that the action of papain takes 
 place best in a neutral medium like that of the fresh juice. 
 
216 PROTEOLYTIC ENZYMES. PAPAIN. [CH. 
 
 When fairly pure it is easily destroyed by hydrochloric acid of 
 greater concentration than "05 "/o, l^ut in a faintly alkaline 
 medium, such as a 25 per cent, solution of sodium carbonate, 
 it is almost if not quite as active as in a neutral one. A 
 higher percentage of alkali destroys it. 
 
 Martin has stated that the optimum temperature for papain 
 digestion is 35° — 40° C, though it is active at lower tem- 
 peratures, such as 15° C. 
 
 When fibrin is subjected to the action of papain in neutral 
 solution it is affected as it is by pancreatic trypsin, being 
 corroded rather than dissolved, and converted into a pultaceous 
 mass, a good deal of turbidity accompanying the action. The 
 products of the digestion Martin determined to be a globulin- 
 like body, apparently intermediate between a true globulin and 
 alkali- albumin, some peptone, and leucin and tyrosin, the leucin 
 being in the greater proportion of the two. 
 
 Martin also examined with considerable minuteness the action 
 of papain on the proteids of the papaw juice. When digested 
 with the globulin or the albumin a small quantity of an insoluble 
 residue was left, which was probably a form of antialbumid, 
 such as Chittenden obtained with bromelin. At the end of the 
 digestion the most prominent body found in solution was the 
 hetero-proteose which Martin called /8-phytalbumose, while 
 traces of peptone and both leucin and tyrosin were also present. 
 
 Digestion of the a-phytalbumose or proto-proteose yielded 
 a little antialbumid, a quantity of a deutero-proteose, no 
 peptone, and a certain amount of both leucin and tyrosin. The 
 )8-phytalbumose showed the same decomposition. 
 
 It seems probable from Martin's experiments that the course 
 of proteolysis in the papaw is the same as that brought about 
 by bromelin. The juice as it is extracted from the plant 
 contains the two albumoses or proteoses, but it seems quite 
 likely that these are formed by the action of the enzyme before 
 the juice is extracted and are not the primary proteids of the 
 plant. Both yield the deutero-albumose when subjected to 
 the action of the separated papain. 
 
 The native albumin and globulin are both capable of giving 
 I'ise to the hetero-proteose in the course of laboratory experi- 
 
XIIl] PROTEOLYTIC ENZYMES. PAPAIN. 217 
 
 ments, and it is not improbable that the proto-proteose may be 
 formed at the same time, but may undergo further hydrolysis 
 as fast as it appears. If this is the case the course of proteolysis 
 proceeds on exactly the same lines as that set up by bromelin. 
 The primary proteids, albumin and globulin, yield at the outset 
 the two proteoses, proto- and hetero-proteose, which may come 
 from both the hemi- and anti-residues of the primary cleavage. 
 The anti-residue also gives rise to the small amount of anti- 
 albumid, which occurs in this case as it does in bromelin digestions. 
 The proto- and hetero-proteoses on further digestion yield deu- 
 tero-proteose, which Chittenden, as already mentioned, has shown 
 to be a stage nearer to peptone. The deutero-proteose is then 
 hydrolysed to peptone. Being itself derived from the proteoses 
 of both the anti- and the hemi-group, the peptone as formed is 
 a mixture of antipeptone and hemipeptone. The latter is 
 split up into leucin and tyrosin, while the former remains as 
 peptone at the end of the digestion. 
 
 Martin's results have since been confirmed by other ob- 
 servers. Davis has found further that the action of the enzyme 
 is slightly improved by the presence of 'OOo per cent, of 
 hydrochloric acid, or '25 per cent, of sodium carbonate, but is 
 inhibited entirely by "05 per cent, of hydrochloric acid. 
 
 Sharp agrees with Martin that proto- hetero- and deutero- 
 proteoses are formed during the digestion of albumin, but he 
 doubts the production of peptone. He says that a little dys- 
 proteose also occurs. 
 
 Halliburton says that papain converts animal proteids into 
 proteoses and peptone, but that when acting on vegetable 
 proteids it stops short at the proteoses, no peptone being 
 formed. 
 
 It is difficult to reconcile these statements as to the non- 
 formation of peptone with the appearance of leucin and tyrosin. 
 The latter have not been determined to arise from the direct 
 decomposition of proteoses. We must conclude that in the 
 experiments of these writers the peptone was altogether decom- 
 posed. Martin notes the same disappearance of the peptone 
 when digesting some of the albumoses. 
 
 Helbing and Passmore found peptones in the papain diges- 
 
218 PKOTEOLYTIC ENZYMES. PAPAIN. [CH. 
 
 tion of both myosin and egg-albumin, and separated them from 
 the other proteids by saturation of the liquid with ammonium 
 sulphate according to the method adopted by Chittenden. 
 
 Rideal also claims to have shown the existence of peptone 
 in papain digestions by determining the rate of dialysis of the 
 proteids through a parchment membrane. When experimenting 
 with egg-albumin he placed the whole product of the digestion 
 in a dialyser and found evidence of proteid in the water outside 
 it in 10 minutes. Though the proteoses are capable of dialysing 
 through a parchment membrane the rate of their diffusion is 
 very slow compared with that of peptone. Eideal concludes 
 that the rapidity of the passage points unmistakeably to the 
 presence of peptone as well as proteose. He confirmed this 
 result by the method of saturating the liquid with ammonium 
 sulphate. 
 
 Recent work carried out in Chittenden's laboratory fiilly 
 confirms the statement of previous writers that true peptone is 
 produced by papain. Neumeister makes the same assertion. 
 
 Kideal calls attention to the fact that papain is most 
 active when working in a relatively small amount of fluid. 
 Dilution of the digestive solution exercises a very marked 
 retarding influence on the progress of the hydrolysis. He says 
 the most advantageous quantity of liquid to use is from 1^ 
 to 3 times the weight of the proteid submitted to the action of 
 the enzyme. 
 
 He finds the optimum temperature for the working of 
 papain to be 40° C. 
 
 Harlay has stated recently that the digestive action of 
 papain points to its being intermediate between pepsin and 
 trypsin. It forms much proteose but very little tyrosin. In 
 papain digestions he observed the occurrence of the same 
 chromogen as he found produced by pepsin, the body, that is, 
 which with tyrosinase gives a red colouration slowly turning 
 green. Papain has a slight action on this body, producing from 
 it a small quantity of tyrosin. According to Harlay papain is 
 not destroyed by heating unless the temperature of its solution 
 is raised to 80° C. 
 
XIIl] PROTEOLYTIC ENZYMES. VEGETABLE TRYPSINS. 219 
 
 Other Vegetable Trypsins. 
 
 The occurrence of proteolytic enzymes in those seeds whose 
 reserve stores consist largely of proteid materials was suspected 
 almost as soon as the existence of diastase was demonstrated 
 in them. The search for them was first undertaken by von 
 Gorup-Besanez in 1874. He detected such an enzyme in the 
 seeds of the Vetch, and subsequently in those of Hemp, Flax, 
 and Barley. He describes it as having power to convert fibrin 
 into peptone, but apparently he did not investigate its action 
 upon the proteids with which it is associated in the seeds. 
 Beyond pointing out its existence his work does not give us 
 very much information about it. Krauch writing in 1878 
 opposed the views of von Gorup-Besanez and denied the 
 existence of such an enzyme. Von Gorup-Besanez says that 
 the body he prepared from the vetch seed dissolved the fibrin, 
 and that the solution when filtered gave a good biuret reaction, 
 which he attributed to peptone. Krauch insisted that the 
 biuret reaction was due to something present in the extract of 
 the seeds and was readily yielded by the latter alone. He 
 attributed the diminution of the fibrin to shrinkage of its 
 substance and not to solution. Blrauch's work however appears 
 untrustworthy, for von Gorup-Besanez says the diminution of the 
 fibrin went on to the point of disappearance, and this Krauch 
 does not explain. Neither does he show that there was no 
 further formation of a body giving the biuret reaction, though 
 the extract itself may have shown the. same. Krauch's 
 own control experiments were somewhat scanty. Von Sorup- 
 Besanez did not determine that the decomposition of the 
 fibrin was carried beyond the stage of peptone, but he points 
 out, that under certain conditions large quantities of amido- 
 acids, such as leucin and asparagin, could be detected in the 
 shoots of very young vetch plants. 
 
 A series of investigations was carried out by the writer in 
 1886 upon the seed of a species of Lupin {Lupinus hirsutus). 
 The experiments were made with germinating seeds, germina- 
 tion being allowed to proceed for four days, when the radicles 
 were nearly 3 inches long. The cotyledons were separated, 
 
220 PROTEOLYTIC ENZYMES. VEGETABLE TBYPSIXS. [CH. 
 
 ground in a mill, and extracted with glycerin. The extract was 
 strained and dialysed till free from crystalline amido-acids. It 
 contained such of the proteids of the germinating seeds as were 
 soluble in neutral fluids. 
 
 This extract was found capable of digesting fibrin in 
 a faintly acid solution. The fibrin used was boiled in water 
 and subsequently in "2 per cent, hydrochloric acid solution, 
 in which it swelled up and became transparent. Control 
 experiments, in which the fibrin was suspended in dilute acid 
 of the same degree of concentration as was used with the 
 extract, showed that no change took place during the digestion 
 unless the latter was present. In the acid extract of the seeds 
 the fibrin became corroded, causing the liquid to become very 
 turbid. It was then gradually dissolved, with the exception of 
 a small amount of granular residue. Further control experi- 
 ments were conducted in which the extract was boiled. 
 
 The digestions were generally carried out in parchment 
 dialysing tubes and the liquid outside them was frequently 
 changed. This liquid contained the same percentage of acid as 
 that inside the tube. After a short time the dialysates showed 
 the presence of a peptone, or at any rate of a body giving the 
 biuret reaction, and appropriate treatment separated from them 
 crystals of leucin and tyrosin, the latter in small amount only. 
 When the digestion had proceeded for some time the liquid 
 inside the dialyser contained a proteid which was precipitated 
 upon neutralisation, and was soluble in dilute acids and alkalis ; 
 a considerable amount of proteoses, chiefly hetero-proteose, was 
 also present. The course of digestion thus appeared to be the 
 same as that brought about by papain. 
 
 The boiled controls showed the fibrin intact at the end of 
 the experiment. 
 
 The action of the enzyme was further tested upon the 
 proteids of the resting seed. These were three, a globulin, 
 and two albumoses or proteoses, apparently proto- and hetero- 
 proteose. The action of the glycerin extract on these was to 
 transform them into what appeared to be peptone, and to cause 
 the appearance of amido-acids, chiefly asparagin, but a certain 
 amount of leucin as weU. 
 
XIIl] PROTEOLYTIC ENZYMES. VEGETABLE TRYPSINS. 221 
 
 Comparing the digestive effects upon animal and vegetable 
 proteids the course of the action seemed to be the decomposition 
 of a primary proteid, such as fibrin, into primary proteoses and 
 acid-albumin, with a small quantity of antialbumid ; the 
 primary proto- and hetero-proteoses, whether formed from the 
 fibrin or preexisting in the seed, were then changed into what 
 was taken to be peptone, but which was probably a mixture of 
 the latter with deutero-proteose. This seems probable in the 
 light of Chittenden's work on bromelin, but no certain state- 
 ment can be made, as his methods of separating the two 
 were at that time unknown. Part of the peptone was then 
 decomposed, yielding the amido-acids mentioned. 
 
 The enzyme was found to work most advantageously in a 
 liquid containing "2 per cent, of hydrochloric acid, which 
 corresponded approximately to the natural reaction of the 
 germinating seeds. When the acid was increased the diges- 
 tion became much slower. The ferment was totally inactive in 
 weak alkalis ; indeed exposure to the presence of '5 per cent, of 
 sodium CEirbonate destroyed it entirely. Neutral salts such as 
 sodium chloride impeded the action but did not destroy the 
 enzyme. The optimum temperature for the activity of the 
 ferment was about 40° C. ; it then worked about twice as 
 quickly as at the usual temperature of the soil. 
 
 Examination of the resting seed showed that it did not yield 
 any active enzyme to glycerin. The glycerin extract became 
 active however on warming it for some time with a dilute acid, 
 in which particular it agreed with the extract of the pancreas. 
 The experiments therefore indicate the existence of a zymogen 
 in the seed which is converted into an enzyme in the presence 
 of a feeble acid. It may be noted that just such a change in 
 the reaction of the parenchjrma of the seed takes place when 
 germination commences. It is difficult, though not impossible, 
 to prove the presence of the zymogen, as the digestion of the 
 proteid must take place in the presence of such an acid. It is 
 possible however to confirm its existence by a method adopted 
 by Langley and Edkins in their study of the zymogen of 
 pepsin. A description of this method will be given in a sub- 
 sequent chapter. 
 
222 PKOTEOLYTIC ENZYMES. VEGETABLE TRYPSINS. [CH. 
 
 The writer subsequently detected the existence of this 
 vegetable trypsin in the germinating seed of the Castor-oil 
 plant (Ricintts communis). 
 
 The presence of this enzyme in the Lupin seed has recently 
 been observed by Butkewitsch. 
 
 Neumeister has shown that a similar enzyme is not un- 
 common in seedlings. He has extracted it from those of the 
 Barley, Poppy, Wheat, Maize and Eape. It does not occur in 
 the early stages of germination but is developed as the plantlet 
 grows, and is plentiful when it has attained a length of from 
 15 to 20 centimetres. He prepared it by a somewhat novel 
 method. Having obtained an extract of the seedlings he 
 soaked moist fibrin in it. This proteid appears to have the 
 property of absorbing the ferment from the solution. Fibrin so 
 impregnated with the enzyme, was removed and placed in acid 
 or alkaline liquids and the results observed, control experiments 
 being carried out in which fibrin that had not been in contact 
 with the extract was placed in similar fluids. Neumeister says 
 that the enzyme is active only in acid liquids, and that the 
 acid must be organic, oxalic being the best. Mineral acids 
 such as hydrochloric destroy it. 
 
 Fembach and Hubert have shown that a similar enzyme 
 exists in malt and can be precipitated by alcohol with the other 
 enzymes which have already been described. It shows a similar 
 behaviour to that of diastase in the presence of phosphates. 
 Laszcynski states that kiln-dried malt contains the enzyme but 
 that it is absent from barley seedlings. He could not detect its 
 action upon fibrin till it had been digested with it for six hours 
 at 40° C. 
 
 Few observations have been made on the digestion of 
 proteids in the living plant. Schulze has stated that during 
 germination a mixture of nitrogenous compounds results from 
 the decomposition of the proteids, among which aromatic and 
 fatty amido-acids and arginin are probably invariably present. 
 
 The course of proteolysis in the cereals has not been fully 
 investigated at present, but it appears to present some differ- 
 ences from that afready described. Osborne and Campbell have 
 examined the proteids of barley and of malt with a view to 
 
XIIl] PROTEOLYTIC ENZYMES. VEGETABLE TRYPSINS. 223 
 
 seeing what changes can be traced during the preparation of 
 the latter. Barley contains nearly 11 per cent, of proteid 
 matter; of this a globuhn, edestin, with a small inseparable 
 amount of proteose, constitutes about 2 per cent. ; an albumin, 
 leucosin, is present to the extent of -3 per cent. ; and a peculiar 
 proteid, kordein, amounts to 4 per cent. The latter differs from 
 all proteids of animal origin by being freely soluble in 75 per 
 cent, alcohol, while it is insoluble in water. The remainder of 
 the barley proteids, about 4'5 per cent., is insoluble ia either 
 water, potash, or alcohol. On examining malt, hordein was found 
 to have disappeared, being partly replaced by another proteid, 
 also soluble iu alcohol, to which the investigators gave the name 
 hynin. Another globulin, hynedestin, replaced the edestin. Two 
 proteoses were present, amounting together to about 1'3 per 
 cent, of the total. A great deal of the insoluble proteid also was 
 present, though nearly 1 per cent, less than the corresponding 
 body in the barley. The total amount of proteid matter in 
 malt was a little less than 8 per cent., so that during germi- 
 nation about 3 per cent., or nearly one-third of the whole, had 
 disappeared. The authors conclude that the proteids change 
 extensively during germination before acquiring the properties 
 of proteoses. The proteids replacing hordeiu and edestin are 
 richer in carbon and poorer in nitrogen. The authors did not 
 discover any enzyme capable of effecting these changes. 
 
 It was mentioned above that the proteolytic power of 
 pine-apple juice was first observed by Marcano. Some years 
 earlier he foimd that the expressed sap of the leaves of certain 
 species of Agave was capable of digesting meat, if the latter 
 was soaked in it and exposed to a temperature of 35° — 40° C. 
 The digestion was somewhat slow, but after it had continued for 
 26 hours Marcano ascertained that about 20 per cent, of the 
 meat used had been converted into "peptone," which was 
 probably a mixture of peptone and proteoses. The results of 
 the experiments show therefore that the Agave contains a 
 proteolytic enzyme. The digestion proceeded equally well in 
 the presence of chloroform, so that it was not due to putre- 
 factive changes. Marcano says he has found this proteolytic 
 power in the juices of a great number of fruits. 
 
224 PROTEOLYTIC ENZYMES. CEADINA- [CH. 
 
 In 1880 Bouchut carried out some reseai-ches upon the juice 
 of the common fig-tree (Ficus Cai-ica), and found it to contain 
 a powerful enzj-me capable of dissolving proteid substances. 
 Hansen investigated the same plant in 1883 and 1884 and 
 confirmed Bouchut's results. He described the enzyme as 
 working most advantageously in an acid medium, but as not 
 being without proteolytic power in an alkaline liquid. The 
 enzyme was the subject of a more extended research in 1890 
 by Mussi. He took the juice expressed fi-om the branches, 
 leaves, and finiit of the fig-tree, and after filtration to remove 
 the ddbris accompanying the liquid he precipitated certain of 
 its constituents by the addition of absolute alcohol. The 
 resulting precipitate was insoluble in water but dissolved easily 
 on the addition of a trace of acid or alkali. When either solution 
 was placed in contact with moist fibrin it dissolved it readily. 
 Mussi gave the name Cradina to the enzyme. It is inactive in 
 neutral fluids. Mussi gives several reactions for it, but as by 
 the method of preparation used it must have been very impure 
 these have no particular importance. 
 
 Another Indian fixdt, the Kachree gourd (Cucumis utilis- 
 simus), was exannned by the writer in 1892 with a similar 
 result. The fioiit is in appearance much like a small vegetable- 
 marrow, about 6 inches in length. It is yellow in colour, and 
 when cut has an aroma similar to that of the melon. Its pulp 
 is extremely succulent and the expressed juice is faintly acid in 
 reaction. Both the juice and the pulp of the pericarp contain 
 an enzyme which is associated with a glflbuUn-like proteid. 
 The ferment is most completely extracted fix)m the pulp by a 
 dilute salt solution, containing 3 per cent, of sodium chloride. 
 Such an extract acts slowly on coagulated egg-albumin, yielding 
 proteoses, peptone, and leucin. It is most efiiective in a faintly 
 alkaline medium, less so in a neutral one, and acts still more 
 feebly in the presence of acid. 
 
 A proteolytic ferment was described in 1892 by Daccomo 
 and Tommasi as obtainable fi-om Anagallis arvensis. It can be 
 prepared as a white amorphous substance, easily soluble in 
 water. The authors say that if the fi-esh plant is reduced to 
 powder and kept in contact with fi:esh meat or moist fibrin for 
 
XIIl] PROTEOLYTIC ENZYMES. VEGETABLE TRYPSINS. 225 
 
 4 or 5 hours at a temperature of 60° C, the proteid is consider- 
 ably softened, though complete disintegration is not effected in 
 less than thirty-six hours. The enzyme is stated to have the 
 property of destroying fleshy growths and homy warts. 
 
 The normal action of the proteolytic enzymes so far described 
 is presumably intracellular. This is certainly the case with 
 the trypsin of germinating seeds, which indeed is formed in the 
 cells in which the reserve-proteids He. We find however in 
 the vegetable kingdom cases in which a proteolytic secretion is 
 formed and poured out upon the surface of the plant, or into 
 particular receptacles, to carry on there a digestive process 
 remarkably similar to that of animals. The plants in question 
 are the so-called insectivorous plants, which by various methods 
 capture, kill, and digest various insects which alight upon them. 
 The most conspicuous of them are the pitcher-plants. Nepenthes, 
 Sarracenia, Darlingtonia, and others, certain of the leaves of 
 which are in part or altogether transformed into large re- 
 ceptacles, containing in life a considerable amount of fluid. 
 Insects attracted to the plants are enticed into entering the 
 pitchers and are drowned in the Hquid they contain. Some of 
 these plants, particularly Sarracenia and Darlingtonia, have 
 nothing but water in the pitchers and the insects drowned 
 therein undergo ordinary putrefaction, the products of which 
 are absorbed by the plant. Nepenthes stands out conspicuously 
 from these in possessing a series of glandular structures in the 
 lower portion of the pitcher. The glands secrete into the 
 pitcher a proteolytic enzjrme by whose agency the organisms 
 which are captured undergo a process of true digestion, in the 
 absence of putrefactive changes. The liquid of the pitcher 
 indeed has- antiseptic properties. The enzyme has generally 
 been compared to pepsin, chiefly because during digestion the 
 reaction of the liquid is acid. It has recently been shown by 
 Vines to have features which associate it preferably with 
 trypsin. 
 
 The existence of the proteolytic property in the liquid of 
 Nepenthes was first ascertained in 1874 by Hooker, who 
 however did little more than determine that it is capable of 
 dissolving boiled white of eg^. 
 
 0. F, 15 
 
226 PROTEOLYTIC ENZYMES. VEGETABLE TRYPSINS. [CH. 
 
 Lawson Tait in the following year prepared the enzyme in 
 a crude form and spoke of it as resembling pepsin. A little 
 later von Gorup-Besanez made a detailed study of its action on 
 fibrin. He found it to be capable of dissolving fibrin with the 
 formation of a soluble body giving the reactions of peptone so 
 far as they were then known. He ascertained that for the 
 formation of the enzyme it was necessary that the pitchers 
 should be stimulated by the absorption of some digested 
 matter, much as is the case with the gastric glands of a 
 mammal. Such stimulation was followed by the secretion of 
 an acid liquid which had well-marked proteolytic powers. 
 The latter were only manifested while the reaction remained 
 acid and were considerably greater at a temperature of 40° C. 
 than at 20° C. These results of von Gorup-Besanez were 
 confirmed by other observers ; Vines in 1877 showed that the 
 pitchers yield a proteolytic extract when pounded up and 
 mixed with glycerin. Such an extract is not quite so active 
 as the actual secretion of the glands as it is poured into the 
 pitcher. If an unstimulated pitcher is extracted with glycerin 
 the resulting preparation is inactive, but it develops proteolytic 
 powers if warmed for a time with a dilute acid. The pitcher 
 wall therefore contains a zymogen, just as do the gastric and 
 pancreatic glands. 
 
 Kecently a certain controversy has arisen as to these 
 proteolytic powers, Dubois and Tischutkin separately asserting 
 that they have entirely failed to obtain digestion of animal 
 matter by the aid of Hquid in the pitchers when the experi- 
 ments have been carried out under antiseptic precautions. 
 They attribute the digestion observed by von Gorup-Besanez, 
 Vines and others to the agency of bacteria. On the other hand 
 Goebel maintains that he found the liquid fi-om a pitcher, when 
 made acid with hydrochloric acid, capable of readily digesting 
 fibrin, and that when a gelatin film was inoculated with some 
 of the same liquid no micro-organisms of any kind made their 
 appearance upon it. 
 
 It is difficult to explain the discrepancy between these 
 statements; possibly the age of the pitcher, the condition of 
 the plant, or some other condition may affect tlie formation 
 
XIIl] PROTEOLYTIC ENZYMES. VEGETABLE TRYPSINS. 227 
 
 of the enzyme. At the same time it is impossible to avoid 
 laying greater stress on positive than on negative results. 
 
 More recently Vines has re-investigated the subject, with 
 the result that he has proved the enzyme to exist and to be 
 tryptic in character, breaking down the digested proteid par- 
 tially to the state of amide crystalline bodies. His experiments 
 were carried out under strict antiseptic precautions. A tj^ical 
 experiment carried out with the secretion of Nepenthes Masters- 
 iania, may be quoted here : 
 
 " Two test-tubes were prepared, each containing 5 c.c. of 
 neutral pitcher-Uquid and a shred of fibrin; to the one {A), 
 5 c.c. of "25 Vo HCl were added ; to the other (B), 5 c.c. of 
 distilled water : the tubes were placed in the incubator (about 
 35° C.) at 11.30 a.m. At 2.30 p.m. the fibrin in tube A was 
 completely dissolved, the liquid giving a good biuret-reaction. 
 The fibrin in tube B was still undissolved at 9 a.m. on the 
 following morning ; 5 c.c. of '26 "/, HCl were then added to it, 
 with the result that the fibrin had completely disappeared by 
 11.30 a.m." 
 
 In considering this experiment it is difficult to avoid ad- 
 mitting the presence of the enzyme ; fibrin is not dissolved by 
 •125 °/o HCl except with extreme slowness; bacterial action 
 would not be sufficiently rapid to dissolve it in anything hke 
 the time in which it disappeared. 
 
 Vines quotes several other experiments in which the diges- 
 tions were carried out under the influence of such antiseptics 
 as potassium cyanide, thymol, chloroform, and corrosive subli- 
 mate. In all these cases digestion of the fibrin was complete 
 in a few hours. Egg-albumin was digested also, but not quite 
 so rapidly. The two proteids behave similarly under the 
 action of the animal enzymes, the digestion of fibrin being 
 easier than that of white of egg. 
 
 The writer has also examined the question of bacterial 
 agency in the process. Some faintly acid pitcher-liquid was 
 put into a small bottle and a little coagulated egg-albumin 
 added. It was then tightly corked and set aside. The proteid 
 was gradually digested, leaving a little granular residue. 
 The bottle with its contents was kept under observation for 
 
 15—2 
 
228 PROTEOLYTIC ENZYMES. VEGETABLE TBYPSINS. [CH. 
 
 12 months, and at the expiration of that time the liquid it 
 contained waa perfectly limpid and clear, showing not a trace of 
 bacterial contamination. Had the digestion been due to micro- 
 organisms it would have become turbid and shown evidence of 
 considerable growth in a few days, as the amount of proteid in 
 it was fairly large. 
 
 Clautriau has observed the digestion of proteid matter in 
 the liquid of the pitcher while the latter remained attached to 
 the plant. He denies that bacteria were present, for he found the 
 liquid to remain limpid and to possess no odour of putrefection. 
 
 It has been noticed in connection with many of the enzymes 
 we have discussed that they exist in the various juices in 
 association with some form of proteid. So close is the con- 
 nection that many observers have held the view that the 
 enzjTne and the proteid are identicaL It is interesting to note 
 in the case of Nepenthes that Vines says many very active 
 liquids taken from pitchers contained hardly a trace of proteid 
 matter ; they gave only a very faint xanthoproteic reaction, no 
 precipitate with nitric acid, or with acetic acid and potassic 
 ferrocyanide, and no turbidity when boiled. 
 
 The course of proteolysis under the influence of the 
 Nepenthes enzyme appears to be similar to that effected by 
 bromelin and the other trypsins described. There is always 
 a little insoluble residue of the nature of an antialbumid, a 
 neutralisation precipitate which is probably hetero-albumose, a 
 quantity of deutero-albumose, not precipitated by nitric acid, a 
 variable but slight amount of peptone, and some leucin. Some 
 observers question the existence of the peptone, but the balance 
 of evidence is in favour of its presence. 
 
 Clautriau did not find any amido-acids and he consequently 
 held the enzyme to be a form of pepsin. 
 
 Besides the pitcher-plants, there are others which capture 
 and digest small insects. The chief of these are Drosera, 
 Dioncea, and Pinguicula. Our knowledge of the nature of the 
 enzymes which these form is very much less complete than 
 that which we possess in the case of Nepenthes, being chiefly 
 confined to observations on the fate of small pieces of nitro- 
 genous matter placed upon the leaves and subjected there to 
 the action of the secretion which exuded from the latter. 
 
XIIl] PEOTEOLYTIC ENZYMES. VEGETABLE TRYPSINS. 229 
 
 Darwin investigated the behaviour of the leaves of Drosera 
 ia great detail. These structures are provided vdth long-stalked 
 glands which when stimulated pour out upon the surface of the 
 leaf a peculiar viscid secretion having an acid reaction. When 
 an insect, or a small piece of nitrogenous matter, is placed upon 
 the leaf, the glands or tentacles bend over slowly and enclose it, 
 at the same time pouring out the viscid fluid. The surface and 
 the margins of the leaves are alike provided with the tentacles, 
 but the secretion of the central ones is more acid than that of 
 those at the periphery. The imprisoned matter, living or dead, 
 is slowly dissolved by the secretion, and the resulting products 
 are absorbed by the leaf-surface. Darwin found that the 
 secretion could dissolve not only proteid matters, but also 
 connective tissue, cartilage, and gelatin, while it had no action 
 on mucin. The secretion of the enzyme like that of the animal 
 glands is dependent upon the absorption of nitrogenous matter 
 by the leaf, and the acid is only developed under the same 
 condition. If the leaf is stimulated by putting upon it a piece 
 of indigestible matter, less secretion takes place and the liquid 
 poured out has no proteolytic powers. 
 
 Darwin discovered that the same enzyme exists also in the 
 leaves of Dionsea. These differ in the arrangement of then- 
 glands from those of Drosera; the leaves have their upper 
 surfaces covered with small, almost sessile, secreting glands of a 
 purplish colour. Like the leaves of Drosera those of Dionsea do 
 not secrete any enzyme until they are excited by the absorption 
 of nitrogenous matter. Then they pour out a fluid which is 
 colourless and slightly mucilaginous. It is more acid than 
 that of Drosera and like the latter dissolves coagulated egg- 
 albumin. Pinguicula also secretes a similar digestive fluid on 
 the edges of the upper surface of the leaf, which folds over to 
 enclose its captives. 
 
 No examination has been made at present of the products 
 of digestion by these enzymes. It is consequently uncertain 
 whether they are peptic or tryptic. From analogy with the 
 pitcher-plants it seems probable that they belong to the latter 
 category, but the point must for the present be left imdecided. 
 
 Similar considerations affect certain enzymes which have 
 
230 PEOTKOLYTIC EXZTMES. VEGETABLE TRYPSINS. [CH. 
 
 been found to be secreted by several of the FungL One of the 
 earliest known of these is the ferment which Krukenberg 
 found to be procurable from the plasmodium of JEthcdium, one 
 of the Myxomycetes. A glycerin extract of the plasmodium 
 was found to have very marked proteolytic powers in the 
 presence of lactic or hydrochloric acid. KJrakenberg's statement 
 has been confirmed by Miss Greenwood, who has stated that 
 the Plasmodium of another member of the same group yielded 
 to "4 per cent, hydrochloric acid an extract which showed 
 marked solvent action on fibrin. 
 
 Boiuquelot states that an extract of the mycelium of 
 Aspergillus niger has the property of dissolving both fibrin 
 and coagulated egg-albumin when digested with them for 
 2 hours at 40° C. There is an unmistakeable formation of 
 peptone during the digestion, which takes place best in a 
 neutral medium. The extract of Aspergillus has also the power 
 of liquefying gelatin. Malfitano has found that the secretion 
 of the en2yme is constant during the life of the fungus, but is 
 chiefly associated with the death of the cells which form it. 
 
 Bouiquelot has also obtained a feebly proteolytic enzyme 
 bom. Polyporus sulfureus, and Zopf and Bourquelot and Herissey 
 state that ferments of this kind exist in several other fimgi. 
 Bourquelot's results have been confirmed by Hjort. 
 
 Descending still lower in the scale we find evidence of the 
 probable existence of proteolytic enzymes in Teast. If this 
 organism is kept in a medium which supplies it with no 
 nourishment, such for instance as a liquid containing no sugar, 
 and if it is at the same time deprived of oxygen, a process of 
 true digestion of its own proteid reserve-material takes place. 
 If yeast is pressed till it is dry, and the exuding liquor collected, 
 it will be fotind to contain a great deal of proteid material, 
 which forms a bulky coagulum when the fluid is heated to 
 45° C After standing a few days under antiseptic precautions, 
 this form of proteid will have disappeared, so that the liquid 
 gives but a slight coagulum even on boiling. These facts point 
 to the presence of a proteolytic enzyme in the yeast cells. 
 Hahn has recently prepared it by the following method. He 
 pressed yeast till it was dry and then carefully ground it up 
 
XIIl] PEOTEOLTTIC ENZYMES. VEGETABLE TRYPSINS. 231 
 
 vdth kieselguhr and sand till the cells were disintegrated, 
 when he prepared an extract by making the fine powder into 
 a paste with water. On pressing this till the liquid was 
 squeezed out, a yellowish solution resulted, which contaiaed 
 a quantity of proteid material. Hahn treated a small volume 
 of this extract with chloroform and added to it some solid 
 gelatin containing a trace of phenol. He found that an 
 appreciable amount of gelatin dissolved in 24 hours and that 
 the process of solution went on till the whole was liquefied. 
 The absence of either yeast-cells or micro-organisms was ensured 
 by the antiseptics used. 
 
 The same author in conjunction with Geret proved the 
 presence of the enzyme by another method. It was stated 
 above that the extract of the yeast contains a quantity of 
 proteid matter. The authors added chloroform to some of this 
 extract and kept it at a temperature of 37° C. for several weeks. 
 The chloroform served two purposes ; it kept the liquid free firom 
 microbes, and it slowly precipitated the proteids. The bulky 
 precipitate so formed slowly but gradually diminished, and at 
 the end of several days the extract was almost clear. After this 
 stage was reached the liquid again became turbid and the 
 turbidity increased for several days. The second precipitate 
 was found to consist chiefly of crystals of tyrosin, while some 
 leucin was ascertained to be in solution in the mother liquor. 
 
 The course of action has thus been shown to be comparable 
 with that taken by pancreatic trypsin. 
 
 Geret and Hahn have shown more recently that the enzyme 
 is capable of digesting added albumin as well as the yeast 
 proteids. They say that hypoxanthin occurs as a product of 
 the digestion. 
 
 Various forms of Bacteria also are known to form proteolytic 
 enzymes. In 1887 Bitter showed by sterilising a culture at a 
 temperature of 60° 0. that one of these organisms excretes the 
 enzyme into the medium in which it is growing. He found 
 this treatment killed the micro-organisms, but did not destroy 
 the enzymes, which continued able to liquefy gelatin and to 
 peptonise albumin. Hankin has extracted firom the bacillus 
 of anthrax (Bacillus anthracis) an enzyme which is capable 
 
232 PROTEOLYTIC ENZYMES. VEGETABLE TRYPSINS. [CH. 
 
 of forming albumoses from fibrin. Several toxic bodies of 
 this class have been traced to similar agency, an extract 
 prepared from the organisms being capable of effecting the 
 proteolysis ia the absence of the cells. The ordinary putre- 
 factive bacteria may excrete or yield an enzyme resembling 
 trypsiii in. its action on proteids. Sirotinin has shown that 
 culture fluids in which certain microbes have been growing 
 have been able to liquefy gelatin after they have been filtered 
 through porcelain. The conditions of life of these bacteria 
 frequently influence the secretion of the enzyme. Lauder 
 Brunton and MacFadyen found that a particular microbe 
 secreted a proteolytic enzyme when it was cultivated in meat 
 broth, but not otherwise. Indeed the secretion showed a very 
 definite relationship to the culture medium, having diastasic 
 powers when the latter consisted of starch-paste. The two 
 enzymes were quite distinct, the proteolytic one being most 
 easily extracted when present. Acids favoured and alkaHs 
 impeded its activity. 
 
 The relative amounts of trypsin and diastase in the, pancre- 
 atic JTiice have also been shown to be largely influenced by 
 diet. Vassihef established this fact in the case of dogs. 
 
 Among the microbes which secrete proteolytic enzymes may 
 be named four which are well known. These are Koch's 
 cholera hacillus, Deneke's cheese-bacillus, Finkler's cholera- 
 nostras-hadllus, and Miller's bacillus. The enzymes have been 
 extracted by "Wood from culture fluids in which the microbes 
 had grown, and which had subsequently been sterilised by 
 antiseptics. Wood found that the enzymes from the different 
 bacilli varied a good deal in their power of resisting the 
 influence of acid media, those from Koch's bacillus being 
 destroyed by very little acidity, while those from Finkler's and 
 Miller's bacilli could act in distinctly acid solutions. Wood 
 noticed that the bacilli themselves showed a varying sus- 
 ceptibility to acids exactly corresponding to that of the 
 enzymes. Vignal discovered that B. mesentericus vulgatus 
 secretes a peptonising ferment, in addition to those already 
 mentioned. 
 
 Babcock and Eussell have shown that Bacillus svitilis and 
 
XIIl] PROTEOLYTIC ENZYMES. CASEASE. 233 
 
 two other species of Bacillus carry the proteolytic decompositioD 
 as far as the liberation of ammonia. 
 
 The proteolytic enzyme in all these bacteria is associated 
 ■with at least one other, generally rennet. 
 
 Fermi has ascertained that the proteolytic enzymes secreted 
 by several Schizomycetes have the power of liquefying gelatin, 
 and that this corresponds to their ability to form peptone from 
 fibrin. He has found such enzymes in cultures of Bacillus 
 subtilis, B. anthracis, B. megatherium, B. pyocyaneus, Vibrio 
 cholerae asiaticae, V. Finkler-Prior, Micrococcus prodigiosus, 
 M. ascoformis, M. ramosus, and a few others. Fibrin is dis- 
 solved by these enzymes as well as gelatin, though with a little 
 greater difficulty. Egg-albumin and coagulated serum-albumin 
 are still more resistant, so that the enzymes hardly correspond 
 to either pepsin or trypsin. Indeed Fermi gives reasons for 
 supposing that several enzymes exist in these plants, pointing 
 out that they are destroyed at different temperatures. Thus 
 the enzyme of Micrococcus prodigiosus is unable to work at 
 a temperature of 55° C, while those of Bacillus pyocyaneus, 
 B. anthracis and Vibrio Finkler-Prior are not rendered inactive 
 till heated to 60° C, 65° C. and 70° C. respectively. These 
 enzymes resemble trypsin in working most advantageously in 
 faintly alkaline solutions, though they will attack a solution of 
 gelatin containing '5 per cent, of hydrochloric acid. 
 
 Another proteolytic enzyme has been described by Duclaux 
 which is associated with the digestion of casein or tyrein both 
 in milk, and in cheese during the operation of ripening. He 
 gave it the name of casease. It is secreted by several species 
 of bacteria belonging to the genus Tyrothiao, which also contain 
 a rennet ferment such as will be described in a subsequent 
 chapter. 
 
 This proteolytic enzyme can be precipitated from a culture 
 of any of these species by the addition of a large excess of 
 alcohol. It acts not only on the clotted proteid produced by the 
 rennet, but on the unchanged caseiu of the milk, and its action 
 can be traced by the disappearance of the opalescence of the 
 liquid, a fat-splitting enzyme being associated with it. This 
 enzyme occurs also in the hepato-pancreatic secretion of Sepia, 
 
234 PROTEOLYTIC ENZYSfES. VEGETABLE TRYPSINS. [CH. 
 
 which has the same efifect on milk. Casease is a tryptic ferment, 
 leucin and tyrosin being formed among the products of its 
 activity. When it is working in the cheese it produces also 
 the amido-bodies already mentioned which occur together with 
 other products that are traceable to the metabolic activity 
 of the micro-organisms. Weigmann states that casease can 
 be prepared from bacterial cultures, and that when added to 
 fresh cheese it accelerates its ripening just as do the organisms 
 themselves. 
 
 On a review of all these vegetable proteolytic enzymes it 
 will be seen that our knowledge is not at present sufficiently 
 definite for us to say whether we have to do with one or many. 
 Some of them may be peptic only, though it seems probable 
 that they are all tryptic. Those which have been at all ex- 
 haustively examined undoubtedly carry the proteolysis to the 
 stage of crystalline amido-acids, some of those secreted by bac- 
 teria even liberating a little ammonia. We do not yet know 
 again whether there is one enzyme only, varying somewhat in its 
 features according to the conditions of its secretion, or whether 
 the different plants discussed yield different varieties of trypsin. 
 Bromeltn and papain certainly show very little difference in 
 their behaviour, and one is tempted to pronounce them iden- 
 tical. For the present however it is perhaps advisable to leave 
 this question undecided. 
 
 The identity of the trypsins of animal and vegetable origin 
 is not established. From a consideration of the composition of 
 the proteoses and peptone formed during their respective action 
 Chittenden has concluded them to be different bodies. Speaking 
 of bromelin he says, " We are impressed with the exceptionally 
 low percentage of nitrogen in the deutero-albumose and pep- 
 tone. In fact this is so contrary to our general experience 
 with digestion products formed by animal ferments that we are 
 forced to consider it as something peculiar to the vegetable 
 ferment. As is well known, secondary proteoses and peptone 
 formed by the action of pepsin and trypsin usually show a much 
 lower content of carbon than the proteid undergoing digestion, 
 as in the bromelin products, but the percentage of nitrogen is 
 ordinarily increased in proportion to the decrease of carbon.... 
 
XIIl] PROTEOLYTIC ENZYMES. VEGETABLE TRYPSINS. 235 
 
 The vegetable ferment bromelin is peculiar in giving rise to 
 secondary proteoses and peptones with a much lower percentage 
 of nitrogen than is contained in the mother proteid, thus im- 
 plying a cleavage of a nitrogen-containing radical as part of 
 the proteolysis. This, if true, would constitute a good ground 
 of distinction between the animal and vegetable proteoljrtic 
 ferments." 
 
236 
 
 CHAPTER XIV. 
 
 FAT-SPLITTING ENZYMES. LIPASK {PIALTN, STEAPSIN.) 
 
 The transformations which attend the digestion of fat in 
 the mammalian alimentary canal are twofold. A variable 
 quantity of the fat of a meal — considered by some physiologists 
 the greater part — appears suspended in the liquid contents of 
 the intestine in the form of very fine globules which show no 
 tendency to run together. Such a condition constitutes what 
 is known as an emulsion. It is apparently a purely physical 
 condition, and the unaltered fat can be separated from the 
 liquid by shaking the latter with ether. This is the condition 
 in which the fat remains permanently in milk. But beside 
 this physical change a chemical alteration also occurs, the 
 molecule of the fat being hydrolysed, with the result that it is 
 decomposed into fetty acid and glycerin. How much of the 
 fat undergoes hydrolysis is at present chiefly a matter of 
 speculation, many observers thinking that only a small per- 
 centage is so changed and that the greater amount is only 
 emulsified and absorbed by the intestinal epithelium otherwise 
 unaltered. The two processes occur together in the duodenum 
 and there is little doubt that the emulsification is largely 
 helped by the coincident hydrolysis. A small amount of soap, 
 which is formed when free fatty acid combines with an alkali, 
 materially accelerates the process of emulsion. If a neutral 
 fat is shaken with a dilute alkali no emulsion occurs, but if 
 the fat is ranctd,' that is if a little fatty acid is present, an 
 emulsion is veiy readily produced. 
 
 Claud Bernard was the first physiologist to point out that 
 in the digestion of fat the pancreatic secretion plays a most 
 important part. He called attention to the fact that both the 
 
CH. XIV] FAT-SPLITTING ENZYMES. LIPASE. 237 
 
 phenomena mentioned occur under its influence, showing that 
 when neutral oil and pancreatic juice are shaken up together 
 an emulsion rapidly results, and further that the action 
 of pancreatic juice on oil produces free fatty acid. In his 
 opinion the chemical change took much longer to set up than 
 the physical one. By many physiologists these have both been 
 held to proceed from the action of enzymes, which have been 
 called emulsive and saponifying respectively. Twenty years 
 after Bernard wrote it was established that there is no emulsive 
 enzyme but that the emulsion is due to the free fatty acid which 
 results from the action of the saponifying one. Among the 
 workers upon whose investigations this view is based may be 
 mentioned Brucke and Gad. The enzyme has been variously 
 named by different writers, having been called Pialyn, Steapsin, 
 and more recently Lipase. The decomposition it sets up is 
 expressed in the following equation 
 
 C3H,(C,8H350,)a + 3H,0 = C3H,(HO)3 + 3(C,3H3,OHO) 
 
 stearin glycerin stearic acid 
 
 which represents the hydrolysis of stearin, one of the neutral 
 fats. 
 
 The enzyme which, adopting the most recent terminology, 
 we may call lipase, can be detected in the secretion of the 
 pancreas when this is collected by means of a cannula inserted 
 into the duct of the gland, or it can be prepared from a fresh 
 gland by extracting it with glycerin or water. It is important 
 to use a neutral solvent, as the enzyme is very easily injured by 
 acids. Lipase has also been found in the vegetable kingdom, 
 where it plays an important part in the utilisation of the fatty 
 reserve-products which are stored in many seeds. During the 
 germination of such seeds the fats undergo decomposition with 
 the liberation of free fatty acids. The process of emulsion does 
 not appear to occur as it does in animals. 
 
 Lipase has a fairly wide distribution in the animal kingdom. 
 In mammalia the great seat of its formation is the pancreas, 
 from which it is easy to extract it by either of the methods 
 already mentioned. It is said to occur also in the stomach, but 
 in much smaller quantity. 
 
238 FAT-SPLirriNG enzymes. [ch. 
 
 Hanriot, who has detected its occurrence in the blood of 
 several vertebrates, has tested its action on monobutjnrin. He 
 says this body is readily saponified by blood serum in neutral or 
 slightly alkaline solution, but a cautious addition of alkali is 
 necessary as the action proceeds, for it is inhibited by the free 
 fatty acid produced as soon as the latter reaches a certain per- 
 centage. The serum loses its power of hydrolysing monobutyrin 
 if it is heated to 90° C. 
 
 Cohnstein and Michaelis have also observed that the blood has 
 the power of causing the disappearance of the fat of the chyle, 
 which they attribute to a lipolytic substance in the red corpuscles. 
 
 The power of hydrolysing fats is found also among the 
 invertebrata. Fredericq extracted from sponges a substance 
 which formed an emulsion with neutral fats and finally de- 
 composed them into fatty acids and glycerin. The same body 
 is present in Uraster, one of the Echinodermata : also in the 
 so-called liver of the spider, and of the edible snail, the latter 
 only containing it in summer. The "liver" of several of the 
 Crustacea yields an extract which appears to have a similar 
 action, as it renders milk transparent. The corresponding organ 
 in certain of the Cephalopoda has the same action, and it has 
 been shown to hydrolyse neutral tats. Lipase has been prepared 
 from the " liver " of Sepia. Biedemann has found a lipase in the 
 secretion of the intestine of the larva of Tenebrio molitor, the 
 Mealworm. The eggs of several of the Crustacea have also 
 been shown by Abelous and Heim to contain this as well as 
 other enzymes. 
 
 The presence of lipase in a solution, secretion, or extract 
 can be tested by preparing an emulsion of a neutral oil, with 
 the aid of a little powdered g;um arabic and a very little water. 
 The emulsion should then be mixed with the extract and the 
 whole neutralised with care, as the gum has sometimes an 
 acid reaction. The whole may then be digested at 40° C. 
 together with a minute quantity of neutral solution of litmus. 
 If the enzyme is present the fatty acid it liberates from the oil 
 causes the liquid to turn pink or red as it affects the litmus. 
 The rapidity of the change of colour gives some idea of the 
 amount of lipase present. 
 
XIV] LIPASE. 239 
 
 Another method which may be used to test smaller 
 quantities of solution is based upon Gad's experiments on 
 the spontaneous emulsions set up wheu free fatty acids and 
 oil are added in appropriate quantities to dilute solutions of 
 carbonate of sodium. In this case no shaking is necessary, but 
 a beautiful emulsion results as soon as the liquids are brought 
 into contact. Gad's method may be described as follows : 
 
 Carbonate of sodium solution of "25 per cent, strength is placed 
 in a series of watch-glasses, and drops of oil containing different 
 percentages of fatty acids placed carefully upon the surface of 
 the alkaline solution. With a certain percentage of free fatty 
 acid in the oil, an emulsion at once results. If the optimum 
 percentage is present, this emulsion is instantaneously complete : 
 with less or more than this amount, the emulsion is more or 
 less imperfect. With the percentage of sodium carbonate 
 mentioned and at a temperature of about 18° C. the oil must 
 contain 5'5 per cent, of free fatty acid to give the best results. 
 The emulsifying agent in this method is the soap produced by 
 the combination of the free fatty acid with the alkali In the 
 presence of this no gum arable is necessary. 
 
 The formation of a spontaneous emulsion is therefore a very 
 satisfactory test for the presence of free fatty acid. If perfectly 
 neutral oil is taken and digested for a time with a solution 
 containing lipase the enzyme will set free sufficient acid to 
 make the emulsion, while the completeness of the latter will 
 depend upon the proportion so liberated. 
 
 This method has been employed by Rachford in a series of 
 researches upon the lipase of pancreatic juice, and incidentally 
 upon certain problems connected with the digestion of fat in 
 the small intestine. Rachford obtained his pancreatic juice 
 by inserting a cannula into the pancreatic duct of a rabbit 
 and collecting the secretion which exuded therefrom. He 
 mixed a small quantity of the juice with twice its volume of 
 neutral olive oil in a small test-tube and after shaking the 
 mixture he allowed it to settle. Separation of the two fluids 
 was almost immediate. Then taking a drop of the floating oil he 
 placed it upon the surface of a small quantity of '25 per cent, 
 solution of carbonate of sodium, and noticed whether any 
 
240 FAT-SPLITTING ENZVMES. [CH. 
 
 emulsion resulted. After a few minutes' interval he repeated 
 the operation and continued to experiment in the same manner 
 for some time. By taking a series of such observations, with 
 definite intervals of a few minutes after each, he was able to 
 see that the power of forming an emulsion was gradually 
 developed in the oil. It follows necessarily that lipase was 
 present in the pancreatic juice and that it gradually de- 
 composed part of the neutral oil used in the experiment. 
 Eachford says that the alkalinity of the juice employed was 
 not sufficient to absorb the free fatty acid liberated, so that the 
 mixture did not form an emulsion in the test-tube. 
 
 The lipase of the pancreas does not appear to be inhibited 
 by the free fatty acid as Hanriot says is the case with the 
 enzyme in blood. It will decompose most of the neutral 
 fats, but its action on castor-oil is comparatively feeble. It 
 hydrolyses not only the fats which are fluid at the temperature 
 of the body, but also solid ones, such as spermaceti, though 
 its action on these is very slow. 
 
 In the process of the digestion of fats in- the intestine the 
 action of lipase is complicated by the fiee acid which the 
 chyme contains as it leaves the stomach and by the bile which 
 is poured into the duodenum coincidently with the pancreatic 
 juice. Rachford found that the effect of the addition of an 
 equal volume of bile to the extracted juice and the oil in a 
 test-tube is to increase the action of the lipase in the proportion 
 of three and one-fifth to one. If "25 per cent, solution of 
 hydrochloric acid is substituted for the bile the action is 
 materially retarded. If however both are added simultaneously 
 the hydrolysis of the oil is increased in the proportion of four 
 to one. The influence of the bile is chiefly associated with the 
 glycocholate of sodium which it contains. Rachford found the 
 action of lipase was considerably impeded by excess of alkali. 
 
 The extent to which lipase takes part in the process of the 
 digestion of fat appears now to be more considerable than was 
 formerly supposed. It has been held by many observers that 
 fat is absorbed by the intestinal epithelium in the form of an 
 emulsion, and that the work of lipase is completed when enough 
 fat has been decomposed to form the quantity of soap required 
 
XIV] LIPASE. 241 
 
 to set up such an emulsion. Recent investigations have led 
 many physiologists to question the accuracy of this view and 
 to hold that the fate of the great bulk of the fat is to be 
 hydrolysed, and absorbed either as free fatty acid or as soap. 
 The emulsion may then be regarded as helping the further 
 hydrolysis and saponification. It is however outside the 
 scope of the present work to enter upon a discussion of this 
 point. 
 
 The activity of lipase like that of other enzymes is remarkably 
 influenced by temperature. The optimum point for its working 
 is not that of the body, as would be expected, but 55° C It 
 has not so wide a range as some ferments, being very materially 
 slowed by increasing the temperature to 60° C, and it ceases to 
 be active at 72° C. When the conditions are so arranged that 
 the action is rapid, the amount of change is proportional to the 
 amount of lipase present. 
 
 Hanriot has published the results of experiments made 
 with the view of comparing the activity of lipase prepared 
 from different sources. The most interesting comparison is that 
 between the enzyme of the serum and that of the pancreatic 
 juice of the dog. In his researches he prepared solutions 
 of lipase from the two sources in such a way that under 
 standard conditions they had the same hydrolysing action on 
 monobutyrin in the presence of a little sodium carbonate. 
 When his solutions were neutralised and left to work for 
 20 minutes the quantity of fatty acid liberated by the serum 
 was nearly twice as much as that set free by the pancreatic 
 solution. It appears therefore that serum lipase is more 
 energetic than pancreatic in the presence of the products of the 
 action. Solutions of the two lipases that showed equal hydro- 
 lytic power at 15° C. were examined at different temperatures. 
 Serum lipase liberated one and a half times as much fatty 
 acid at 30° C. and twice as much at 42° C. Pancreatic lipase 
 showed no change of activity through that range of temperature. 
 Serum lipase retained its activity for several months under 
 antiseptic precautions, while the pancreatic enzyme was inert 
 after a few days. 
 
 Hanriot says that the lipase prepared from the serum of 
 
 G. F. 16 
 
242 FAT-SPLITTING ENZYMES. [CH. 
 
 the horse is materially assisted by weak alkalL In one case 
 he found that the presence of "2 per cent, of sodium carbonate 
 increased the production of fatty acids fourfold. 
 
 Lipase exists in the vegetable as well as in the animal 
 kingdom. The very frequent occurrence of oils in seeds suggests 
 that a good deal of carbonaceous matter is stored in this form 
 for the nutrition of the young embryo. It is noteworthy that 
 oil and starch seldom occur together in seeds. The trans- 
 formations of the oily reserves in many endosperms during 
 germination are however very quickly followed by the appear- 
 ance of starch in the young plant, to which the products of the 
 germinative changes have made their way. The view that 
 this indicated an actual transformation of oil into starch 
 was advanced by many observers, based of course upon the 
 elementary facts of the disappearance of the oil and the 
 formation of the starch. This view was held by Sachs in 18-59 
 and by other writers who followed him. More accurate views 
 were introduced in 1871 by Mtlntz, who pointed out that 
 during germination a fatty acid appears in the seed, pointing 
 to a process of hydrolysis of the oil. Schiltzenberger in 1876 
 showed that when an oily seed is bruised in water, an emulsion 
 is obtained in which careful observation will soon show the 
 presence of glycerin as well as fatty acid. He pointed to the 
 evident hydrolysis going on and suggested that it is due to an 
 enzyme. Detmer in 1880 attempted to reconcile the new 
 theory with the old one by the suggestion that the fatty acid 
 arising during the hydrolysis is the immediate antecedent of 
 the starch, and put forward the following theoretical equation to 
 explain the process : 
 
 C,8H340, + 270 = 2CeH,„05 -I- 600^ + 7H,0. 
 
 It is evident however that no suggestion involving the 
 direct transformation of oil into starch is at all adequate to 
 explain the course of events, for neither oil nor starch is 
 diffusible, and as the two bodies appear at a considerable dis- 
 tance from each other, separated by a number of cell-membranes, 
 there can be no very direct connection between the disappear- 
 ance of the one and the formation of the other. 
 
XIV] VEGETABLE LIPASE. 243 
 
 The enzyme whose presence was suggested by Schiitzen- 
 berger was discovered by the writer in 1889 in the germinating 
 seeds of Ricinus, the castor-oil plant. Some of these seeds 
 were germinated for about .5 days, until the embryo was of some 
 considerable size, its hypocotyledonary portion being 2^ inches 
 long and a fair root system developed. The endosperm was 
 swollen and semi-mucilaginous in appearance where it was in 
 contact with the cotyledons. Such endosperms were ground up 
 in a mortar with a solution containing 5 per cent, of sodium 
 chloride and "2 per cent, of potassium cyanide, the latter being 
 used as an antiseptic. After standing 24 hours the liquid was 
 filtered till it was nearly clear, retaining only a slight opalescence. 
 A thick emulsion of castor-oil was then prepared and a little 
 of the extract carefully stirred into it, a little neutral litmus 
 solution being added at the same time. A boiled control was 
 prepared in the same way, and the two were placed in an in- 
 cubator at 35° C. In about half-an-hour the litmus in the 
 unboiled preparation began to redden, indicating the liberation 
 of fatty acid. This was extracted by shaking the digestion with 
 dilute soda solution, when the acid formed a soluble soap with 
 the alkali. On decomposing the soap solution with a mineral 
 acid a quantity of fatty acid soon rose as a scum to the surface. 
 
 In a subsequent experiment a larger quantity of the 
 emulsion treated in the same way was allowed to digest in a 
 dialysing tube, suspended in distilled water. The digestion was 
 carried on for a week; during this time the reaction in the 
 dialyser became more and more acid, while that of the sur- 
 rounding fluid remained unchanged. At the end of the week 
 the dialysate was concentrated and examined for the presence 
 of glycerin, which was detected in it by the acrolein test. A 
 control experiment with boiled extract, which was carried on 
 side by side with the other, showed no change of reaction and 
 no glycerin was present in the dialysate. The experiments 
 showed that lipase was present, that it hydrolysed the oil, 
 forming fatty acid and glycerin, the latter passing into the 
 dialysate, while the former was not able to do so. Many 
 subsequent experiments confirmed these results. 
 
 An examination of the properties of the lipase showed that 
 
 16—2 
 
244 FAT-SPLITTING ENZYMES. [CH. 
 
 it worked most advantageously in neutral solution, but was 
 also active in dilute alkalis, and to a much less extent in dilute 
 acids. The activity was tested by noting the amount of a 
 standard alkaline solution which was necessary to neutralise 
 the fatty acid formed. Comparing three digestions, the first of 
 which was neutral, the second containing 066 per cent, of 
 sodium carbonate, and the third 066 per cent, of hydrochloric 
 acid, the relative activities were as 78 : 69 : 21. Hydrochloric 
 acid when present in the proportion of "13 per cent, stopped 
 the action almost entirely; sodium carbonate needed to be 
 present in the proportion of "6 per cent, to lessen its power 
 one-half. The effect of prolonged action of both acid and alkali 
 was the destruction of the lipase and not merely an inhibition 
 of its working. Neutral salt such as sodium chloride impeded 
 the working of the enzyme, but did not destroy it. 
 
 Lipase is present only in the endosperm of Ricinus, the 
 embryo containing none. This is what would be expected, as 
 the oil does not pass from the one to the other and the 
 carbonaceous food . material temporarily appearing in the 
 embryo is in great part starch. 
 
 There appears to be no lipase present in the resting seed of 
 Ricinus, but ground seeds when kept at 35° C for a few hours 
 in the presence of very dilute acetic acid develop the power of 
 hydrolysing oil. An extract of the resting seeds, made with 
 salt solution, and then faintly acidulated with acetic acid and 
 kept warm, also undergoes the same change. At first quite 
 inactive, it gradually develops the enzyme, just as the pancreas 
 under the same treatment develops trj'psin. The lipase there- 
 fore may be regarded as existing in the form of a zymogen in 
 the resting seeds, the latter being converted into the enzyme 
 when germination begins. The same transformation takes 
 place without acid, if an extract of resting seeds is allowed to 
 stand some days under adequate antiseptic precautions. 
 
 The further changes which the products of hydrolysis under- 
 go may be briefly alluded to here. The fatty acid is not 
 further affected by lipase ; after a prolonged exposure of the 
 latter with ricinoleic acid no change can be detected. During 
 germination however the fatty acid does disappear from the 
 
XIV] VEGETABLE LIPASE. 245 
 
 cells in which it is formed, and as it cannot dialyse through a 
 membrane it is evidently decomposed. As the germination 
 proceeds, other acids, crystalline in character, make their 
 appearance in the cells, the quantity being approximately 
 proportional to the diminution of the fatty acid. There is 
 every probability that the ordinary oxidative processes going 
 on in the cells under the influence of the protoplasm transform 
 the heavy fatty acids iuto others which can leave the cells by 
 dialysis. There is a continuous production of sugar during the 
 germination which must come from the oil. It is most probable 
 that its immediate antecedent is the glycerin which is the other 
 product of the hydrolysis. This travelling through the tissue is 
 absorbed by the embryo, and is the forerunner of the starch 
 which the latter soon exhibits in its cells. 
 
 The formation of sugar from the fat or oil has been shown 
 recently by Maz6. He ground up germinating seeds of Ricinus 
 with clean sand and made a friable paste, which was easily 
 penetrable by the air. This paste was spread out in a thin 
 layer over a water bath kept at a temperature of 53° C. A 
 control was prepared and the paste heated to 100° C. for 
 10 minutes. They were allowed to stand for several hours, 
 at the end of which time the liquid was drained off from 
 both. The control gave an emulsion, the other a limpid 
 liquid which filtered clear. The latter contained sugar in 
 abundance, the former scarcely a trace. Maz^ determined the 
 quantity of sugar present twice, after 7 hours and again after 
 22 hours. The amount was nearly doubled in the interval. Maz6 
 attributes the formation of the sugar to an enzyme, but he does 
 not indicate its course of action. In his experiments he found 
 he was able to obtaia a quantity of sugar equal to about 3'5 per 
 cent, of the dry weight of the ungerminated seeds or about 
 7 per cent, of the weight of its oil. 
 
 The existence of lipase was demonstrated two years later 
 by Sigmund, who found it in both resting and germinating 
 seeds of the Rape, the Opiwm Poppy, Hemp, Flax and Maize. 
 His mode of experiment was to crush the seeds with water, and 
 estimate the free fatty acid in the resulting emulsion. Com- 
 paring the amount of standard alkali needed to neutralise part 
 of the emulsion immediately on crushing and a further part 
 
246 FAT-SPLITTING ENZYMES. [Cfl. 
 
 after allowing it to stand for 24 hours at 30" C, he found in all 
 cases a larger quantity was required after the interval. Free 
 fatty acid was accordingly developed on standiag, and this 
 was due no doubt to the presence of lipase. Sigmund says 
 that the resting seeds he examined contained a certain amount 
 of enzyme, and that this was increased at the onset of germi- 
 nation. 
 
 It has already been mentioned that Sigmund attributes the 
 power of hydrolysing oils to myrosin and to emulsin. Causing 
 what he took to be a preparation of these enzymes to act upon 
 olive oil in closed glass vessels at a temperature of 38° to 40° C, 
 he found that there was a gradual and continuous formation of 
 free fatty acid in the mixture, its presence being demonstrated 
 by both litmus and phenol-phthalein. 
 
 On looking over his experiments it does not appear to be at 
 all certain that the results were due to emulsin or to myrosin ; 
 it seems far more likely that he was dealing with lipase 
 present in the seeds in addition to the other enzymes. The 
 occurrence of more than one enzjrme in a tissue is very common, 
 both in vegetable and animal organisms, and many instances 
 of it have already been given. Sigmund prepared his two 
 glucoside-splitting enzymes by bruising in water seeds of the 
 mustard in one case and of the almond in the other, and allowing 
 them to digest with excess of water for 12 — 14 hours. He 
 then decanted the supernatant fluid and added to it an excess 
 of alcohol. This caused the formation of a bulky precipitate; 
 removing the latter by filtration he washed it with alcohol and 
 dried it at about 40° 0. This method would certainly not 
 prepare any enzyme pure ; his precipitate would contain all the 
 soluble proteids of the seed and all the enzymes that might be 
 present. The action of a solution of this powder at once 
 suggests that it contained lipase, and the natural deduction 
 from the experiment would be that mustard seeds aad almonds 
 must be added to the list of those in which the existence of 
 this enzyme has been established, rather than that glucoside- 
 spUtting enzymes possess the power of hydrolysing oils. The 
 constitution of oil on the one hand and of glucosides on the 
 other opposes very strongly the view that there is one 
 hydrolytic agent which is capable of decomposing them both. 
 
XI V] VEGETABLE LIPASE. 247 
 
 It is interesting to note that Sigmund attributes to his 
 lipase the power of splitting up amygdalin and salicin. On his 
 hypothesis therefore lipase and emulsin are practically the same 
 body. This appears equally unlikely. 
 
 The germiaation of several oily seeds has been examined 
 also by Leclerc du Sablon, whose work has embraced the 
 Gastor-oil plant, Hemp, Golza and Flax, besides one or two less 
 conspicuously of an oily nature. Du Sablon found the oil of 
 these seeds to diminish markedly during germination, and the 
 diminution to be accompanied by the formation of sugar of 
 both the cane-sugar and glucose types. In his study of the 
 processes of germination he relied chiefly upon analyses of the 
 contents of the seeds from time to time. He pronounces 
 agaiost the hydrolysis of the oil on the ground that he has 
 not been able to detect the presence of glycerin. This however 
 is not very surprising, as most other observers have shown that 
 no appreciable quantity of it is present in the tissue of the 
 seeds during the progress of the germination. It should not 
 however be hastily inferred that none is ever formed; it is 
 quite conceivable that it may undergo further decomposition as 
 fast as it is set free. Du Sablon did not attempt by any 
 method to secure an accumulation of it, and hence his denial of 
 its existence cannot be accepted as decisive. He did not attempt 
 either to bring about the decomposition of the oil by any 
 extract containing lipase, but relied entirely on his analyses. 
 
 In his discussion of his results he suggests the existence of 
 a modified lipase which sets free fatty acid from the oil, but does 
 not liberate glycerin ; such fatty acid however represents but a 
 small part of the oil decomposed. The glycerin radical of the 
 whole of the oil together with so much of the fatty acid as is 
 not set free goes directly to form sugars, of which those of the 
 cane-sugar type appear first and give place subsequently to 
 those resembling glucose, in which latter form they are ab- 
 sorbed. Transitory starch ' also appears in the seeds during 
 these changes ; and ultimately this also is converted to dextrin 
 and sugar. 
 
 Both the cane-sugar and the starch he is inclined to con- 
 sider as indications that the oil is decomposed more rapidly 
 
248 FAT-SPLTTTTNG ENZYMES. [CH. 
 
 than it is required by the young plantlet, and hence he regards 
 them as secondary and temporary reserve-stores. He identifies 
 both invertase and diastase as present ia the watery extracts 
 of his seeds. In some cases, especially in the seed of Colza 
 {Brassica oleracea), he thinks the dextrins occur as inter- 
 mediate bodies between the oil and the non-reducing sugar. 
 
 One difficulty in the way of accepting du Sablon's views on 
 the course of the transformation is the great unlikelihood of 
 fatty acid serving directly as an antecedent of carbohydrates. 
 No instance of a similar transformation is known, and no 
 laboratory method can at present bring it about. The great 
 stress he lays on the presumed absence of glycerin is also 
 noteworthy, for it is not difficult with proper methods to prove 
 its occurrence, as the writer has already shown in the course of 
 his own work on Ricinus. Its presence or absence appears very 
 important as indicating whether or no the enzyme which he 
 predicates is identical with lipase. 
 
 It has already been noticed that Penicilium glaucum is the 
 source of several enzymes. To those already mentioned, lipase 
 must be added. Gerard has found that the watery extract of 
 this fungus is capable of hydrolysing monobutyrin. The best 
 means of showing its power is the process of cultivating it in 
 Raulin's fluid to which a little monobutyrin has been added. 
 Butyric acid is very quickly liberated, just as with Hanriot's 
 Hpase fi-om blood. It may be noticed in view of Sigmund's 
 work discussed above, that Gerard finds emulsin does not 
 hydrolyse monobutyrin. 
 
 The conclusions of Gerard have been confirmed inde- 
 pendently by Camus, who has also found lipase in another 
 fungus, Aspergillus niger. 
 
 Lipase has been extracted by Biffin from a fungus which 
 sometimes attacks Coco-nuts during germination. The fungus 
 in question has not been completely identified but it appears to 
 be a member of the Hypocreacese, a division of the Pyrenomy- 
 cetes. It grows fi:eely on vegetable tissue which is rich in oil. 
 
 In his experiments Biffin cultivated the fungus in sterilised 
 coco-nut milk till the liquid became acid to Utmus. The 
 mycelium was then taken out of the flasks, washed rapidly 
 
XIV] VEGETABLE LIPASE. 249 
 
 in distilled water and ground to a thin paste with kieselguhr 
 and water in a bacterium-mill. On filtering the paste under 
 pressure through several thicknesses of filter-paper a faintly 
 brown opalescent fluid was obtained which gave a slight acid 
 reaction. It was neutralised and made sterile by the addition 
 of potassium cyanide. 
 
 Sections of coco-nut endosperm containing oil, and cover- 
 slips coated with a thiij layer of oil, were placed in some of the 
 extract and in all cases the oil was decomposed. Control 
 experiments in which the extract had been boiled gave negative 
 results. The extract also decomposed monobutyrin liberating 
 butyric acid. 
 
 Biffin found that the lipase could be precipitated by alcohol 
 from the extract of the fungus. It was thrown down as a 
 flocculent whitish grey substance, which powdered readily after 
 being dried over calcium chloride. A solution of the powder 
 had the same power of decomposing fats as was exhibited by 
 the original extract. 
 
250 
 
 CHAPTER XV. 
 
 THE CLOTTING ENZYMES. RENNET. 
 
 The enzymes which next present themselves for exam.uiation 
 form a group which at first sight appears well marked and 
 sharply separated from those we have so far discussed. The 
 prominent feature of their action is that it results in the forma- 
 tion of a semi-gelatinous clot or jelly, which soon after its 
 production undergoes a species of contraction or shrinkage and 
 ultimately becomes semi-fibrous in character. Instead therefore 
 of producing from a primary body, often insoluble and indiffusible, 
 a more freely soluble and diffusible material, they appear to 
 invert this order. In two cases however the clot or coagulum is 
 accompanied by another product more soluble than the original 
 one, or at any rate not less so. Another feature which is 
 common to three of them is that the action of the enzyme is 
 peculiarly associated with certain compounds of Calcium, in the 
 absence of which the clotting does not occur. It is however not 
 certain that the resemblance in this particular is more than 
 superficial, for the action of the inorganic salt cannot at present 
 be said to have been proved the same in all cases. 
 
 One of these enzymes, generally known as rennet, occurs in 
 both the animal and vegetable kingdoms ; the so-called fibrin 
 ferment, and the ferment which is instrumental in causing the 
 formation of myosin in muscles, are only found in the former. 
 Another, pectase, is of purely vegetable origin. The three 
 first mentioned may be regarded as related to the proteolytic 
 enzymes: pectase is associated with certain changes in the 
 materials which constitute the cell-membranes of plants. 
 
CH. XV] EENNET. 251 
 
 Rennet (Lab, Chymosin, Presure, Rennin). 
 
 The first of these enzymes which we shall discuss is the 
 so-called Reimet, which is so largely employed in the production 
 of cheese fi:om milk. When a small quantity of this body is 
 added to a relatively large volume of milk, the latter rapidly 
 changes to a stiff jelly ; after standing a shorter or longer time, 
 the jelly shrinks somewhat, and a watery liquid known as whey 
 oozes out of it, and after a little while a firm clot is found 
 floating in a quantity of the watery liquid. 
 
 The formation of this clot, which is really the crude cheese, 
 or curd, is due to an alteration of one of the constituents of the 
 milk. This is a body which is known as caseinogen; it is a 
 member of the group of nucleo-albumius, but is generally 
 spoken of as the principal proteid of the milk. 
 
 As it exists in milk, caseinogen can be readily precipitated 
 fi-om solution by the cautious addition of a dilute acid : it then 
 separates in a loose flocculent form, and can be easily separated 
 firom the liquid by filtration. When so separated it can be 
 redisaolved in dilute alkalis and again reprecipitated. 
 
 When caseinogen either in milk, or ia solution in dilute 
 alkalis, is acted upon by rennet in the presence of a small 
 quantity of calcium phosphate, it is at once chemically altered. 
 It gives rise to the formation of a body which has been named 
 casein, which is the proteid constituent of the clot. When the 
 whey has separated out, it is found to contain a by-product 
 having properties resembling those of a soluble albumin, except 
 that it is not made insoluble by boiling. It is consequently 
 distinct fi:om the serum albumin, or lact-albumin, present in 
 normal milk. The formation of this by-product when solutions 
 of pure caseinogen are treated with rennet shows that it results 
 from the alteration of the caseinogen. The clotting of milk by 
 rennet is consequently not to be confused with the precipitation 
 of caseinogen by acid. In the latter case the separated body 
 can be redissolved and is found to be unchanged in constitution ; 
 in the former case it has been decomposed, with the formation 
 of an insoluble curd (consisting chiefly of casein) and a certain 
 quantity of a variety of albumin. The curd differs in composi- 
 
252 THE CLOTTING ENZYMES. [CH. 
 
 tion from caseinogen by containing a relatively large amount of 
 calcium phosphate, which on ignition of the proteid remains as 
 ash. If this calcium salt is removed from it and it is then 
 dissolved in dilute alkalis it cannot again be clotted by rennet 
 as precipitated caseinogen can. Halliburton suggests that it is 
 caseate of lime. 
 
 The chief source of rennet in the animal body is the mucous 
 membrane of the stomach, and it is present in largest quantity 
 in the stomach of yoimg animals, particularly the calf It can 
 be obtained however from the stomach of almost any animal 
 and may be regarded indeed as one of the normal constituents 
 of gastric juice. It exists in the pancreas of several animals, 
 especially the pig, ox, sheep, horse, dog and cat; also in the 
 human pancreas. Halliburton and Brodie found it present in 
 pancreatic juice obtained from the dog by means of a cannula 
 inserted in the pancreatic duct. Edmunds prepared it from 
 various tissues of the mammalian body, including the testis, 
 liver, lung, kidney, spleen, thymus, thyroid, brain, intestine and 
 ovary, but it was only in very small amount in any of these 
 organs. He also obtained evidence of the presence of a small 
 quantity in the blood. In animals belonging to groups below 
 the Mammalia, Benger has prepared it from the stomach 
 of the cod-fish, and appropriate treatment has shown that 
 it is formed in the stomach of the pike. Roberts obtained 
 it from the digestive organs of the fowl, and Harris and Gow 
 extracted it from the pancreas of the eagle. It is stated that a 
 small amount may be present in urine, but this is probably an 
 indication of its excretion from the body. Babcock and Russell 
 have found that milk itself contains a very small quantity. It 
 has a wide distribution in the vegetable kingdom, but this will 
 be treated of in greater detail in a subsequent section. 
 
 The preparation of rennet in an impure form from the 
 stomach or pancreas is very easy.. All that is necessary is to 
 mince the organ finely, either in the fresh state, or after 
 dehydration by alcohol, and then to extract the pulpy mass with 
 glycerin, water, or solutions of neutral salts. If the reaction of 
 the solvent is made faintly acid, a more active preparation is 
 obtained than if it is perfectly neutral. The most eflBcient 
 
XV] RENNET. 253 
 
 extractive is a solution of sodium chloride, containing about 
 5 — 10 per cent, of the salt. 
 
 Many attempts have been made to prepare the enzyme free 
 from admixture with other ferments or -with proteids. The 
 first experiments in this direction were made by Deschamps in 
 1840, but they were subsequently shown to be unsuccessful. 
 The isolation was however carried out by Hammarsten, who 
 published his method in 1872. It consists in the first place 
 of a fractional precipitation of an acid aqueous extract of the 
 stomach by means of magnesium carbonate or a solution of 
 acetate of lead. Both pepsin and rennet are thrown down by 
 these reagents, but pepsin is precipitated with the greater readi- 
 ness. It is possible therefore to free the liquid from pepsin 
 while the greater part of the rennet remains in solution. After 
 filtering this off, further addition of the lead acetate throws 
 down the rennet, the process being facilitated by the simul- 
 taneous addition of a little ammonium hydrate. The precipi- 
 tate is then filtered off, suspended in water and decomposed by 
 very dilute sulphuric acid. This acid solution, which contains 
 mere traces of albumin, is mechanically precipitated according 
 to Briicke's method for the separation of pepsin, with the aid of 
 cholesterin, as already described. 
 
 This method is useful in cases where the rennet exists in 
 considerable quantity, but it always involves the loss of a good 
 deal of material. 
 
 A few years later the rennet and the trypsin of pancreatic 
 extract were separated by Roberts by the following method. 
 A salt-solution extract of a pancreas was slightly acidulated 
 with hydrochloric acid and kept for 3 hours at 40" C. When 
 neutralised it was found that the trypsin had been destroyed 
 while the rennet was unharmed. Roberts says that he found 
 that when some of the same extract was filtered through porce- 
 lain under pressure, the rennet passed through the porcelain 
 and the trypsin remained behind. 
 
 In 1886 a different method was introduced in America by 
 Blumenthal. In his process, the stomach of the calf is cut 
 into small pieces and macerated for 24 hours in a solution of 
 common salt of "5 per cent, concentration, kept at about 30° C. 
 
254 THE CLOTTING ENZYMES. [CH. 
 
 The solution is then filtered and 'l per cent, of mineral a«id is 
 added. This produces a thick precipitate of mucous matter 
 which can be separated mechanically, leaving the enzymes ia 
 solution. The acidity is next raised to -5 per cent., and the 
 liquid saturated with powdered sodium chloride. It is then 
 kept for 2 or 3 days at 25°— 30°C. with constant stirring, 
 and the temperature is gradually raised to 30° — 35° 0, After 
 standing for a day or two, a white flocculent scum separates out 
 which can easily be removed by skimming or filtration and 
 dried at about 28° C. This is an amorphous white gelatinous 
 substance, greatly resembling aluminium hydrate in appearance. 
 It is without taste or smell, and dissolves readily in water, 
 forming a clear solution. This precipitate is nearly pure ren- 
 net, a small portion of it readily inducing coagulation in milk. 
 The mother liquid firom which it is removed has no curdling 
 action but is possessed of very considerable peptic powers. 
 
 Blumenthal says that the same treatment will separate the 
 two enzymes fi'om the so-called pepsin essence of commerce, 
 which is very impure. 
 
 Friedburg found on repeating Blumenthal's experiments 
 that the prolonged standing after saturation with salt was not 
 always necessary, but that if comparatively small amounts of 
 material were used, the rennet separated almost immediately. 
 He recommends its immediate removal, for on standing it 
 shows a tendency to sink in the liquid. 
 
 Hammarsten quotes the following reactions as characteristic 
 of his purified product : — 
 
 (1) it does not show the xanthoproteic reaction. 
 
 (2) its watery solution does not coagulate on boiling. 
 
 (3) it is not precipitated by alcohol, nitric acid, tannin, 
 
 iodine, or normal lead acetate. 
 
 (4) it is precipitated by basic lead acetate. 
 Friedburg found his product obtained by Blumenthal's 
 
 method gave exactly the same reactions. 
 
 The effect of heat on the rennet enzyme is to some extent 
 dependent on the reaction of the solution. Its optimum point 
 is about 40° 0. Neutral preparations will remain active after 
 a short exposure to 70° C, but if kept long at that temperature 
 
XV] EENNET. 255 
 
 they are rendered inert. A momentary exposure to a higher 
 point than this is destructive of the enzyme. If the preparation 
 contains "3 per cent, of hydrochloric acid it will not survive 
 heating to 63° C. Prolonged exposure of such an acid solution 
 to only 40° C. is very deleterious. The presence of a little 
 alkali has a similar influence. 
 
 Kennet is active iu neutral, or in faintly acid or alkaline 
 solutions, but the presence of only a little more than the 
 optimum quantity of either acid or alkali speedily destroys the 
 enzyme at the ordinary temperature. Hammarsten found that 
 clotting occurred most rapidly when a faintly acid extract was 
 used, and Ringer has confirmed the observation, care being 
 taken that the acid was added in insufficient quantity to pre- 
 cipitate the casein. The clotting was not due to the action of 
 the acid on the caseinogen, as the addition of the same amount 
 of acid without the rennet produced no effect upon the milk. 
 Roberts says that pancreatic rennet differs from gastric in being 
 active m the presence of larger amounts of alkali. Alcohol in 
 small quantities does not injure the enzyme, but large quan- 
 tities or prolonged exposure destroy it. 
 
 The effect of neutral salts such as sodium chloride, or 
 magnesium sulphate in greater concentration than 4 per cent., 
 is to impede the action, but not to destroy the rennet. Small 
 quantities of less than 1 per cent, are beneficial. 
 
 The action of rennet is materially impeded by the presence 
 of peptone or some kinds of proteose. Edmunds has published 
 an account of some experiments on this point which are very 
 interesting in the light of the effect of the same proteids on the 
 process of the coagulation of the blood. He mixed in several 
 tubes 10 c.c. of milk, a varying quantity of Witte's " peptone," 
 containing a certain amount of proteoses, and 150 cubic milli- 
 metres of a preparation of rennet and exposed them to a 
 temperature of 40° 0. In the absence of any " peptone " 
 clotting took place in 10 minutes ; with '625 per cent, present, 
 it did not set in for 20 minutes ; with 2'5 per cent, no clot was 
 formed till after 45 minutes, and in the presence of 5 per cent, 
 coagulation was delayed for several hours. 
 
 It has already been mentioned that the salts of calcium 
 
256 THE CLOTTING ENZYMES. [CH. 
 
 play an important part in the action of this enzyme. The most 
 pronounced effect is produced by calcic phosphate; indeed, 
 according to most observers, the clotting of the milk will not 
 take place in the absence of this salt. The experiment can be 
 made by dialysing the milk till all the salts have disappeared, 
 when rennet is inoperative upon it. The same effect can be 
 seen when experiments are carried out on a pure solution of 
 caseinogen prepared by precipitating it from milk with dilute 
 acetic acid, filtering and washing quickly with distilled water. 
 The solution of this in water gives no clot with rennet. If 
 however it is dissolved in lime water and the solution carefully 
 neutralised with dilute phosphoric acid, a milky-looking liquid is 
 obtained which behaves exactly like milk itself, curdling indeed 
 even more readily on the addition of rennet. It is noteworthy in 
 this connection that cheese always contains a certain amoimt 
 of calcic phosphate. Einger states that other salts of calcium, 
 especially the chloride, can replace the phosphate. 
 
 Hammarsten, to whom our knowledge of this peculiar 
 behaviour is due, explains the action by saying that the 
 enzyme induces the conversion of caseinogen into casein, and 
 the calcic phosphate makes the latter separate out in the 
 condition of the clot or curd. He quotes in support of his view 
 the following experiment. 
 
 Some pure caseinogen prepared as described above, and 
 ascertained to be ash-free, was dissolved in dilute hydric-disodic- 
 phosphate and the solution divided into two parts, A and B. 
 A was treated with rennet and both were digested for half-an- 
 hour at 30° C. A was then boiled to destroy the enzyme, and 
 B was boiled also that the two might be capable of comparison. 
 A quantity of the rennet solution equal to what had been 
 added to A was then boiled and added to B. After both had 
 been cooled the same amount of calcium chloride was added 
 to each. A, in which the rennet had acted for thirty minutes 
 without producing a visible change, at once gave a thick 
 precipitate of casein. B, in which rennet though present 
 had been prevented from acting, remained unchanged. The 
 curd appears to be therefore not casein, but a compound 
 of casein and the calcic salt. Ringer has published an account 
 
XV] RENNET. 257 
 
 of similax experiments which completely confirm those of 
 Hammarsten. 
 
 The part played by calcium salts may perhaps explain the 
 inhibitory action of peptone. This body can combine with such 
 salts, and it may be that no clot is formed owing to their being 
 taken out of the sphere of action by means of such a combina- 
 tion. The subject needs however further research before definite 
 opinions can be formed upon the point. 
 
 The proportion of water to caseinogen in the milk must be 
 taken into account in considering the curdling. Mayer found 
 that milk which gave a normal clot in 25 minutes took 30 
 minutes when 5 per cent, of water was added ; farther addition 
 of water retarded it longer, and when 20 per cent, had been 
 added the time taken was 73 minutes, the proportion of rennet 
 and the temperature of the experiment being alike in all cases. 
 The clot produced in diluted milk is much more flocculent than 
 a normal one. 
 
 The dilution of the rennet extract itself has still greater 
 effect in the same direction. Soxhlet found that when one 
 part of a particular preparation of rennet was made to act upon 
 ten thousand parts of milk, curdling took place in 40 minutes ; 
 but when the same quantity of rennet was mixed with five 
 hundred parts of milk, the clot was formed in two minutes and 
 six seconds. From his experiments he inferred that the time 
 taken to form the clot was inversely proportional to the amount 
 of rennet used. When however smaller quantities of the 
 enzyme are employed, this relation does not hold. The writer 
 found that with a particular preparation of the enzyme firom the 
 germinating seeds of Ricinus, while 1 c.c. clotted 5 c.c. of milk 
 in two minutes, '2 c.c. did not produce its effect till after four 
 hours. In another series of experiments in which the extract 
 was diluted so that the total amount of fluid added to the milk 
 in each case was the same, the same result was obtained. 1 c.c. 
 clotted 5 c.c. of milk in five minutes ; "125 c.c. added to '875 c.c. 
 of water, did not clot 5 c.c. of milk till the expiration of 5|- hours. 
 
 The character of the clot depends also to a large extent 
 upon the source of the milk. When cows' milk is coagulated 
 by rennet, or precipitated by acids, the curd, or the precipitated 
 
 G. F. 17 
 
258 THE CLOTTING ENZYMES. [CH. 
 
 caseinogen, is more solid than when human milk is used. This 
 is particularly noticeable in the case of true coagulation, the 
 curd of human milV always being of a loose, flocculent con- 
 sistency. From what has already been said we can see that the 
 solidity or compact condition of the curd formed by the rennet 
 of the stomach is mainly determined by three factors : — 
 
 (1) The concentration of the caseinogen solution. 
 
 (2) The amount, of soluble calcium phosphate. 
 
 (3) The acidity of the milk 
 
 Comparing the milV of the cow with the human secretion 
 we find it contains about twice as much caseinogen, and six 
 times as much calcium, while it has about three times the 
 acidity of human milk. It is natural therefore that the 
 coagulum should be flocculent in the case of the latter, while 
 in cows' milk it is more compact and leatheiy. By dilution 
 with water and appropriate adjustment of reaction, cows' milk 
 can be so altered that it will coagulate very nearly in the same 
 way as human milk. There will always be some difference 
 between the two, on account of the preponderance of calcium 
 phosphate in cows' milk. 
 
 Rennet will induce the formation of a clot in boiled mUk, 
 but the curd is very flocculent and a considerable quantity of 
 the enzyme is needed for the coagulation. The alteration which 
 causes this difference is greater in proportion to the temperature 
 to which the heating has been carried and the time the milk has 
 been exposed to it. 
 
 Before leaving the subject of the action of rennet on milk it 
 seems not inappropriate to allude to some theories of its action 
 which have been advanced, but which are now disproved. The 
 most prominent of these has been that the rennet acted upon 
 the sugar of the milk, converting it into lactic acid, and that 
 the separation of the curd was due to the presence of the latter. 
 Liebig in 1865 advanced this view and suggested that the 
 lactic acid neutralised the alkali which keeps the caseinogen 
 dissolved, thus precipitating the latter. 
 
 Soxhlet had a slightly different conception. He thought 
 that the difference between the coagulation of milk by rennet 
 and the precipitation of the caseinogen following upon its 
 
XV] EENNET. 259 
 
 • 
 
 turning sour on standing, was only that the former took place 
 most rapidly. According to him rennet transformed sugar 
 ■of milk into lactic acid, and this reactiag on the alkaline 
 phosphate existing in milk, converted it into the acid salt. 
 This in turn precipitated the caseinogen. Hallier imputed 
 the process to the micro-organisms present in the stomach of 
 the calf. 
 
 The question was finally decided by Hammarsten. Not 
 only did he show that casein was essentially different from 
 caseinogen, but he also proved that the curdling was quite 
 independent of the presence of sugar in the milk, by pre- 
 paring a solution of caseinogen in the manner already described 
 and seeing it clot in the presence of rennet. In the method of 
 preparing caseinogen which he adopted, the sugar of the milk 
 was lefb behind in the mother liquor from which the proteid 
 was precipitated. Hammarsten's pure rennet, or lab, was further 
 found to be quite -without action on milk sugar, while it clotted 
 milk or casein solutions with great rapidity. 
 
 The theory of the action of the acid is negatived also by 
 the fact that curdling can be brought about quite easily in 
 faintly alkaline solutions. 
 
 Certain peculiarities of the rennet prepared from pancreatic 
 sources have been noticed by several observers. It has already 
 been mentioned that Koberts when examining the proteolytic 
 effect of pancreatic juice or extract upon milk noted a curious 
 change in its proteids which seemed to be different from the 
 ordinary proteolysis brought about by trypsin. When milk 
 was exposed to the action of pancreatic juice for a short time 
 and then boiled, the boiling caused a precipitation of the 
 caseinogen in a curd-like form. Eoberts considered this change 
 to be brought about by the trypsin. Subsequent observers 
 iave however established the fact that it is due to the rennet 
 which is secreted with the trypsin in the pancreas. The 
 product differs from casein in not separating out as a curd 
 at the ordinary temperature. Eoberts called it metacasein, and 
 the name has been retained by subsequent observers. It can be 
 separated from milk that has been subjected to the action of a 
 small quantity of pancreatic rennet, being thrown down by the 
 
 17—2 
 
260 THE CLOTTING ENZTJIES. [CH. 
 
 addition of an equal volume of a saturated solution of sodium 
 chloride. It differs from caseinogen in turn, in that it will not 
 give a clot when subjected to the further action of rennet. 
 
 The conditions that lead to its formation are a large pre- 
 ponderance of milV over the rennet solution. Too little of the 
 latter must be added to bring about the ordinary clotting and 
 then metacasein appears to be formed instead of casein. The 
 by-product, or whey-proteid already mentioned, is formed 
 simultaneously with the metacasein. 
 
 Halliburton and Brodie draw a distinction between gastric 
 and pancreatic rennet which has not been noted by previous 
 observers. According to them the clotting indicated by pan- 
 creatic rennet is not a true coagulation, but a precipitation 
 which takes place in the warm bath at 35° — 40° C. The 
 precipitate is finely granular, and cannot be detected by the 
 naked eye. On cooling it to the temperature of the air, it sets 
 into a coherent curd which can be again broken up by warming 
 to 35° C, when the granular condition returns and the milk 
 appears fluid. This may be repeated several times. Halliburton 
 and Brodie call the proteid in this condition "pancreatic 
 casein," and they say it can be converted into casein by 
 gastric rennet. 
 
 Kennet does not appear to be stored in the cells of either 
 gastric or pancreatic glands in a condition in which it will 
 effect coagulation, but rather to occur in the form of an 
 antecedent body or zymogen. This has been established by 
 Hammarsten and subsequently by Langley. The latter physio- 
 logist examined the gastric glands of the dog, cat, rabbit, mole,, 
 and frog; also the cesophageal glands of the latter; none 
 contained rennet, but the zymogen was present in them all. 
 The zymogen, Kke that of trypsin or pepsin, can be converted 
 into enzyme by warming it for fifteen minutes with a dilute 
 acid. Langley foimd that the zymogen, like the rennet itself, 
 is destroyed by keeping it for a few minutes at 38° C. in the 
 presence of 05 — 10 per cent, of sodium carbonate. 
 
 Lorcher has recently confirmed these observations of the 
 existence of rennet zymogen. 
 
 In addition to mammalian sources Hammai-sten found that 
 
XV] VEGETABLE RENNET. 261 
 
 rennet z3rmogen exists in the gastric glands of certain fishes, 
 among which the Pike is conspicuous. 
 
 The coagulating action of rennet can be inhibited by some 
 substance which is normally present in the serum of blood. 
 Our knowledge of this subject is due to the researches of 
 Camus and Gley and of Biot. The latter observer has stated 
 that when the serum of the horse is added to milk at the same 
 time as a solution of rennet the coagulation is retarded and 
 that there is a quantitative relation between the serum and 
 the rennet. The substance ia the serum which has this 
 property is incapable of dialysing through a moist membrane, 
 is destroyed by heating, and can be precipitated from solution 
 by sulphate of ammonia or by alcohol. Biot suggests that it is 
 itself an enzyme. This seems on the whole unlikely as egg 
 albumin has been found to have the same effect, if added in 
 sufiicient quantity. 
 
 Vegetable Rennet. 
 
 The existence of rennet in the vegetable kingdom has been 
 noted by many observers from the sixteenth century to the 
 present day. Linnseus stated that the power of curdling milk 
 was known in his day to exist in the leaves of Pinguicula 
 vulgaris and that certain of the Lapland tribes with whom he 
 became acquainted always used them for that purpose. Prior 
 ia his Popular Names of British Plants speaks of a curious 
 property of Galium verum, which was noted by Matthioli in the 
 sixteenth century, who wrote of it, " Galium inde nomen sorti- 
 tum est suum quod lac coagulet." In West Somersetshire and 
 certain parts of Herefordshire this property of Galium verum is 
 still recognised and it is customary for dairymen to put the 
 plant into milk to set the curd ready for cheese-making. The 
 active principle seems to be located in the flowers, though 
 the whole plant is used. It is somewhat singular that our 
 other native species of Galium do not appear to contain the 
 enzyme. 
 
 Many other observations of a similar character have been 
 made by various investigators. Pfeffer says that Pinguicula 
 vulgaris is employed in the Italian Alps by the peasantry to 
 
262 THE CLOTTING ENZYMES. [CH. 
 
 prepare curd from milk. Daxwin noticed that the secretion of 
 the glands of Drosera not only digested proteids, but curdled 
 nulk as well. The association of rennet with a proteolytic 
 enzyme is very often met with. Baginsky found that it was 
 present in the juice of the papaw (Carica Papaya) which we 
 have seen is especially rich in vegetable trypsin. Martin con- 
 firmed Baginsky's observation in the course of the researches 
 which we have already discussed. Chittenden has shown that 
 rennet exists side by side with bromelin in the juice of the 
 pine-apple, but is associated with a different proteid, as already 
 mentioned. 
 
 Rennet has a very curious distribution in some plants. It 
 is found in the bast of the stem bundles of Clematis Vitalba, 
 and in the petals of the artichoke (Cynara scolymu^), neither 
 of which localities would appear likely ones for an enzyme. 
 
 The most constant situation for vegetable rennet appears 
 to be the finiit and seed of the plant; at any rate it is these 
 regions which famish most instances of its occurrence. It is 
 more firequently present in the seed than the finiit. 
 
 A very active preparation can be made firom the pericarp of 
 the fruit (pepo) of the singular plant known as " Naras," which 
 is met with in very dry desert places in Namaqua Land and 
 Whalfish Bay in South Africa. Naras {Acanthosicyos hoiTida) 
 is a typical creeping desert plant, of very wiry habit and well 
 furnished with spines. It has squamiform leaves which are 
 very quickly deciduous, and while they are attached to the stem 
 they are closely adpressed to it, so that at a little distance it 
 appears to be leafless. There are two sharp spines at the base 
 of each, which do not fall off. The flowers are developed 
 without stalks between these spiaes and therefore in the aiils 
 of the frequently abortive leaves. The fruit is an almost 
 globular pepo with a hard tough skin enclosing a very pleasant 
 acid pulp something like that of an orange, in which a large 
 number of seeds are embedded. Though the pulp is very 
 palatable, strangers eating it are often very singularly and 
 painfully affected by it. It is said to produce an almost in- 
 tolerable itching of the anus. The rennet is confined to the 
 succulent parts of the fruit, the seeds strangely being without 
 
XV] VEGETABLE RENNET. 263 
 
 it. The enzyme is not destroyed by drying but can be pre- 
 served almost indefinitely by exposing the pulp to the sun. 
 like other enzymes it is destroyed by boiling. According to 
 Marloth it is soluble in alcohol of 60 per cent, strength. It is 
 developed only as the fruit ripens. 
 
 In seeds rennet seems to be of very common occurrence. 
 Its preparation from this source was first successfally carried 
 out by Lea, who found it in the resting seeds of Wiiliania 
 coagulans, a shrub belonging to the Solanaceae, which grows 
 freely in Afghanistan and Northern India. Withania has a 
 capsular fruit containing a large number of small seeds of a 
 dark brown colour. It can be extracted from the ground seeds 
 by means of glycerin or a moderately strong solution of common 
 salt. It is however much contaminated by the colouring matter 
 of the seed-coats, which dissolves in the extracting liquid, 
 causing it to have a dark brown hue. Its activity is about the 
 same as that of most commercial samples of animal rennet and 
 the details of its behaviour are undistinguishable from those of 
 the latter. It is similarly affected by temperature, but it can 
 withstand a moderately prolonged exposure to alcohol without 
 destruction. 
 
 The writer has met with rennet in the seeds of Datura 
 Stramonium, Pisum sativum, Lupinus hirsutus, and Ricinus 
 commimis; in the two former in the resting, and in the two 
 latter in the germinating condition. Though in Eicinus it does 
 not exist in the resting state, the seed contains a zymogen 
 which is soluble in water, and which readily develops the milk- 
 curdling property on being warmed with dilute acids. The 
 rennet itself can be extracted from the germinating seeds by 
 either salt-solution or glycerin. It exists in the endosperm, 
 in close association with both lipase and trypsin. There is 
 generally a considerable quantity present, or it has very 
 energetic powers ; in one experiment a glycerin extract curdled 
 two and a half times its volume of milk in 5 minutes. The 
 salt-solution extract acts more slowly than the glycerin one, 
 sodium chloride being a hindrance to the activity of rennet as 
 it is to trypsin. Different seeds however contain very varying 
 quantities of the enzyme. 
 
264 THE CLOTTING ENZYMES. [CH. 
 
 Ricinus rennet is capable of acting in either acid, neutral, 
 or alkaluie solutions. Too great a degree of acidity obscures 
 the action, as the acid itself tends to precipitate the caseinogen 
 of the milk. 
 
 In the germinating lupin seed rennet exists side by side 
 with trypsin, but there is much less of it present. 
 
 The enzyme exists also in many of the more lowly forms of 
 plants. Duclaux has demonstrated that it is produced by 
 several of the filamentous fiingi, and many micro-organisms 
 secrete it. The bacilli examined by Wood which we have seen 
 produce proteolytic enzymes also furnish rennet. That two 
 enzymes are present in the secretion from these plants is shown 
 by their different powers of resisting different destructive 
 reagents. When they are exposed to the action of gradually 
 increasing quantities of mineral or organic acids the power of 
 peptonisation disappears before the destruction of the rennet, 
 while when carbolic acid is used the reverse is the case. Vignal 
 has shown that rennet exists in Bacillus mesentericus vulgatus, 
 side by side with four other enzymes. Conn separated the 
 rennet from the proteolytic enzyme in some of these Bacilli by 
 a method resembling those already described. He mixed the 
 secretion of the microbes with TnilV and allowed coagulation to 
 take place. The clot was then broken up, shaken well with 
 distilled water and the whole filtered through porcelain. Sul- 
 phuric acid was added till the liquid contained "1 per cent., and 
 it was then saturated with sodium chloride. A white granular 
 looking scum floated to the surface, which on removal was 
 found to be almost pure rennet, the proteolytic enzyme being 
 left in solution in the brine. Conn found that the rennet 
 enzyme was secreted most plentifully by the microbes when the 
 culture of the bacillus was carried out at about 20° C, while 
 the proteolytic one preponderated if the temperature was main- 
 tained at about 35° C. 
 
 The action of rennet is most probably proteolytic, but the 
 details of its action are still very obscure. That it splits up 
 the caseinogen seems probable when we consider the coincident 
 appearance of the whey proteid and the casein. Neither of 
 these bodies however falls into Une with the course of proteolysis 
 
XV] VEGETABLE EENNET. 265 
 
 carried out by pepsin or trypsin, and the relation of the two 
 groups of enzymes must at present be left undecided. Nothing 
 at all is known at present as to its action in the course of 
 vegetable metabolism. Its constant occurrence in the stomachs 
 of young animals whose chief food is milk points to its being 
 of use in the preliminary digestion of proteid, but why the clot 
 of rennet should be more beneficial from this point of view than 
 a precipitation of the caseinogen by the acid of the gastric juice 
 is obscure. It may be however that the combination of proteids 
 with small quantities of free acid which is known to take place 
 in the stomach would delay the acid precipitation so long 
 that a certain quantity of the milk might make its way out 
 of the stomach into the intestine unchanged. 
 
266 
 
 CHAPTER XVI. 
 
 THE CLOTTING ENZYMES (continued). 
 
 THEOJIBASE, {tUEOMBIN, PLASMASE), THE FIBRIN-FERMENT. 
 
 The phenomena which are noticeable when mammalian 
 blood, freshly drawn from an animal, is collected in suitable 
 vessels, have received the name of coagulation. The blood, 
 preferably drawn from an artery, leaves the blood-vessel in a 
 perfectly fluid condition ; after an exposure of a minute or two 
 to contact with the sides of the collecting receptacle it becomes 
 viscid and gradually sets into a firm jelly. After some hours 
 a yellow liquid exudes in drops from the surface, and gradually 
 increases in quantity till the jelly, now become much firmer, 
 is floating in a considerable bulk of the liquid. The exudation 
 of this fluid, known as serum, is due to a shrinkage of a 
 certain constituent of the jelly which thus squeezes out of itself 
 the liquid part, leaving a firm clot of a leathery consistency. 
 This clot is of a deep red colour, owing to the presence in it 
 of the red corpuscles of the blood, which are mechanically 
 entangled in it owing to the rapidity with which the change 
 sets in. 
 
 It was originally supposed that the coagulation or formation 
 of the clot was due to a mechanical coherence of the corpuscles, 
 which can be seen to run together and form rouleaux when a 
 drop of blood diluted with water or a weak solution of common 
 salt is examined under the microscope. It was first shown by 
 Hewson in 1772 that the process is independent of the presence 
 of the red corpuscles and is due to the separation of a peculiar 
 fibrous-looking substance from the plasma or liquor sanguinis. 
 
CH. XVl] THROMBASE (THE FIBRIN-FERMENT). 267 
 
 This substance is now called fibrin, and coagulation is known 
 to resiilt from its formation from some antecedent sub- 
 stance which exists in the fluid portion of the blood. The 
 separation of this antecedent of fibrin was attempted in 1859 
 by Denis. He saturated the liquor sanguinis with sodium 
 chloride and obtained a sticky precipitate of a proteid nature. 
 When this was separated from the liquid and redissolved in a 
 dilute solution of the same salt, the solution underwent coagula- 
 tion, in the same way as normal blood. _ Denis called his 
 precipitated proteid-matter plasmine. A little later Schmidt 
 showed that certain serous fluids that would not clot spon- 
 taneously could be made to do so by adding serum to them. 
 From one of these, hydrocele fluid, he prepared by Denis' 
 method a proteid which he called fibrinogen. The serum which 
 set up the coagulation in the hj'drocele fluid jdelded another 
 proteid when similarly saturated. Schmidt called this fibrino- 
 plastin. Neither serum nor hydrocele fluid will clot spon- 
 taneously, nor will solutions of the precipitates obtained from 
 them in this manner. The mixed liquids or the mixed solutions 
 of their saturation precipitates will however give a true 
 coagulum. Schmidt found that the formation of the clot 
 depended on the presence of a third factor which was present 
 with his fibrino-plastin when prepared by saturation of the serum 
 with salt. When this proteid was prepared by passing a stream 
 of CO2 through considerably diluted serum, its solution did not 
 always cause clotting when added to a solution of fibrinogen. 
 Schmidt held that the presence of this third body was necessary 
 for the interaction or union of the other two. He sought 
 for it in the serum, and in 1872 separated it in a crude 
 condition by precipitating the proteids of the serum with a 
 large excess of alcohol. The precipitate was allowed to stand 
 under alcohol for a long time till the proteids were rendered 
 almost entirely insoluble ; it was then separated by decantation 
 or filtration and dried. A watery extract of the powder so 
 produced was found to be capable of bringing about coagulation 
 in such of his preparations of fibrinogen as would not clot 
 spontaneously. Schmidt gave this third factor the name of 
 fibrin-ferment. His fibrino-plastin was subsequently named 
 
268 THE CLOTTING ENZYMES. [CH. 
 
 paraglobulin or sei-um-globulin, its reactions showing that like 
 fibrinogen it was a member of the globulin class of proteids. 
 Schmidt explained coagulation by saying that these two glo- 
 bulins interacted with each other under the influence of the 
 fibrin-ferment to form fibrin. 
 
 Some years later, Hammarsten showed that the presence 
 of serum-globulin is not necessary for the formation of fibrin, 
 but that the part which it takes in the process can be sustained 
 by other substances, such as calcium chloride and impure 
 casein. Serum-globulin itself when pure is without effect 
 He was thus led to the view that the formation of fibrin is due 
 to the action of the fibrin-ferment upon fibrinogen only, under 
 certain favourable conditions, which are usually realised in the 
 presence of serum-globulin. 
 
 The process of the coagulation of shed blood is thus found 
 to be a ferment action, and the fibrin-ferment has within the 
 past twenty years been the subject of much careful investigation. 
 Its behaviour indicates that it belongs to the great group of 
 enzymes, more closely approaching rennet than any other so far 
 discovered. It shows the same dependence upon temperature 
 as the other enzymes, having a minimum activity at a very low 
 point, rising thence to an optimum at about 38° — 40° C, and 
 being destroyed at about 50° C. It does not apparently enter 
 into the reaction by uniting with fibrinogen, nor does it undergo 
 destruction in the course of its activity. The decomposition 
 it sets up is not certainly known, but it appears to behave after 
 the manner of rennet, decomposing the fibrinogen with the 
 formation of an insoluble proteid, fibrin, and a soluble one of 
 globulin character which remains in solution. This decompo- 
 sition recalls the formation of casein and the whey proteid. 
 
 We have seen that the fibrin ferment, which has recently 
 been named thrombose, or thrombin, was originally prepared by 
 Alexander Schmidt from defibrinated blood or from the serum 
 which separates out from a normal clot. This is not however its 
 only possible source, for it may be obtained with equal ease and 
 in much greater amount from the fibrin which is formed. An 
 indication of this fact was first obtained in 1835 by Buchanan, 
 who found that both hydrocele and pericardial fluids, which ai-e 
 
XVl] THROMBASE (THE FIBEIN-FERMENT). 269 
 
 not spontaneously coagulable, would produce a clot on the 
 addition of some fibrin. Buchanan diluted blood with a con- 
 siderable quantity of water, thereby delaying the process of 
 coagulation, and causing the clot when formed to be of a loose 
 fibrous character. The fibrin could be removed from the blood 
 as it was formed by stirring the latter constantly with a bundle 
 of twigs to which the fibrin adhered. Buchanan called his 
 preparation "washed blood clot.'' Many years later Garages 
 ascertained that the washed blood clot when extracted with 
 a solution of sodium chloride containing about 8 per cent, of 
 the salt, yielded a very active ferment. Subsequently other 
 observers showed that normal fibrin, obtained from blood with- 
 out dilution, yielded an equally active extract. The thrombase 
 can consequently be prepared from the fibrin of the clot or from 
 the serum which exudes from it after standing. 
 
 Thrombase does not exist in normal blood before coagulation 
 has taken place. If blood is shed from a vein or artery directly 
 into alcohol and subsequently treated in the way Schmidt 
 treated his serum or defibrinated blood, the solution or extract 
 of the precipitated proteids will not cause coagulation of serous 
 fluids. The appearance of the thrombase appears to be almost 
 coincident with the act of coagulation. Schmidt stated 
 that just at this moment there occurs a disintegration of the 
 colourless corpuscles or leucocytes, and suggested that this was 
 the decomposition which gives rise to the enzyme. This is 
 supported by an earlier observation of Buchanan that when 
 coagulation is retarded to such an extent that the red corpuscles 
 sink to the bottom of the liquid before it takes place, the 
 supernatant plasma, known as the huffy coat, yields a more 
 active " washed-blood-clot " than the rest of the blood. This 
 bufify coat contains the greatest abundance of the leucocytes, 
 which sink much more slowly than the red corpuscles. He 
 therefore concluded that the power of the washed clot to set up 
 coagulation depended on its cellular elements. 
 
 The nature of the fibrin-ferment as obtained from the two 
 sources mentioned does not appear at first sight to be the same. 
 When prepared by Schmidt's method it seems improbable that 
 it is of a proteid nature, as all the proteids of the blood he used 
 
270 THE CLOTTING ENZYMES. [CH. 
 
 may be supposed to be coagulated and rendered insoluble by a 
 prolonged exposure to the action of the alcohol. As extracted 
 from fibrin however it appears likely to belong to the class of 
 globulins, proteids which are insoluble in water, but soluble in 
 solutions of sodium chloride or other neutral salts. There is 
 reason to think however that the difference is more apparent 
 than real, for Schmidt says he never obtained his ferment free 
 from proteids, and he found it most active when the admixture 
 with proteid was greatest. It is not difficult to suppose that 
 his proteids were not completely rendered insoluble by the 
 alcohol. He says moreover that he was never able to obtain 
 his fibrino-plastin (serum globulin) free from ferment. 
 
 These considerations led most observers to entertain the 
 opinion that the ferment was proteid in nature and was closely 
 associated with the serum-globulin. 
 
 On the other hand, the globulins are always precipitated or 
 coagulated by being heated. Heat coagulation of a proteid 
 must not be confused with the coagulation of the blood or of 
 milk, as it only indicates a conversion of the proteid into a 
 peculiarly insoluble form and not the formation of a clot. It 
 is a little unfortunate that the same term is applied to two 
 different processes. When an albumin or a globulin in solution 
 is heated to a temperature usually about 70° C, it is converted 
 into the insoluble body in question, which is known as coagulated 
 proteid. Schmidt says that when aqueous solutions of his fer- 
 ment were boiled they did not throw down any of this proteid, 
 though the specific action of the enzyme was destroyed at a 
 temperature of 70° C. The same observation was niade by 
 Sheridan Lea, in conjunction with the writer, in the course of 
 some experiments made upon the preparation of thrombase 
 from fibrin. 
 
 The view that the enzyme is a proteid was very strongly 
 urged by Halliburton, who prepared it from the leucocytes of 
 the lymphatic glands. He associated it with a particular 
 proteid, much resembling serum-globulin, but differing from the 
 latter in a few particulars, one of which is its heat-coagulation 
 point. The change on heating in 5 — 10 per cent, sodium 
 chloride solutions takes place at 60 — 65° C, while serum- 
 
XVl] THROMBASE (THE FIBRIN-FERMENT). 271 
 
 globulin is not affected below 75° C. Halliburton originally 
 called this proteid "cell-globulin." 
 
 The probability that the serum-globulin and the ferment 
 are distinct is confirmed by Hammarsten's statement that he 
 obtained from hydrocele fluid, which is not spontaneouly coagu- 
 lable, a pure paraglobulin, (serum-globulin) which was free from 
 ferment and which had no fibrino-plastic activity. 
 
 In serum obtained from a normal clot these two globulins 
 exist together, and the presence of two rather than one escaped 
 detection until Halliburton's researches. 
 
 More recently Halliburton has found reason to doubt that 
 his cell-globulin is a true proteid, as it leaves on gastric 
 digestion a residue of nuclein, a body containing phosphorus. 
 In his latest work he includes it in a group of bodies allied to 
 proteids and known as nucleo-proteids. Pekelharing has come 
 independently to the same view. Halliburton says further that 
 when thrombase is prepared according to Schmidt's method, 
 it gives reactions which show that it belongs to this group. 
 Thrombase is therefore a nucleo-proteid, or is associated very 
 closely with such a body. 
 
 The story of the coagulation of shed blood is however much 
 more complicated than would appear from what has so far 
 been advanced. It is found that by many methods it may be 
 very greatly delayed or altogether inhibited. It may be hin- 
 dered considerably by keeping the shed blood at a very low 
 temperature, the blood of the horse being peculiarly susceptible 
 to this influence. If the blood when shed is at once mixed 
 with strong solutions of various neutral salts it will remain 
 fluid for a very long time, the most efficient salts being the 
 sulphates of magnesium or sodium. On copious dilution, so as 
 to make the percentage of salt very small, coagulation slowly 
 takes place. If a certain amount of commercial peptone, which 
 is very largely mixed with proteoses, is injected into the veins 
 of a dog previously narcotised with chloroform, and the blood 
 then withdrawn from the animal, it remains fluid indefinitely. 
 In some cases the same result follows when solution of peptone 
 is added to the blood as it is being shed. If an extract pre- 
 pared from the head and pharynx of the common leech is mixed 
 
272 THE CLOTTING ENZYMES. [CH. 
 
 with shed blood, the process of clotting does not take place. 
 This extract contains a peculiar proteose. Other methods of 
 maintaining its fluidity when shed will be referred to subse- 
 quently. 
 
 Blood kept fluid by such methods yields thus different 
 plasmas which are not spontaneously coagulable but which by 
 different treatment can be made to yield a clot. These are 
 generally referred to as "salted plasma," "peptone-plasma," 
 "leech-extract plasma," etc. 
 
 The influence of the inorganic salts present in the liquor 
 sanguinis must be taken into account in the study of the 
 phenomena of coagulation. If blood is kept fluid by any of 
 the methods in vogue and dialysed till quite free from salts, no 
 coagulation can be induced. Of all the salts present in the 
 plasma, the compounds of calcium invite particular attention. 
 It was first demonstrated by Brucke that the ash of fibrin 
 always contains this metal in some combination. The writer 
 found in 1884 that fibrin prepared by whipping blood always 
 contains a certain quantity of calcium sulphate which can be 
 extracted by appropriate means. As already stated Ham- 
 marsten showed that calcium chloride could take the place of 
 paraglobulin in aiding the action of thrombase. 
 
 The writer found in 1885 that when a salted plasma was 
 diluted so that its normal coagulation took place in about an 
 hour, it could be made to clot in a few minutes by the addition 
 of a few drops of calcium sulphate solution. The salted plasma 
 itself of course contained some of the enzyme, the action of 
 which was temporarily inhibited by the magnesium sulphate 
 used. Addition of calcium sulphate to a serous fluid such as 
 hydrocele which contains no ferment caused no coagulation. 
 
 The conclusion was obvious, that in some way or other the 
 thrombase was dependent upon a calcium salt, possiblv the 
 sulphate, for its manifestation. 
 
 This conclusion was borne out by depriving horse's blood of 
 its salts by a long process of dialysis, which was carried out in 
 tubes surrounded by ice. When the blood of the horse is 
 maintained at a temperature of 0" C. as already stated, coagu- 
 lation may in favourable cases be indefinitely postponed. After 
 
XVl] THEOMBASE (THE FIBRIN-FERMENT). 273 
 
 a long period of dialysis all the salts were removed except 
 sodium chloride, the presence of which is necessary to keep 
 the globulins from precipitation. This was therefore kept in 
 the blood by causing the dialysis to take place into '6 per cent, 
 sodium chloride solution. Such plasma removed from the ice 
 and subjected to the temperature of the body refused to clot, 
 either with or without dilution. On the addition of a little 
 solution of calcium sulphate solution clotting speedily took 
 place. Precipitation of calcium sulphate from the blood by the 
 addition of barium chloride materially hindered coagulation 
 but did not altogether prevent it. It seemed from these 
 experiments probable that calcium sulphate was a necessary 
 factor in the process. Its removal by barium chloride was not 
 complete. 
 
 In 1888 Arthus and Pages discovered that coagulation may 
 be almost entirely prevented by mixing the blood with a 0'2 
 per cent, solution of potassic oxalate. This reagent forms an 
 insoluble compound with calcium salts and thereby removes 
 nearly all that metal from the liquid. It is much the best 
 precipitant, as no other throws the calcium out of solution to 
 so great an extent. 
 
 It is stated by Schafer that even oxalated plasma slowly 
 coagulates, so that even rendering the greater part of the 
 calcium insoluble does not entirely prevent its interaction with 
 fibrinogen. 
 
 Other observers, especially Ringer, have found that other 
 salts of calcium than the sulphate are able to cause coagulation 
 in salted plasmas. Of these may be mentioned the chloride 
 and the phosphate. Neither acts however so rapidly as the 
 sulphate. Whether the latter is the actually interacting body 
 must be left at present uncertain, but it is not impossible, for 
 the blood contains sulphates of other metals, which would 
 cause the formatiob of calcic sulphate on addition of any other 
 soluble calcium salt. The occurrence of calcium sulphate in 
 fibrin may be remembered in this connection. 
 
 The theory of coagulation already stated needs amplifying 
 in the light of these facts ; the conversion of fibrinogen into 
 fibrin occurs only under the influence of certain salts of calcium. 
 a. F. 18 
 
274 THE CLorriNG enzymes. [ch. 
 
 The part played by the salt of calcium is at present still ob- 
 scure. When we consider that calcium is always present in the 
 ash of fibrin it seems at first sight very possible that the latter 
 is produced by the union of the fibrinogen and either calcium 
 or some compound of it, and that the work of the thrombase is 
 to secure their combination. But experiment and analysis 
 alike negative this supposition. With a considerable quantity 
 of fibrinogen in a solution the weight of fibrin formed does not 
 vary with the amount of calcium salt introduced. A very 
 small quantity induces coagulation, and a larger quantity does 
 no more. Hammarsten has shown recently that fibrinogen and 
 fibrin both contain the same amount of calcium, so that the 
 latter cannot be a calcium compound of the former. 
 
 From the experiments already referred to the writer was 
 led to advance the view that the thrombase might exist in the 
 blood in the form of a zymogen, and that under the influence of 
 the calcium salt it might be converted into the active enzyme. 
 He was unable however to obtain any evidence in support of this 
 hjrpothesis. More recently the same view has been put forward 
 by Pekelharing, who has been able to bring forward some 
 experiments which tend to establish its accuracy. He has 
 prepared from various foiins of extra-vascular plasma a sub- 
 stance which possesses no fibrino-plastic properties, but which 
 by treatment with a salt of calcium, acquires the power of 
 setting up coagulation in solutions of fibrinogen. This sub- 
 stance appears to be identical with Halliburton's so-called " cell- 
 globuHn," which is really a nucleo-proteid, as already shown. 
 Hammarsten also has shown that the nucleo-proteid can be pre- 
 pared firom plasma, and that it is incapable of acting as a fer- 
 ment until it has been exposed to the action of soluble salts of 
 calcium. Pekelharing has suggested that the interaction of 
 the calcium salt with the nucleo-proteid zymogen, which has 
 been called prothrombin, is really a union of the two, but 
 this view has not at present met with general acceptance by 
 physiologists. 
 
 Thrombase is thus seen to possess the properties of an 
 enzyme. Its activity shows the same dependence on tempera- 
 ture that has been noted in the cases of the other soluble 
 
XVl] THROMBASE (THE FIBRIN-FERMENT). 275 
 
 ferments which have so far been discussed. A very small 
 quantity will induce clotting in a very large amount of blood, 
 the time it takes to bring about the change being approxi- 
 mately in inverse ratio to the amount present. It exists in 
 the living body in the state or condition of a zymogen, but it is 
 peculiar in its mode of conversion from the latter, calcium in 
 one or other of its combinations being necessary for the trans- 
 formation. The reason why the interaction of the two does not 
 normally take place in the living blood-vessels has not been 
 satisfactorily ascertained. 
 
 Thrombase is peculiar in many ways. It shows a very 
 great susceptibility to the influence of certain neutral salts. 
 Its action is impeded by the sulphates of magnesium and 
 sodium, and to a less degree by the chloride of the latter metal. 
 Coagulation is almost entirely inhibited by soluble oxalates, 
 but on the other hand it is aided conspicuously by the presence 
 of salts of calcium, especially the sulphate ; indeed the action 
 of the enzyme is only possible in their presence. Whether all 
 these affect the formation of thrombase from its zymogen, or 
 whether they influence rather its activity, are points which 
 are still matters of controversy. We have seen reason to 
 believe that the salts of calcium are concerned in the forma- 
 tion rather than the working of the enzyme. 
 
 According to Dastre a neutral reaction is the most favour- 
 able for the exhibition of its activity. Indeed he says that 
 when working with any artificial plasma, neutralisation always 
 accelerates coagulation. Hammarsten found that the neu- 
 tralisation of many serous transudations not spontaneously 
 coagulable, often led to the formation of a clot. 
 
 The action of thrombase is inhibited by certain organic 
 bodies, of which the chief are certain albumoses or peptones. 
 The secretion of the buccal glands of the leech also prevents its 
 action. 
 
 The reason of the inhibitory effect of some of these bodies 
 has been ascertained. The soluble oxalates form a compound 
 with the calcium which is insoluble and which consequently 
 withdraws this metal in great part from the solution. Schafer 
 has shown that this removal does not in all cases prevent the 
 
 18—2 
 
276 THE CLOTTING ENZYMES. [CH. 
 
 ultimate clotting of the plasma. The interaction however be- 
 tween the oxalates and the soluble calcium salts does not effect 
 the total removal of the latter. When two soluble salts which 
 can exchange their metals with one another are mixed in a solu- 
 tion, the interchange between them is never complete, but takes 
 place up to a certain point at which a dynamical equilibrium is 
 reached between all four of the possible combinations. This 
 point will vary according to the relative amounts of the 
 originally interacting bodies, but the interchange is never 
 complete unless one of the new products is withdrawn as 
 fast as it is formed. Even on the addition therefore of consider- 
 able excess of the oxalates, some soluble salt of calcium will 
 remain. This agrees with Schafer's observation that the onset 
 of coagulation is very materially postponed but not altogether 
 prevented. 
 
 The action of peptone and leech-extract is considered by 
 some physiologists to be of the same order. They hold that 
 these bodies enter into combination with the calcium salts, and 
 so prevent the formation of the enzyme. It is noteworthy that 
 if a stream of carbon-dioxide is passed through a peptone- 
 plasma which is not spontaneously coagulable, it soon clots. 
 The carbon-dioxide appears to break up the union between the 
 peptone and the calcium salt, and so restores the conditions 
 necessary for clotting. Addition of more calcium salt to the 
 peptone-plasma will, in like manner, enable coagulation to take 
 place. 
 
 Other physiologists, with less probability, attribute to the 
 peptone or albumose a power of destroying the enzyme alto- 
 gether. Dickinson claims that this is undoubtedly the effect 
 of the leech-extract. 
 
 It is not possible at present to explain the inhibitory effect 
 of magnesium and sodium sulphates. It seems probable that 
 they interfere in some way with the enzyme and not with the 
 calcium salt. Sodium chloride in excess may be supposed to- 
 act like the oxalates, but much less completely, calcium chloride 
 being soluble. 
 
 A further condition of the action of thrombase is that the 
 blood shall be withdrawn from the body. In some way or 
 
XVl] THROMBASE (THE FIBRIN-FERMENT). 277 
 
 other the blood in the living vessels, or the vessels themselves, 
 either destroy it or altogether inhibit its action. Most ob- 
 servers have found that the injection of a powerful ferment- 
 extract into the veins of a living animal does not produce any 
 intravascular clotting, even if considerable quantities are used 
 and if soluble salts of calcium are simultaneously introduced. 
 Halliburton says that if very large quantities of thrombase are 
 injected into the vessels, the blood coagulates more rapidly than 
 normal blood when it is subsequently withdrawn. There appears 
 to be a destruction of the enzyme on its introduction into the 
 body. Such a destruction moreover may be normally taking 
 place in the blood. We have already seen that one source of 
 the enzyme is the leucocytes of blood or lymph. Schmidt was 
 of opinion that the cause of its formation was the disintegration 
 of these leucocytes, which he found to take place as soon as 
 blood is shed. As these cells are continually being decomposed 
 in the living body, we must suppose that they do not under 
 these conditions form thrombase, or that it is destroyed as soon 
 as formed, or that the living blood or the vessels in which it 
 flows prevent the action of the enzyme on the fibrinogen. 
 The absence of coagulation cannot be due to a deficiency of 
 calcium salts, for the mere shedding of the blood cannot affect 
 the amount of these which it contains. The observation of 
 Schmidt that when normal blood is allowed to flow into alcohol, 
 no enzyme can be extracted from the precipitate which is 
 formed in the spirit, suggests that an actual destruction of 
 thrombase takes place in the blood-vessels. 
 
 The action of thrombase on fibrinogen may be compared 
 with that of rennet on caseinogen. In both cases we have the 
 formation of a very insoluble body, which constitutes the solid 
 matter of the clot, together with the appearance of a soluble 
 proteid which appears to be formed simultaneously. Denis 
 found that when a solution of his "plasmine" coagulated, a 
 new proteid substance was present in the solution. He gave it 
 the name of " fibrine soluble," to distinguish it from the normal 
 fibrin of the clot. Hammarsten has observed that the decom- 
 position is attended by the appearance of this new body, which 
 he says is a globulin coagulating when heated to 64° C. More 
 
278 THE CLOTTING ENZYMES. [CH. 
 
 recently Arthus has confirmed the observation. He finds thaf, 
 in an oxalated plasma the weight of fibrin produced by the 
 addition of a soluble salt of calcium is less than the weight of 
 the fibrinogen which can be obtained by heating an equal 
 volume of the plasma to the point at which this proteid is 
 precipitated, and is therefore considerably less than the total 
 weight of the fibrinogen present, for many observers have 
 noticed that this proteid does not all separate out when its 
 solutions are heated to 56° C. If this observation is correct, 
 it follows that in the formation of fibrin fi-om fibrinogen there 
 must be a splitting or decomposition of the latter. Coagulation 
 cannot therefore be due to a combination of fibrinogen with 
 calcium ; a conclusion which we have seen already to be reached 
 by a different method of experiment. 
 
 Hammarsten holds that the change in the fibrinogen is not 
 one of splitting into two other proteids, but is rather one of 
 intro-molecular rearrangement. 
 
 Besides being obtainable fi:om the serum and fibrin of shed 
 blood, thrombase can be prepared firom many of the tissues of 
 the body. Halliburton has extracted it firom leucocytes fi:om 
 lymphatic-glands, firom the glands themselves, fi:om the thymus, 
 and fi:om the stromata of the red corpuscles. He says that a 
 proteid which can be obtained firom muscle-juice, to which he 
 has given the name myosinogeii, has precisely the same action 
 on diluted salted plasma as his cell-globulin or nucleo-proteid 
 has, though he hesitates to identify it with fibrin-ferment. 
 Buchanan found that many of the tissues of the body pos- 
 sessed the power of setting up clotting in serous fluids that 
 were not spontaneously coagulable. He instances muscle, 
 connective tissue, and certain parts of the central nervous 
 system. Pekelharing has prepared a nucleo-proteid firom muscles 
 by macerating them in a '25 per cent, solution of sodium 
 carbonate, and adding acetic acid to the extract till a precipitate 
 falls. He says this causes coagulation of the blood if it is in- 
 jected into a vein. 
 
 The question of intravascular clotting and its relation to 
 thrombase is still very obscure. Though most observers agree 
 that the injection of a very active preparation of the ferment 
 
XVl] THROMBASE (THE FIBRIN-FERMENT). 279 
 
 into the blood-vessels of an animal is not followed by coagulation 
 of its blood, some exceptions have been noticed. 
 
 Edelberg has shown that intravenous iajection of Schmidt's 
 fibrin-ferment may produce coagulation of the blood in the 
 vena cava, the right side of the heart, and the pulmonary 
 arteries. General intravascular clottiag may be caused by the 
 injection into the vessels of certain proteids which were first, 
 prepared by Wooldridge and named by him " tissue fibrinogens." 
 These now appear to be nucleo-proteid bodies, comparable with 
 Pekelharing's product obtauied fi-om muscle, and perhaps also 
 with the prothrombin which can be prepared firom plasma and 
 other sources as already described. If these bodies are injected 
 rapidly in large quantity, almost instantaneous coagulation of 
 the blood takes place. If on the other hand the injection is 
 carried out slowly and not much of the nucleo-proteid is in- 
 troduced, the blood becomes less coagulable. The nature of the 
 changes which are thus set up and their relation to the form- 
 ation or the action of thrombase need further investigation. 
 Schafer suggests that when these nucleo-proteids are either 
 naturally formed in circulating blood, or are artificially in- 
 jected into the vessels, they tend to interact at once with the 
 calcium salts of the plasma and to form thrombase, intravascular 
 coagulation being the result. It is difficult however to recon- 
 cile this with the observation that the injection of active 
 thrombase into the vessels generally fails to produce such 
 coagulation. 
 
 Lilienfeld has also associated nucleo-proteids with the phe- 
 nomena of coagulation. He holds that a somewhat complex 
 substance to which he gives the name of nucleo-histon is one of 
 the products of the destruction of the leucocytes of the blood, 
 and that this itself is capable of decomposition into two other 
 substances one of which he terms leuco-nuclein and the other 
 histon. The latter possesses anti-coagulant properties but the 
 leuco-nuclein aids coagulation by splitting fibrinogen into two 
 new proteids, one of which, called by him thrombosin, becomes 
 fibrin by combining with a calcium salt. The action of the 
 leuco-nuclein he attributes to the nucleic acid it contains. Other 
 acids, particularly acetic, are said by him to have the same action. 
 
280 THE CLOTTING ENZYMES. [CH. 
 
 If a solution of pure fibrinogen, prepared by precipitating it 
 from blood plasma by semi-saturation with sodium chloride, is 
 dissolved iu a dilute saHne solution and acetic acid is added, 
 a precipitate is formed which is Lilienfeld's thrombosin. A 
 solution of this in a dilute solution of sodium carbonate yields 
 fibrin on the addition of some calcium chloride. 
 
 Lilienfeld's conclusions have not met with entire acceptance. 
 They have been criticised particularly by Schafer, who has 
 repeated his experiments without entirely confirming his results. 
 Schafer is of opinion that the so-called thrombosin is only 
 fibrinogen. 
 
 Crustacean Fibrin-ferment. 
 
 A coagulation which somewhat closely resembles that of the 
 blood of Vertebrate animals can be observed to take place in 
 the hsemolymph of many of the decapod Crustacea, the most 
 convenient animals for examination being the lobster {Homarus 
 vulgaris), the crab (Garcinus memos), and the marine and fi-esh- 
 water cray-fishes. The fluid in question, which is often spoken 
 of as blood, is of a very pale red colour or nearly colourless. It 
 is somewhat opalescent fi'om the presence of numerous amoeboid 
 corpuscles. 
 
 When this blood or hsemolymph is shed fi-om the animal 
 and collected into a vessel it very quickly forms a clot in which 
 the appearance of a network of fibres can be traced. The clot 
 very soon contracts and squeezes out a clear fluid, which ia 
 a few more minutes sets into the form of a jelly. The process 
 of coagulation appears to take place in two stages, the first clot 
 only being fibrous. The translucency of the second jelly-like 
 coagulum shows that the corpuscles of the blood are entangled 
 in the first fibrous formation. After a few hours the second 
 clot also shrinks and squeezes out a liquid which has no 
 further power of coagulatiug, but which is the serum of the 
 blood. 
 
 It was originally held that the two clots had different 
 origins; that the first was made up of the corpuscles, which 
 were thought to coalesce together after the manner of the 
 
XVl] CRUSTACEAN FIBKIN-FEEMENT. 281 
 
 Plasmodium of a Myxomycetous fungus, while the second was 
 due to the formation of a true fibrin, identical with that 
 produced in the blood of the Vertebrata. This view was 
 advanced by Fredericq and supported by Geddes and other 
 writers. 
 
 Other observers suggested that the second process was only 
 a continuation of the first and that both were due to a normal 
 formation of fibrin, the chief difference between the clots being 
 that the earliest-formed fibrin entangled the cells or leucocytes 
 in its fibrils as they were produced. The accuracy of this view 
 was established in 1885 by Halliburton, who made a very 
 complete examination of the blood or hsemolymph and the 
 phenomena of its coagulation. 
 
 The blood contains two principal proteids, one of which is 
 a form of fibrinogen which differs but little fi:om the fibrinogen 
 of the blood of the Vertebrata ; it possesses a somewhat higher 
 heat-coagulation point, remaining unaltered up to a tempera- 
 ture of 65° C. It is not precipitated by sodium chloride until 
 the liquid is saturated by the salt. Except for this peculiarity 
 it can be precipitated from its solutions in exactly the same 
 way as the fibrinogen of the higher animals. When extracted 
 from the blood and dissolved in a dilute salt-solution it will 
 undergo coagulation on the addition of a little thrombase, 
 prepared from the blood of a vertebrate animal. 
 
 The process of coagulation presents the same features and 
 peculiarities which are found in connection with the same 
 phenomena in the blood of the Vertebrata. 
 
 It can be delayed or prevented by appropriate addition of 
 neutral salts such as magnesium sulphate to the blood as it is 
 shed. Usually a considerable quantity of the salt must be 
 employed. It is affected by cold in the same way as the 
 coagulation of the blood of a vertebrate animal. 
 
 The correspondence in behaviour with ordinary salted 
 plasma, and especially the reaction with thrombase, suggests 
 that the coagulation of crustacean blood is due to an enzyme, 
 and the first occurrence of the fibrin in direct relationship with 
 the leucocytes points to the latter bodies as its probable 
 source. 
 
282 THE CLOTTING ENZYSIES. [CB^- 
 
 Halliburton made a series of experiments upon these points 
 and found that the results bore out the conclusions suggested. 
 He was able to prepare an enzyme from either the blood or 
 the serum separated out from it after coagulation. He used 
 Schmidt's method of preparation, and found that either the 
 powdered precipitate or an extract of it was easily able to set 
 up coagulation in a solution of crustacean fibrinogen. He 
 ascertained moreover that the enzyme which he thus prepared 
 was so much like thrombase as to be able to cause coagulation 
 in a salted plasma prepared from the blood of a cat, and in a 
 solution of fibrinogen prepared from hydrocele fluid. 
 
 Halliburton says that the amoeboid corpuscles of the blood 
 are the source of the enzyme, but he does not quote any 
 experiments which definitely lead to that conclusion. Hardy 
 has made a very careful examination of the different kinds of 
 corpuscles which the blood of the Crustacea contains and among 
 them he finds some pale ones, oval in shape, which contain a 
 small number of fine granules. When these, which occur in 
 considerable numbers, are watched under the microscope they 
 show some very peculiar features. From each a number of fine 
 pseudopodia are shot out with some suddenness and little 
 portions of the protoplasm travel rapidly along them, and burst, 
 after swelling up to form a kind of vesicle. The pseudopodia 
 vary in number and in shape j indeed the protoplasm of the 
 surface of the cell sometimes swells and bursts, without any 
 formation of them at all. These changes are associated with 
 remarkable alterations of the nucleus of the corpuscle. Hardy 
 has called them "explosive corpuscles," and has associated the 
 formation of the fibrin-ferment with them, on account of the fact 
 that they are the only ones which are disintegrated at all rapidly 
 after the shedding of the blood. He says that there is a marked 
 correspondence in time between their solution and the solidifi- 
 cation of the plasma, and that substances such as very dilute 
 solutions of iodine, which keep the corpuscles from exploding, 
 delay in about the same degree the coagulation of the blood. 
 He calls attention to the fine granules described as appearing 
 in these cells, and shows that if the cells are prevented from 
 breaking up while the granules are allowed to dissolve out of 
 
XVl] MYOSIN-FERMENT. 283 
 
 them slowly, the onset of coagulation corresponds with the time 
 of the discharge of these granules from the corpuscles. There 
 is thus a certain amount of evidence that these explosive cor- 
 puscles secrete a form of fibrin-ferment, which is probably 
 identical with thrombase prepared from the higher animals. 
 
 The coagulation as we have said is seen to take place in two 
 stages, the first of which is almost instantaneous and results in 
 all the corpuscles being enclosed in threads of fibrin. This 
 must be due to the rupture of the explosive cells and the 
 liberation of their ferment, which causes the very rapid for- 
 mation of the fibrin, the coagulation taking place before 
 diffusion of the. liberated ferment can go on to any great 
 extent. The contraction of the fibrin threads of the clot 
 squeezes out what plasma has not coagulated and at the 
 same time the ferment spreads more fully through the liquid, 
 hence the second slower phase of the coagulation is seen. 
 Although the two coagula seem to the eye to be quite distinct 
 from each other it is very doubtftil whether the second process 
 is not a continuation of the first one without any break or 
 interruption, for the most careful efforts to extract the first clot 
 from the liquid before the formation of the second have been 
 entirely unsuccessful. 
 
 Myosin-ferment. 
 
 The formation of myosin, which is characteristic of the onset 
 of rigor mortis, is a phenomenon which presents many features 
 in common with the formation of fibrin in the blood. The 
 similarity of the two processes has been pointed out by Kuhne, 
 who worked with the muscles of frogs, and by Halliburton, 
 whose experiments were conducted on rabbits, cats and pigeons. 
 Both observers were able to prepare from muscles by strong 
 pressure a liquid or plasma which quickly became gelatinous 
 in consistency and formed a clot. On standing the clot shrank 
 and separated from a fluid comparable to serum. It was 
 necessary in making the experiments to carry out all the 
 operations at a very low temperature, scarcely above 0°0. 
 
 The two clots, obtained from blood and muscle plasma 
 
284 THE CLOTTING ENZYMES. [CH. 
 
 respectively, differ however considerably in appearance and in 
 properties. While that from blood is stringy and its fibrin 
 portion almost insoluble after its formation, that from muscle is 
 gelatinous and soft, and readily dissolves in salt solutions of 
 moderate concentration. The substance which is formed and 
 which is compaxable to fibrin is known as myosin. 
 
 Halliburton showed a further difference between the two in 
 that the salt solution of myosin can be made to clot a second 
 time with comparative ease. In some cases he found that the 
 process of redissolution and recoagulation could be repeated 
 three or four times. A salt solution extract of rigid muscles, 
 in which the myosin had been formed before thefr removal 
 from the body, behaved similarly. 
 
 When a muscle plasma is prepared by squee2ing it from 
 a fresh muscle caxefuUy freed from blood and manipulated at 
 0° C, it can be kept fluid for a long period in the same way as 
 blood plasma by admixture with neutral salts, magnesium or 
 sodium sulphate and sodium chloride being most advantageous. 
 If such a salted plasma is diluted, coagulation slowly takes place. 
 
 The coagulation is due to the action of an enzyme which is 
 present in the muscle, and which according to Halliburton can 
 be extracted from it by the following method : — 
 
 Muscle is allowed to undergo rigor and is then minced 
 finely and kept under absolute alcohol for a long time. The 
 small pieces are dried over sulphuric acid and powdered. An 
 aqueous extract of the powder contains the ferment. 
 
 If such an extract is added to a diluted salted plasma 
 which normally clots in 12 hours, the coagulation takes place 
 in less than one quarter of the time. Addition of the powdered 
 muscle substance produces a similar effect. 
 
 The myosin-ferment is associated with a proteid which has 
 the properties of a deutero-proteose, and which can be extracted 
 from the muscle plasma or from coagulated muscle kept for a 
 long time under alcohol. Halliburton is rather inclined to the 
 view that this substance is itself the enzyme, much as he 
 contends the nucleo-proteid or cell-globulin afready described 
 is the fibrin-ferment. 
 
 This enzyme is quite distinct from the fibrin-ferment, not 
 
XVl] MYOSIN-FERMENT. 285 
 
 being able to cause coagulation of salted blood plasmas ; nor can 
 fibrin-ferment set up clotting in muscle plasma. They differ 
 moreover in the temperature at which they are destroyed ; while 
 thrombase is decomposed at 70° — 80° C, the myosin-ferment is 
 not destroyed till a temperature of nearly 100° C. is reached. 
 
 The change which the ferment induces differs fi'om that 
 which is involved in the formation of fibrin. Muscle plasma 
 contains four proteids which can be converted into coagu- 
 lated proteid by heating. These have been investigated by 
 Halliburton, whose description of their reactions may be briefly 
 summarised as follows : 
 
 1. Paramyosinogen — a globulin coagulating on heating to 
 47° C; precipitated by magnesium sulphate of 50 per cent, 
 concentration or by 26 per cent, of sodium chloride. 
 
 2. Myosinogen — a globulin coagulating at 56° 0. ; precipi- 
 tated by 94 per cent, of magnesium sulphate or 36 per cent, of 
 sodium chloride. 
 
 3. Myoglobulin — a globulin coagulating at 63° 0. ; precipi- 
 tated by saturation of its solution with either magnesium 
 sulphate or sodium chloride. 
 
 4. Myoalbumin — an albumin coagulating at 73° C. and 
 not separated from the plasma by saturation with either salt. 
 
 On removal of these a deutero-proteose remains in the 
 solution. 
 
 When the serum fi-om the clot is examined it is found to 
 contain no proteid coagulating below 63° C. When the clot is 
 dissolved in dilute salt solution and the latter gradually heated. 
 coagula occur at 47° C. and at 56° C. 
 
 The clot so appears at first sight to consist of the two 
 myosinogens. But on separating them by fi-actional precipita- 
 tion with magnesium sulphate and dissolving them separately, 
 clotting only occurs in the solution of the second of them. 
 With care this myosinogen can be prepared quite free fi:om 
 ferment, when its solution remains fluid indefinitely. Addition 
 of ferment causes coagulation to set in. The myosin formed 
 in the clot of the normal plasma appears to entangle the 
 paramyosinogen in its substance. Paramyosinogen itself will 
 not clot either with or without addition of the enzyme. 
 
286 THE CLOTTING ENZYMES. [CH. XVI 
 
 The action of the latter is therefore, according to Halliburton, 
 to convert myosinogen into myosin. It is not a splitting like 
 the conversion of fibrinogen into fibrin, no second proteid being 
 formed coincidently with the clotting. There is, however, a 
 simultaneous formation of a form of lactic acid which appears 
 to arise from the same or an accompanying decomposition of 
 the proteid. The formation of the myosin is also attended by 
 the liberation of a considerable quantity of carbon dioxide. 
 
 Vesiculase. 
 
 Camus and Gley have stated that they have discovered 
 another enzyme of this class in the secretion of the prostate 
 glands of the guinea-pig, rat and mouse. The liquid coming 
 fi:om this gland is clear and limpid and has a neutral reaction. 
 When a drop of it is added to the semi-liquid contents of the 
 vesiculae seminales a coagulation sets in instantaneously. The 
 secretion of the prostate loses the coagulating property if it is 
 heated for 15 minutes to 70° 0. Camus and Gley give the 
 enzyme the name of vesiculase. The proteids of the vesicular 
 fluid have not been ascertained, but they do not appear to 
 include fibrinogen. 
 
287 
 
 CHAPTER XVII. 
 
 THE CLOTTING ENZYMES (continued). PECTASE. 
 
 The formation of the vegetable jellies which can be prepared 
 from so many ripe fruits is due to the action of an enzyme upon 
 a certain substance which can be extracted from many vegetable 
 tissues, in some cases from cell-walls, in others from the sap 
 contained in the cells. This substance, which is known as 
 pectine, is a member of a series of compounds which are very 
 widely represented in plants, being usually associated with 
 cellulose in the young cell-wall. The enzyme which is con- 
 cerned in its transformations was originally investigated by 
 Fremy in 1840. He says in his account of his researches 
 that he finds a substance in cell-walls, which differs in many 
 important respects from cellulose, and he gives it the name of 
 pectose : it is easily converted into a closely related body, pectin, 
 by the action of an acid. By the action of an enzyme, which he 
 terms pectose, and which he extracted from certain cells of 
 various plants, pectose or pectin can be converted into two 
 gelatinous bodies, pectosic and pectic acids. The transformation 
 takes place in two stages, the two acids being formed suc- 
 cessively. They differ from pectin chiefly in the amount of 
 water which they contain. Pectase according to his observations 
 exists in two conditions in the vegetable organism, sometimes 
 in solution in the neutral sap of such roots as the carrot or 
 beet, sometimes in an insoluble state, as in the juice of acid 
 fruits. If the juices or the pulp of these plants is added to a 
 solution of pectin, a rapid gelatinisation of the latter takes 
 
288 THE CLOTTING ENZYMES. [CH. 
 
 place and, as stated above, pectosic and later on pectic acids are 
 formed. Fremy says further that the enzyme can be prepared 
 from the juice of young carrots by precipitation with alcohoL 
 The solid precipitate possesses the ferment power, but will 
 not give it up to water. Its optimum working temperature 
 is 30° C, and it is destroyed by boiling. It can work in the 
 absence of oxygen. 
 
 Since Fremy's work was published the occurrence of pectic 
 compounds in plants has been the subject of careful investigation 
 by many observers. In particular Mangin has contributed sub- 
 stantially to our knowledge of the several members of the 
 group and their transformations under different reagents. The 
 peculiarities of the action of pectase have also been studied 
 and its distribution more completely determined. 
 
 The general properties of the pectic compounds show that 
 they differ considerably from the celluloses, though they have 
 often been confounded with them. They are uncrystallisable 
 bodies, which can be precipitated from their solutions by various 
 reagents, and then appear very often in a colloidal or gela- 
 tinous condition. They are however very rapidly altered by 
 the solutions used for their extraction. Fremy, Scheibler and 
 Beichardt considered them to be carbohydrates, allied to 
 mucilages and gums. Mangin holds that their reactions 
 separate them from the carbohydrate group; when oxidised 
 by dilute nitric acid they give rise to mucic acid, while the 
 carbohydrates are converted into oxalic acid. Pectic bodies 
 are insoluble in ammonio-cupric-oxide, which dissolves the 
 celluloses readily. The latter are coloured violet or blue by 
 iodine in the presence of sulphuric or phosphoric acids; the 
 pectic bodies yield no such coloration mth any combination 
 of iodine. 
 
 De Haas and Tollens from their analyses of pectine prepared 
 from various sources support the view of Fremy and others that 
 they are related to carbohydrates if not actual members of the 
 group. They show that they contain hydrogen and oxygen 
 approximately if not definitely in the proportion of two to one, 
 while their carbon amounts to about 43 per cent. Hydrolysis of 
 these pectines by dilute mineral acids yields sugars contaiuing 
 
XVU] PECTASB. 289 
 
 either five or six carbon atoms. Tollens suggests that they are 
 carbohydrates chemically combined with acids. 
 
 A great number of these pectic bodies have been identified 
 and described by various writers from 1825 onwards', and a 
 certain confusion has been unavoidable in comparing those of 
 one investigator with those described by another. Mangin's 
 memoirs have put the whole group upon a surer foundation. 
 
 The pectic compounds can now be arranged in two series, 
 one of the latter comprising bodies of a neutral reaction, while 
 those of the other are feeble acids. In each there are probably 
 several members which show among them every stage of physical 
 condition between absolute insolubility and complete solubility 
 in water, the intermediate bodies exhibiting gelatinous stages, 
 characterised by the power of absorbing water in a greater or 
 less degree. 
 
 Two of these bodies are of particular interest in connection 
 with the study of the enzyme. One, pectine, belongs to the 
 neutral, the other, pectic acid, to the acid series. According 
 to Mangin their reactions are the following : — 
 
 Pectine. This substance swells up and dissolves in water, 
 yielding a viscid liquid which is very difficult to filter and 
 which tends to gelatinise when its solutions become at all 
 concentrated. It is soluble also in dilute acids, from which it 
 can be precipitated by alcohol. It gives no precipitate with 
 neutral acetate of lead but is thrown down by the basic acetate 
 in the form of white flocculi. If boiled for several hours in water 
 it is converted into an isomer, parapectine, which is precipitated 
 by the neutral plumbic acetate. Further boiling with dilute 
 acids converts it into metapectine, which is precipitated by 
 barium chloride. 
 
 Pectic acid. This body is insoluble in water, alcohol, and 
 acids ; it forms soluble pectates with alkalis, and insoluble ones 
 with the metals of the alkaline earths, of which calcic pectate 
 is most widely distributed. It dissolves in solutions of alkaline 
 salts, such as the carbonates of sodium and potassium, stannates, 
 alkaline phosphates, and most organic ammoniacal salts, forming 
 
 1 For a r€sumS of these investigations see a paper by the writer on " The 
 Cell Membrane," Science Progress, Vol. vi. p. 344. 
 
 G. P. 19 
 
290 THE CLOTTING ENZYMES. [CH. 
 
 with them double salts, which gelatinise more or less freely 
 with water. Its solutions in alkaline carbonates are mucilagiaous 
 and difiScult to filter, while when oxalate of ammonia is the 
 solvent, the liquid remains perfectly fluid and filters readily. 
 
 Fremy's discovery of the enzyme has been confirmed by 
 more recent investigators, and the nature of the changes set 
 up by its action are now more fully known. 
 
 The most complete information upon the peculiarities of 
 pectase has been furnished by Bertrand and Mallevre, who 
 published several memoirs upon the subject in 1895 and the 
 following years. 
 
 Their first preparations of the enzyme were obtained fi-om 
 the juice of carrots, which were cultivated for the purpose and 
 gathered at the period of most vigorous growth. As already 
 mentioned, Fremy ascertained that the roots of these plants 
 contain pectase. 
 
 In its preparation Bertrand and Mallevre separated the 
 central cylinders of the roots from the cortex, and carefully 
 reduced them to pulp, afterwards extracting the sap by 
 pressure. About 70 — 80 per cent, of the bulk of the pulp 
 was thus squeezed out in the form of a turbid liquid, which 
 was then saturated with chloroform and filtered through 
 Bezelius filter-paper. 
 
 This juice when added to solutions of pectine quickly caused 
 the production of a jelly, just as Fremy had previously observed. 
 
 The change thus brought about was considered by Fremy 
 and his immediate successors to be the conversion of pectine 
 into pectic acid. The latter gelatinises more readily than 
 pectine and in solutions of less concentration. A perfectly 
 limpid preparation of pectine which shows no tendency to 
 gelatinise can be made to undergo the change readily, and 
 to set into a soft clot on the addition of a solution of 
 pectase. 
 
 But though confirming Fremy's work to the extent already 
 mentioned, Bertrand and Mallevre came to different conclusions 
 as to the nature of the product formed. They found that when 
 they dissolved the jelly in dilute hydrochloric acid, the solution 
 contained a certain quantity of calcium, which was not 
 
XVIl] PECTASE. 291 
 
 precipitated on saturation with ammonia. Continued investi- 
 gation satisfied them that the jelly was not pectic acid but a 
 compound of this body with calcium ; — in fact calcic pectate. 
 
 Further researches were directed towards ascertaining 
 whether the clotting was altogether dependant on the presence 
 of calcium. 
 
 A purified solution of pectase was prepared from the crude 
 one already described by adding to it a certain amount of the 
 oxalate of one of the alkali metals to precipitate any calcium 
 that the juice of the carrots might contain. After standing for 
 a time till all sediment had settled down it was again filtered, 
 when it yielded a clear liquid. 
 
 Pectine was then prepared from the residue left after the 
 expression of the juice from the pulpy tissue of the root. This 
 material was washed with alcohol and boiled for 1.5 minutes; 
 and the mixture filtered before allowing it to cool. 
 
 The debris on the filter was thus freed from any adherent 
 or soluble pectase, and was next extracted with a 2 per cent, 
 solution of hydrochloric acid, in which pectine is soluble. 
 Twenty-four hours' maceration was considered sufficient to 
 extract the latter substance; at the end of that time the 
 liquid was filtered from the residue and an equal volume of 
 alcohol was added to it. This precipitated the pectine in the 
 form of gelatinous flakes, which were collected and dried on a 
 porous surface. To ensure their freedom from calcium salts 
 they were then steeped in 50 per cent, alcohol containing 2 per 
 cent, of hydrochloric acid. This treatment was continued till 
 the pectine gave on incineration only a trace of ash which was 
 tree fi:om calcium salts. It was then freed from acid by 
 dissolving it in water, and then precipitating it with alcohol. 
 Repeated treatment in this way eliminated most of the acid, 
 and it was freed from the remainder by the addition of a few 
 drops of dilute caustic potash. 
 
 Bertrand and Mallevre found that when prepared in that 
 way a solution containing 2 per cent, of the pectine remained 
 limpid and was in the best condition for further experiments. 
 When they added to it the decalcified juice of carrots prepared 
 as described, it remained indefinitely liquid, though such 
 
 19—2 
 
292 THE CLOTTING ENZYMES. [CH. 
 
 decalcified solution contained large quantities of pectase. The 
 further addition of only a trace of a soluble calcic salt caused 
 the formation of a jelly, the time taken depending upon the 
 amount of calcium so added. The addition of a calcic salt 
 alone, or of a calcic salt and some boiled carrot juice, always 
 failed to induce the gelatinisation. 
 
 Barium and strontium were found to play the same part as 
 calcium, in cases where excess of either was added to the pectine 
 solution. Magnesium on the other hand had no action, or at 
 most a very feeble one. 
 
 The clot formed by the action of pectase on pectine ia 
 therefore composed of pectate of calcium and not free pectic 
 acid, as the earlier observers thought. 
 
 It will be remembered that, in the case of rennet, a similar 
 combination of calcium with caseinogen takes place, the casein 
 formed being according to Halliburton a caseate of lime. 
 
 Bertrand and Mallevre found that if a large quantity of a 
 soluble calcic salt was added to the solution of pectine a 
 gelatinisation took place without the presence of pectase. This 
 was not however due to the formation of pectic acid or calcic 
 pectate but to the production of another compound which they 
 called a pectinate. The latter was also produced simultaneously 
 with the calcic pectate if pectase was present and the calcium 
 salt was in excess. The two gelatinous bodies can be dis- 
 tinguished from each other by the action of a dilute acid, such 
 as a 2 per cent, solution of hydrochloric acid. This dissolves 
 the pectinate, so that the jelly due to the presence of the latter 
 disappears. It decomposes the calcic pectate by robbing it of 
 the calcium, and leaves free pectic acid, which is insoluble in 
 acids and consequently persists in the gelatinous condition. 
 
 Pectase is materially hindered in its working by the presence 
 of free acid, in fact a neutral medium is almost essential for the 
 production of the jelly. When a mixture of equal volumes of 
 the expressed juice of carrots and a 2 per cent, solution of 
 pectine is taken for experiment, the clot is usually formed 
 in about an hour. If hydrochloric acid is added to such a 
 mixture there is a considerable retardation, the longer as the 
 amount of acid is greater ; "09 per cent, delays the gelatinisation 
 
XVII] PECTASE. 293 
 
 for nearly two days, and -1 per cent, inhibits it entirely. The 
 same results have been observed with sulphuric, nitric, tartaric 
 and citric acids. 
 
 This observation is important, for it supplies a reason for 
 the non-production of jelly in the juice of many fruits, as they 
 contain besides the enzyme a larger percentage of acid than 
 •1 per cent. 
 
 The retarding influence of acid is lessened by the presence 
 of a larger proportion of calcium salts, or of pectase. This 
 explains why the juices of cherries and raspberries coagulate 
 pectine though they are fairly acid in reaction. The gelatini- 
 sation depends ultimately on the relative proportion of pectase, 
 calcium salts, and free acid present in the solution. 
 
 It has already been mentioned that Fremy denied the 
 existence of soluble pectase in acid fruits. He said that in 
 these the enzyme was present in an insoluble form and that it 
 was not separable from the solid pulp. He explained in this 
 way the fact that the latter would clot solutions of pectine, 
 while he failed to get the coagulation with the expressed juice 
 from such pulp. Bertrand and Mallevre repeated his experi- 
 ments with the expressed sap of such fruits and carried them 
 further. Though the juice as squeezed from the fruit will not 
 clot the pectine as Fremy said, they found it would do so readily 
 if a little alkali was added at the same time to neutralise the 
 acid present. The failure was therefore not due to the absence 
 of pectase, but to the presence of free acid which inhibited 
 its action. 
 
 These observers contradict Fremy on another point; they 
 have found that by prolonged maceration pectase can be ex- 
 tracted from the insoluble precipitate produced by alcohol from 
 the expressed juice of the carrot. It is not therefore rendered 
 absolutely insoluble by this treatment, as Fremy supposed. 
 
 Bertrand and Mallevre have discovered that instead of 
 pectase being confined only to such pulpy tissues as have been 
 mentioned, it has a very widespread distribution in the vege- 
 table kingdom. Indeed they go so far as to say that it is 
 universally present in green plants, being especially abundant 
 in foliage leaves, which they think are the seats of its formation 
 
294 
 
 THE CLOTTING ENZYMES. 
 
 [CH. 
 
 and from which they believe it spreads into the rest of the organs 
 of the plant. Leaves which show rapid growth are usually the 
 richest in the enzyme. 
 
 A very active preparation was obtained from the leaves of 
 Lucerne {Medicago sativa) and Trefoil (Trifolium pratense). The 
 plants were gathered when in the condition of most vigorous 
 growth and bruised in a mortar to extract the sap. This was 
 then saturated with chloroform and a flask filled with it, which 
 was allowed to stand for 12 — 24 hours in the dark. A consider- 
 able sediment settled down, which was removed by filtration. A 
 clear liquid was thus obtained which was poured into twice its 
 volume of alcohol of 90 per cent, strength, a copious precipitate 
 resulting. This was removed from the supernatant spirit and 
 macerated for 12 hours in a small quantity of water. The pectase 
 was thus dissolved out from the miscellaneous constituents 
 of the sap, and on filtering the solution a nearly colourless 
 liquid was obtained. The enzyme was again precipitated by 
 the addition of a large excess of alcohol, and was collected on 
 a filter and dried in vacuo. Using this method of preparation 
 a litre of the filtered sap yielded 5 — 8 grammes of a white 
 powder which was not hygroscopic, but dissolved readily in 
 water. Its solution possessed great power of inducing pectic 
 fermentation. A 1 per cent, solution of pectine was coagulated 
 in 48 hours by the addition of j^ of its weight of Lucerne 
 pectase or of j^^ of its weight of Trefoil pectase. 
 
 By similar methods Bertrand and Mallevre ascertained that 
 pectase is present in the following plants : — 
 
 Spirogyra 
 
 Chara fragiUs 
 
 Marchantia polymorpha 
 
 Lolium perenne (leaf) 
 
 Zea mais (leaf) 
 
 Iris florentina (leaf) 
 
 Ginkgo biloba (leaf) 
 
 Thuja occidentalis (leafy shoot) 
 
 Pinus Laricio (needles) 
 
 Cydonia vulgaris (fruit) 
 
 Pynis communis (fruit) 
 
 Malus communis (fruit) 
 Rubus idseus (fruit) 
 Armeniaca vulgaris (fruit) 
 Cucurbita Pepo (stem, leaf, 
 
 flower, fruit) 
 Bheum rhapontiacum (leaf) 
 Beta vulgaris (leaf and root) 
 Plantago media (leaf) 
 Mentha Pulegium (flower heads) 
 Solanum Lycopersicum (ripe 
 
 fruit) 
 
XVTI] 
 
 PECTASE. 
 
 295 
 
 Solanum tuberosum (leaf) 
 Ailanthus glandulosa (leaf) 
 Ampelopsis quinquefolia (leaf) 
 Brassica napus (leaf and root) 
 Brassica oleracea (leaf) 
 Syringa vulgaris (leaf) 
 Helianthus tuberosus (leaf) 
 Sambucus nigra (leaf) 
 Daucus carota (root) 
 
 Ribes rubrum (fruit) 
 Ceratophyllum submersum (whole 
 
 plant) 
 Bobinia pseudacacia (leaf) 
 Medicago sativa (shoot) 
 Trifolium pratense (shoot) 
 Vitis vinifera (leaf, fruit) 
 Acer pseudo-platanus (leaf) 
 Delphinium Staphisagria (leaf) 
 
 Its determination in Thuja was made a little doubtful by 
 the viscous character of the cell-sap. 
 
 The amount of pectase in these different plants was ascer- 
 tained by adding to the extract from a definite weight of tissue 
 an equal volume of a watery solution of pectine of 2 per cent, 
 concentration and noting the time that elaipsed before gelati- 
 nisation took place. The results given by some of the tissues 
 are subjoined: — 
 
 Solanum Lycopersicum (ripe fruit) 48 hours 
 Vitis vinifera (nearly ripe fruit) 24 „ 
 
 Bibes rubrum (white currant fruits) 15 „ 
 Bheum rhaponticum (leaf) 12 „ 
 
 Marchantia polymorpha (thallus) 2^ „ 
 
 Daucus carota (adult cultivated root) 2 ,, 
 
 Delphinium Staphisagria (leaf) 
 
 IJ „ 
 
 Ginkgo biloba (leaf) 
 
 
 35 minut( 
 
 Syringa vulgaris (leaf) 
 
 
 20 
 
 Ailanthus glandulosa (leaf) 
 
 
 20 
 
 Daucus carota (young root) 
 
 
 15 
 
 Zea mais (leaf) 
 
 
 8 
 
 Iris florentina (leaf) 
 
 
 3 
 
 Trifolium pratense (shoot) 
 
 less th 
 
 m 1 minute 
 
 Medicago sativa (shoot) 
 
 
 )» 
 
 Solanum tuberosum (leaf) 
 
 
 )j 
 
 Brassica napus (leaf) 
 
 
 ]> 
 
 Plantago media (leaf) 
 
 
 )) 
 
 Lolium perenne (leaf) 
 
 
 >> 
 
 The activity so denoted probably gives a fairly accurate 
 approximation to the quantity of pectase present. It varies 
 
296 THE CLOTTING ENZYMES. [CH. 
 
 consequently within very wide limits. There are cases when 
 the coagulation is almost instantaneous. In others owing 
 probably to the smallness of the amount of pectase, the clotting 
 is slow and uncertain. In such cases the best results are 
 obtained when the mixture of sap and pectine is exactly neu- 
 tralised and a little calcic salt added. It must not be at once 
 inferred that a slow process undoubtedly indicates a small 
 quantity of the enzyme, for the latter will not always act 
 uniformly, two determining factors being the reaction of the 
 medium and the amount of calcium salt which it contains. 
 
 The distribution of the enzyme varies also in the same 
 plant. In Cucurbita Pepo the time taken to clot the pectine 
 solution by extracts of equal weights of different parts was as 
 follows : — 
 
 Corolla 45 minutes Stem apex 12 minutes 
 
 Young fruit 30 „ Leaf petiole 8 „ 
 
 Stem-base 20 „ Leaf blade 1 „ 
 
 It was mentioned above that the action of pectase was 
 largely influenced by the reaction of the solution in which it 
 was working, as well as by the presence of salts of calcium. 
 Bertrand and Mallevre have found that these determining 
 factors do not act independently of one another. We have 
 seen that calcic salts are essential to the coagulation, — in the 
 first place because they enter into the composition of the clot. 
 But this does not seem to be the only part they play, for the 
 clotting is accelerated by the addition of more calcium salt even 
 when sufficient to combine with the pectic acid is already 
 present. Conversely the presence of free acid has a retarding 
 influence on the action. Bertrand and Mallevre found in one 
 of their experiments that a mixture of equal volumes of the 
 sap of carrots and of 2 per cent, solution of pectine set into a 
 jelly after about one hour. On adding hydrochloric acid to a 
 similar mixture in increasing quantities there was a retardation 
 in the time of the occurrence of gelatinisation, which increased 
 pari passu with the amount of acid added. "088 per cent, of 
 the acid delayed it 40 hours and •! per cent, stopped it 
 altogether. The same effect was noticed with sulphuric, nitric, 
 maHc, tartaric and citric acids. 
 
XVII] PECTASE. 297 
 
 This retarding effect agaia was lessened by the presence of 
 a larger proportion of calcic salts, or of pectase, so that the 
 clotting depends upon the relative proportions of all three, 
 pectase, acid and calcic salts. 
 
 The exact nature of the action remains still unknown, but 
 the facts suggest as a tempting hypothesis that the pectase 
 converts the pectine into pectic acid and that this then reacts 
 with the calcium salt, forming calcic pectate. 
 
 The wide distribution of pectase, which indeed has been 
 found in almost every region where it has been sought for, 
 considered in connection with the universal presence of pectic 
 bodies in conjunction with cellulose in cell-walls, suggests that 
 its function in the living plant is connected with the changes 
 which the cell-membrane undergoes during the life of the cell. 
 The work of various investigators, from Braconnot in 1825 to 
 Mangin during the last few years, has conclusively proved that 
 at no time is the cell- wall a homogeneous membrane consisting 
 of pure cellulose. While the latter enters very prominently 
 into its composition, there are present in it a number of other 
 substances, varying in nature and in relative proportions, which 
 have been somewhat loosely described under the names of 
 pectose, pectine, pectic acid, metapectic acid and their com- 
 pounds. These have been lately investigated by Mangin, who 
 has isolated several of them and described their reactions. 
 They fall into two series, each comprising several members, 
 which show among them every stage of physical condition 
 between absolute insolubility and complete solubility in water. 
 The intermediate bodies exhibit gelatinous stages, character- 
 ised by the power of absorbing water in a greater or less 
 degree. 
 
 The first series, one of whose members is the pectine so 
 often alluded to, is composed of bodies possessing a neutral 
 reaction ; the members of the other are feeble acids. Of these 
 pectic acid is one of the most prominent. The two series are 
 closely related to each other, for by the action of heat, acids 
 or alkalis the various members of both can be prepared from 
 pectose, the most insoluble of the neutral bodies. 
 
 These bodies exist together with cellulose in all the mem- 
 
298 THE CLOTTING ENZYMES. [CH. 
 
 branes which have not undergone change into lignin or suberin, 
 and by treatment of the cell-walls with various reagents they 
 can be separated from it. Pectic acid does not usually exist in 
 the free state, but as pointed out long ago by Payen is usually 
 present in combination with calcium, as calcic pectate. 
 
 Payen pointed out further that the so-called "middle 
 lamella" between contiguous cells is almost or entirely com- 
 posed of calcic pectate, and his opinion has been endorsed by 
 Mangin in the course of his recent work. It is easy to demon- 
 strate that this layer, whatever be its origin, has not the same 
 composition as the rest of the cell-wall as it can easily be 
 dissolved by reagents which leave the cells apparently intact, 
 though isolated from each other. The most active of these 
 reagents is the so-called " maceration fluid " of Schultze, which 
 consists of a solution of potassic chlorate in nitric acid. 
 
 This difference of composition between the middle lamella 
 and the rest of the wall has long been known. Before the 
 name " middle lamella " was given to it by the writers of the 
 time of Naegeli and Sachs it was called "intercellular sub- 
 stance" and was thought to be a kind of cement, binding 
 contiguous cells together. 
 
 Besides the middle lamella other modifications of the original 
 cell-wall have from time to time attracted attention. Chief 
 among these we have the so-called " intercellular protoplasm " 
 of Russow, which he described as forming in certain cases a 
 delicate membrane, or lining layer, coating the intercellular 
 passages. Russow's opinion that this substance is protoplasmic 
 has been controverted by several subsequent observers, who 
 have shown that it is much more probably a derivative of the 
 cell-wall. Schenck held it to be of the same general nature as 
 the middle lamella, and Mangin has suggested that it is composed 
 of a mixture of pectic bodies, including calcic pectate. 
 
 The researches of Mangin have thrown a good deal of light 
 upon the proportion of the neutral pectic bodies and the 
 compounds of pectic acid which are present in various cell- 
 walls. In the young unchanged membranes there is little 
 pectic acid while pectose is present in larger amounts. In older 
 cell-walls, especially in tissues in which intercellular spaces or 
 
XVIl] PECTASE. 299 
 
 passages have appeared the proportion of calcic pectate is more 
 prominent. As said above the middle lamella is almost if not 
 entirely composed of it, and it often collects over the surfaces 
 of the intercellular spaces, being in such cases a continuation 
 of the middle lamella of the wall which has split during the 
 formation of the passage. 
 
 Even the youngest cells can be separated from each other 
 by the reagents which dissolve calcic pectate, so that there is 
 some reason to suppose that the cell-membrane is at no time 
 absolutely homogeneous, but consists of a middle layer of calcic 
 pectate, covered on both faces by a layer of a combination or 
 mixture of cellulose and pectose. As it grows, this layer of 
 calcic pectate becomes more pronounced and prominent till it 
 can be made visible under the microscope. This change must 
 be due to transformations which modify the composition of the 
 layers abutting on the cell cavities and which result in the 
 formation of the increased amount of calcic pectate. 
 
 The mode of deposit of the calcic pectate over the surface 
 of the intercellular spaces may perhaps aid us to form a true 
 conception of what takes place. 
 
 These pectates gradually tend towards the outside of the 
 membrane, possibly passing as soluble pectic acid in its sub- 
 stance and being combined with the metallic base at the 
 external surface, or in the intercellular space. 
 
 In the young growing cell, just behind the zone of cell- 
 division at the growing point, there is the maximum of turgidity 
 or osmotic pressure. It is quite conceivable that in a cell 
 abutting on an intercellular space this is sufficient to cause 
 a stream of soluble bodies to pass across the substance of the 
 cell-membrane from within outwards. This then would lead 
 to the extriision of such soluble pectates or pectic acid as 
 may be present in the wall. Bodies of this series are not 
 unlikely to be formed from the pectine or pectose in the 
 membrane by the action of the dilute acid of the cell sap, 
 or more probably by the pectase which Bertrand and 
 Mallevre have shown to be present in the apical region of 
 the stem in many cases. Such a course of transformation 
 is even more probable in the cells and cell-walls of the young 
 
300 THE CLOTTING ENZYMES. [CH. XVII 
 
 leaves, in which pectase is often present in very considerable 
 amount. 
 
 The same consideration may be applied with even greater 
 probability to the formation of the middle lamella. Where the 
 cells do not abut on an intercellular space, but have their 
 neighbours pressing upon them, as they have in the young 
 part of the growing zone, whether of stem, leaf or root, any 
 of their membranes will be subject to a pressure from each 
 side owing to the turgidity of the contiguous cells. In this 
 case the stream of pectic acid or pectates would not pass out 
 of the cell, but would tend to accumulate in the middle line 
 between the two pressures, in the region, that is, where the 
 middle lamella speedily becomes recognisable. 
 
 Though the transformation of pectine into pectic acid under 
 the action of pectase has not been shown definitely to be the 
 cause of the occurrence of the middle lamella, which belongs at 
 present rather to the realm of hypothesis than of fact, it is 
 certainly supported by the distribution of pectase already 
 established by Bertrand and Mallevre and quoted in the present 
 chapter. They have found it most abundant where cell growth 
 is most vigorous and the more rapid the growth the more 
 plentiful is the enzyme. They have found eight times as 
 much in the leaf-blade as in the petiole, and more than twice 
 as much in the apex of the stem as in the base. In the 
 corolla of Cucurbita, in which scarcely any thickening of the 
 cell-wall can occur, the amount compared with that in the 
 foliage leaf was as 1 : 45. 
 
 The failure of a plant to thrive or even to grow beyond a 
 slight extent in the absence of calcium may be partly connected 
 with the same series of phenomena. 
 
301 
 
 CHAPTER XVITT. 
 
 AMMOXIACAL FERMENTATION. UREASE. 
 
 When urine is first excreted by an animal its reaction is 
 faintly acid ; after standing for some time exposed to the air it 
 becomes alkaline; the strength of the reaction gradually in- 
 creases and after a somewhat longer interval a distinct odour 
 of ammonia is evolved from it. If after being shed it is 
 sterilised and kept from contact with air this change does not 
 take place. 
 
 Chemical examination of the exposed urine shows that the 
 alteration is due to the transformation of the urea into car- 
 bonate of ammonia, which takes place by a simple process of 
 hydrolysis according to the equation 
 
 CO (NH,). + 2H,0 = (NH4), COs. 
 
 This decomposition of urea has been regarded as a fermen- 
 tation almost ever since the time when it was first observed. 
 Before chemical investigation had been made into the com- 
 position of the changing urine, Fourcroy and Vauquelin 
 suggested that the alteration of its reaction was due to a 
 transformation of the urea, and that it was caused by a ferment 
 action set up by the albuminous matter in the urine. Dumas 
 took a similar view, but supposed that the ferment originated 
 in the mucus which urine generally contains. Jacquemart 
 associated the ferment action with the white deposit which 
 gradually forms in the vessels when urine is allowed to stand 
 until it becomes stale. He held the same view as Fourcroy 
 that the ferment principle was amorphous. 
 
 About 1860 more accurate views began to be formulated. 
 
302 AMMONIACAL FERMENTATION. [CH. 
 
 A suggestion was made by a German chemist, Muller, that the 
 action was probably due to a living organism, corresponding to 
 the yeast of beer. Two years later Pasteur discovered such an 
 organism in certain specimens of putrid urine. He described 
 it as consisting of small spherical cells joined together in 
 chains. The cells were in appearance a good deal like the 
 cells of yeast but were much smaller. On account of this 
 resemblance the organism was originally named Torula urew. 
 It was studied with much care in 1864 by Van Tieghem, whose 
 results were confirmatory of those of Pasteur. The organism 
 was subsequently renamed Micrococcus urew by Cohn. It is 
 composed of spherical or globular cells whose mean diameter is 
 1-5/i ; they are united together into long curved chains which 
 are dispersed throughout the liquid as long as the fermentation 
 proceeds. When it is over they sink to the bottom of the 
 vessel and the chains break up, so that a sediment is com- 
 posed of free globules or short chains. The cells show no 
 granulation, their cell-wall is hardly to be distinguished from 
 their contents, and they multiply for the most part by budding. 
 
 Micrococcus urese can be cultivated most easily in urine, 
 but it will grow in any nitrogenous fluid in which urea is 
 dissolved, or in a solution of urea which also contains phos- 
 phates. According to Jaksch it is capable of thriving without 
 urea if other amides or peptones are present instead. 
 
 The organism is aerobic. 
 
 A peculiar feature of its life which was noticed by Van 
 Tieghem is its power of thriving in strongly alkaline solutions. 
 He observed a fermentation to continue until the liquid con- 
 tained 13 per cent, of carbonate of ammonia, a concentration 
 which would be fatal to almost all other forms of vegetable Ufe. 
 
 Van Tieghem has stated that this organism is capable also 
 of decomposing hippuric acid, which is so prominent in the 
 urine of herbivorous animals, the products of the decomposition 
 being benzoic acid and glycin. This process also is one of 
 hydrolysis and may be expressed by the following equation 
 
 0„u,W U3 + M,U - jcoOH JCOOH. 
 
 Hippuric acid Benzoic acid Glycin 
 
XVIIl] UREASE. 303 
 
 When hippurate of ammonia is dissolved in either yeast- 
 water, or a solution of sugar which also contains phosphates, 
 and is exposed to the air, a growth of the Micrococcus soon 
 appears in the liquid, which quickly gives evidence of the 
 decomposition just alluded to. 
 
 Other organisms have been ascertained by Sestini to effect 
 the hydrolysis of uric acid, the resulting products being car- 
 bonate of ammonia and carbon dioxide. 
 
 This Micrococcus is not by any means the only microbe 
 which has the power of decomposing urea. We owe to the 
 researches of Miguel and other writers a knowledge of many 
 other organisms which can effect its hydrolysis. Some of these 
 are ordinary fungi, but most belong to the group of the Schizo- 
 mycetes or fission-fungi. Miguel has described seven species 
 of Bacillus, nine Micrococci, and one Sarcina. The Bacilli 
 appear to act most energetically. 
 
 The organisms are very widely distributed in nature, being 
 found in the air, in spring and river-water, and in the soil. 
 According to Miguel 1 — 2 per cent, of the bacteria present 
 in the soil and 15 per cent, of those present in cow-house 
 manure, are capable of hydrolysing urea. 
 
 The urea does not appear to be normally a nutritive sub- 
 stance for the organisms. If other nitrogenous compounds, 
 particularly proteids, are present in the solution in which they 
 are growing, these are the sources from which they gain their 
 nitrogen. In the absence of such compounds, however, they 
 can use the nitrogen of the urea. 
 
 The activity of the Torula or Micrococcus urese has been 
 ascertained to be due to a soluble enzyme which under certain 
 conditions can be extracted from the cells. The enzyme, to 
 which the name urease has been given, was first described by 
 Musculus in 1874. When urine which was undergoing active 
 ammoniacal fermentation was filtered through fine filter-paper 
 and the paper subsequently well washed and dried, he found 
 that he could excite a similar fermentation by immersing 
 pieces of it in a neutral solution of urea. Musculus tested 
 the progress of the fermentation by staining his paper with 
 turmeric after treating it as already described. After a short 
 
304 AMMONIACAL FERMENTATION. [CH. 
 
 stay in the solution of urea the turmeric became brown owing 
 to the alkalinity of the liquid. The same result was obtained 
 when the paper was washed with strong alcohol before staining 
 with turmeric. Under this treatment the induced fermentation 
 could not be due to living cells left on the filter-paper as these 
 would not survive contact with the alcohol. In a subsequent 
 paper, published in 1876, Musculus described the preparation 
 of the enzyme from some highly alkaline urine which he. 
 obtained from a pathological secretion. He added alcohol in 
 excess to such urine and obtained a viscous precipitate con- 
 sisting largely of mucin, derived from the walls of the bladder. 
 When this precipitate was separated by filtration and dried, it 
 readily yielded to water a solution of the enzyme which was 
 extremely active. This solution when added to alcohol de- 
 posited an amorphous precipitate which possessed the power of 
 setting up the decomposition of urea. The source of the 
 enzyme in this case was apparently the alkaline urine, as 
 Musculus states his mucous urine did not contain any of the 
 cells of the microbe. This does not however prove that the 
 enzyme had any other origin, as they might have been present 
 in the bladder under the pathological conditions existing. 
 
 Musculus found that on acidifying the solution, the enzyme 
 was rapidly destroyed. 
 
 The behaviour of the Micrococcus was the subject of an 
 exhaustive series of experiments by Sheridan Lea in 1885. 
 He cultivated the organism in urine until he had obtained a 
 large quantity which was exciting a very vigorous fermentation, 
 when he poured the whole into an excess of alcohol. A copious 
 precipitate was thrown down, consisting partly of the organism 
 and partly of mineral and other matter contained in the urine, 
 together with a sediment which was already existing in the 
 fluid. The precipitate was thrown into a filter, well washed 
 with more alcohol and dried. A small quantity of the precipi- 
 tate when mixed with a 2 per cent, neutral solution of urea 
 and kept at 38° C. developed a strong alkaline reaction in a few 
 minutes and this was followed a little later by the evolution of 
 a powerful odour of ammonia. 
 
 Some of the precipitate was next extracted with water, and 
 
XVlil] UREASE. 305 
 
 filtered. The reaction of the filtrate was very slightly alkaline 
 but the liquid was clear and limpid. On adding some of this 
 to a quantity of the same solution of urea as was used in the 
 first experiment, the same sequence of phenomena occurred. 
 The alcohol precipitate was in this way shown to contain an 
 enzyme capable of hydrolysing urea and it was found possible 
 to dissolve it out by treating the precipitate with water. 
 
 Lea claims to have been able to obtain the enzyme in an 
 approximately pure condition by repeated solution of the pre- 
 cipitate and reprecipitation by alcohol. This treatment gradu- 
 ally removes the salts which are present as well as the enzyme. 
 So prepared he found it to be a white powder, amorphous in 
 character, giving a clear solution in distilled water, which 
 however always showed the presence of a trace of proteid 
 matter when tested with nitric acid and ammonia. 
 
 Lea's results confirm and extend those of Musculus, in so 
 far as he shows that the enzyme can be prepared from the cells 
 of the Micrococcus. In Musculus' experiments he says the 
 cells were not present and the mucous urine itself was the 
 source of the enzyme he obtained. 
 
 Lea made a further series of experiments to see if the 
 enzyme is excreted by the Micrococcus into the urine or 
 whether the normal action is intracellular. Musculus' results 
 appear to indicate an excretion of the enzyme from the 
 organism in the bladder, as it is hardly likely that the tissue 
 of the latter produced it. 
 
 In these experiments Lea took a quantity of actively 
 fermenting urine and filtered it till it was free from sediment. 
 The organisms were left behind on the filter, very fine paper 
 being used and each filter being composed of 12 — 15 thick- 
 nesses. The filtration was continued till no micrococci were 
 visible under the microscope. The perfectly clear filtrate was 
 then neutralised by very dilute acetic acid, and after the 
 resulting effervescence had subsided, the remaining carbonic 
 dioxide was extracted by exposure to a vacuum. Two per cent, 
 of urea was then added to a measured quantity of the filtrate 
 and the whole placed in a water-bath at 38° C. A control was 
 prepared by taking another equal quantity of the filtrate with- 
 G. F. 20 
 
306 AMMONIACAL FERMENTATION. [CH. 
 
 out adding any nrea. This was placed in a similar vessel in 
 the water-bath side by side with the first one. 
 
 Both quantities remained neutral even after an exposure of 
 six hours. 
 
 In further experiments Lea separated the organisms from 
 the urine by filtering it through a porous earthenware cell, and 
 found the same results as to the absence of the enzyme from 
 the urine so prepared. 
 
 A quantity of similar urine was next filtered till free from 
 cells and precipitated by the addition of an excess of alcohol. 
 The resulting precipitate was soluble in water, but the solution 
 failed to exert any decomposing action on a solution of urea. 
 
 Lea thus failed to obtain any evidence of the excretion of 
 the enzyme from the cells of the organism and came to the 
 conclusion that its action was altogether intracellular. This 
 result was antagonistic to that obtained by Musculus. Only 
 two modes of reconciliation of the two seem possible. Either 
 Musculus' urine must have contained some cells which escaped 
 his observation; or the excretion from the organisms in Lea's 
 experiments was only small and what ferment there was was 
 destroyed by the alcohol used in the precipitation. Neither 
 explanation seems very satisfactory. 
 
 Lea came to the conclusion that the enzyme was incapable 
 of passing out of the cell during life, on account of the cellulose 
 membrane surrounding it. 
 
 When the organism was killed by the alcohol and its 
 protoplasm to some extent decomposed by the action of the 
 reagent, the enzyme could be extracted by a solvent such as 
 water. 
 
 This explanation, however, leaves us in some uncertainty, 
 as the treatment with the alcohol does not destroy the whole 
 of the enzyme, nor does it modify the cell-wall, and it is hard 
 to understand why if the ferment cannot pass the cell-wall 
 during the life of the protoplasm it should be able to do so 
 after its death, tinless the latter modified the physical cha- 
 racters of the cellulose membrane. It seems more likely 
 that the enzyme is retained very strongly by the living proto- 
 plasm, as Buchner has shown to be the case with the enzyme 
 
XVIIl] UREASE. 307 
 
 producing alcohol. After death of the protoplasm it would be 
 much more easily extracted. 
 
 Miguel has extracted the enzyme from fourteen different 
 species of micro-organisms which present distinct morphological 
 characters, and which are all capable of setting up ammoniacal 
 fermentation. He cultivated each of them in peptone solutions 
 containing 2 — 3 grammes of ammonic carbonate per litre. 
 Before inoculation with the microbe, the solutions were steri- 
 lised by being filtered through porcelain. The cultures were 
 continued for some days until the whole liquid had become 
 turbid. The peptone solution was then found to contain a 
 quantity of the enzyme which had been excreted by the 
 microbes. In his experiments Miguel obtained sufficient 
 enzyme per litre of peptone solution to convert 60 — 80 
 grammes of urea into ammonic carbonate in less than an hour. 
 
 The optimum temperature for the working of urease Miguel 
 found to be 50 — 55" C, but even at this temperature the 
 enzyme was gradually destroyed. At a temperature near 0° C. 
 the solution of urease retained its activity for several weeks ; at 
 75° C. it was destroyed in a few minutes and at 80° 0. almost 
 instantaneously. The organisms themselves were not easily 
 killed, surviving an exposure for 2 — 3 hours to a moist 
 temperature of 95° 0. Lea found his preparation of urease 
 was destroyed on heating to 80 — 85° C. 
 
 Bufalini has stated that besides decomposing urea and 
 hippuric acid urease is capable of converting asparagin into 
 succinic acid. 
 
 Schmiedeburg has found an enzyme in the kidney of the 
 pig which he says is concerned in the splitting up of hippuric 
 acid. He has given it the name of histozyme. Whether it is 
 the same as urease is not at present determined, though it 
 does not seem unlikely that they may be identical when we 
 remember that Van Tieghem showed that Micrococcus urese 
 can hydrolyse hippuric acid as already mentioned. 
 
 20—2 
 
308 
 
 CHAPTER XIX. 
 
 OXIDASES, OR OXIDISING ENZYMES. 
 
 The general course of action of the enzymes which we have 
 discussed so far we have seen to be one of hydrolysis, or decom- 
 position of the bodies attacked by them after a preKminary 
 taking up of water into their molecule. This is. fairly satisfac- 
 torily established with regard to most of the ferments which 
 play a leading part in digestive changes in both animal and 
 vegetable organisms. The changes effected by the proteoljrtie 
 enzymes are not so clearly shown to be hydrolytic as are those 
 brought about by ferments which act upon carbohydrates and 
 fats, but we have seen reason to believe that the course of action 
 is the same. The reaction is evidently more complicated in the 
 case of the clotting enzymes, and for the present we must leave 
 the matter doubtful so far as they are concerned. 
 
 A few of the decompositions we have examined do not appear 
 to be concerned with hydration, particularly the action of my- 
 rosin, and we shall see later that the alcohol-producing enzyme 
 of yeast does not initiate such a process. 
 
 During the last few years the existence of another class of 
 enzymes has been indicated, all of which act by promoting 
 direct oxidation of various substances, including various aromatic 
 compounds and sugar. These have been called oxidases ; they 
 are somewhat widely distributed, occurring in both the animal 
 and the vegetable body. They have recently been termed 
 respiratory enzymes as most of those already discussed have 
 been called digestive. 
 
CH. XIX] LACCASE. 309 
 
 Laccase. 
 
 Of these oxidases the earliest to be recognised was laccase, 
 the body which is concerned in the production of lacquer varnish 
 from the crude sap of the lac tree of South-east Asia. 
 
 The existence of this oxidase was first pointed out in 1883 
 by a Japanese chemist, Yoshida, who investigated the latex of 
 that plant and first ascertained the nature of the changes 
 occurring in the production of the varnish. 
 
 The crude juice is obtained by making incisions into the 
 trunk of several species of Rhus, and collecting the viscous 
 matter which exudes. It has the appearance of a nearly white 
 creamy liquid, possessing a faint odour resembling that of 
 butyric acid. On exposure to air it rapidly changes colour, 
 becoming brown, and ultimately black. Spread on a flat surface 
 it dries with a brilliant black lustre. The juice is very difii- 
 cult to experiment with, as it possesses a very irritating property 
 which affects the skin, causing painful eruptions and sores. 
 
 Yoshida states that the juice, known by the name of urushi, 
 consists in great part of a peculiar acid, which he has called 
 urushic acid, and to which he ascribes the formula C14H18O2. 
 Separated by appropriate methods from the crude latex and 
 dried at 110° C. it forms a dark, pasty substance, smelling of the 
 original juice ; it is then soluble in benzol, ether, alcohol, and 
 carbon-disulphide, but is insoluble in water; it has a specific 
 gravity of '9851 at 23° C. When exposed to the air it does 
 not dry nor show signs of change such as the original latex 
 does. 
 
 Besides urushic acid, the crude sap contains a certain pro- 
 portion of gum, and a variable quantity, usually about 3 — 8 per 
 cent., of a peculiar nitrogenous body, which coagulates on 
 heating to 63° C. If the latex is treated with excess of alcohol 
 the gum and the nitrogenous constituent are precipitated. 
 After filtration the latter can be separated from the former by 
 the action of cold water, in which it dissolves, while the gum 
 only becomes swollen. 
 
 The enzyme is associated with this nitrogenous constituent 
 of the juice. If a solution of it is mixed with a small quantity 
 
310 OXIDASES, OB OXIDISING ENZYMES. [CH. 
 
 of free urushic acid, the latter is under certain conditions 
 converted into the varnish. The change does not take place if 
 the solution is heated to 63° C 
 
 The nature of this nitrogenous constituent has not been 
 clearly established, but it seems to differ considerably from the 
 proteids, containing a much smaller proportion of nitrogen, 
 and more carbon than they do. Yoshida's analysis of it gives 
 C 63-44, H7-41, N401, 22-04, Ash 1-2 in 100 parts. 
 
 From his experiments Yoshida has come to the conclusion 
 that urushi jxiice consists essentially of four substances, viz. 
 urushic acid, gum, water, and a peculiar enzyme. The phe- 
 nomenon of its drying is due to the oxidation of urushic acid, 
 CmHisOj, into oxyrurushic acid, CiiHuOs, which takes place by 
 the aid of the enzyme in the presence of oxygen and moisture. 
 
 He supports this conclusion by two series of experiments, 
 which may be quoted here. 
 
 A small quantity of the original juice was put into a covered 
 beaker and subjected to the regulated heat of a water-bath, 
 the water lost by evaporation being subsequently restored. 
 The heating was carried to different temperatures, and subse- 
 quently the heated juice was spread thinly over a glass plate 
 and left to dry in a box the air in which was kept moist. In 
 each experiment the juice was heated for 3^ — 4 hours, and the 
 drying was allowed to take place at a temperature of 20° 0. 
 
 The results were as follows : — 
 
 ap. of exposure. 
 
 Time of drying. 
 
 20° C. 
 
 2 hours 
 
 30° C. 
 
 4 „ 
 
 40° C. 
 
 H „ 
 
 55—59° 0. 
 
 24 „ 
 
 60 63° C. 
 
 Did not dry. 
 
 In the second series of experiments he found that unless 
 moisture was present the latex did not dry ; that in moist air 
 it dried in 4 hours, in moist oxygen in 2 hours, in moist hydrogen 
 or nitrogen it took 36 hours, and in moist COa it was dry only 
 after 2 days' exposure. 
 
 It follows from these experiments that the enzyme works 
 
XIX] LACCASE. 311 
 
 most energetically at a temperature of 20° C, but only when 
 oxygen and moisture are both present ; a rise of temperature 
 above 20° C. is slowly prejudicial to it, and at 60° — 63° C. it is 
 destroyed. It may be noted that it is at this temperature that 
 the nitrogenous matter coagulates. 
 
 Yoshida prepared oxy-urushic acid from urushic acid by 
 the action of strong chromic acid. He says that so prepared it 
 exhibits all the properties of the lacquer varnish. 
 
 The name laccase was given to the enzyme more than ten 
 years later by Bertrand, who made further investigations into the 
 peculiar behaviour of the latex and who ascertained several 
 additional facts about the enzyme. 
 
 In the main he confirms the earlier work of Yoshida as to 
 the constituents of the latex. The body described as urushic 
 acid he prefers to term laccol, but he has not examined it 
 minutely on account of its deleterious properties. 
 
 He prepared the enzyme by treating the latex with a large 
 excess of alcohol ; this precipitated a gummy substance, which 
 he purified by redissolving it after filtration, and again throwing 
 it down by the addition of 10 volumes of alcohol. It separated 
 out in white opaque flakes which yielded on hydrolysis a 
 mixture of galactose and arabinose. 
 
 The enzyme was extracted from the gum by treatment with 
 cold water. 
 
 In the natural juice the laccol exists in the form of 
 an emulsion, which is probably due to the presence of the 
 gum. 
 
 The laccol remains unchanged if it is separated from the 
 latex by solution in alcohol and kept from the air. If a little 
 water is added to the solution in alcohol a white emulsion 
 results, which keeps for a considerable time unaltered ; but if a 
 solution of laccase is substituted for the water, the resulting 
 emulsion turns brown at once and rapidly becomes black, 
 especially if air is admitted. With a boiled solution of laccase 
 no such change can be observed. 
 
 So far as Bertrand has investigated the properties of laccol, 
 it appears to be allied to certain polyatomic phenols. On this 
 account he has examined the action of laccase on several of 
 
312 OXIDASES, OR OXIDISING ENZYMES. [CH. 
 
 the latter, especially hydroquinone and pyrogallol. When the 
 former is submitted to its influence, the colour of the solution 
 quickly becomes rose-red, and after a short interval crystalline 
 scales with a green metallic lustre appear, the quantity rapidly 
 increasing. When this operation is carried out in a sealed tube 
 the oxygen present is almost completely absorbed. The liquid 
 gives off a strong characteristic odour, and quinone can be ex- 
 tracted from it by shaking it with ether after removal of the 
 solid matter. The precipitate is quinhydrone. 
 
 In the absence of the laccase, the hydroquinone does not 
 absorb oxygen, nor undergo alteration. The hydroquinone is 
 therefore oxidised by the free oxygen under the influence of the 
 laccase, according to the equation 
 
 2CeH,(0HX + 0, = 2H3O -I- 2CeHA. 
 
 Hydroquinone Qainone 
 
 The colour given to the liquid is due to the formation of the 
 quinone. Some of the latter, combining with the excess of 
 hydroquinone not oxidised, produces the less soluble crystals of 
 quinhydrone. 
 
 When pyrogallol is used instead of hydroquinone similar 
 results are obtained, a precipitate of purpurogalline being thrown 
 down in the form of a powder which sublimes on heating, 
 forming orange-red needles which are soluble in alcohol and 
 acetic acid. 
 
 Laccase attacks many other polyphenols, but chiefly those 
 whose hydroxyl groups are in the ortho- and para-positions. 
 The con-esponding meta-compounds are affected only with 
 diflSculty. The oxidisability of these bodies by laccase seems to 
 depend on the facility with which they can be transformed into 
 quinones. The monophenols are not oxidised by the enzyme, 
 but it attacks gallic acid and tannin. 
 
 Bertrand's observations on the behaviour of laccase at diffe- 
 rent temperatures do not agree with those of Yoshida, as he 
 finds it still active after heating it to 70° C. 
 
 Bertrand has sought for laccase with some success in other 
 plants and has indicated a rather wide distribution for it. In 
 his researches he has employed the guaiacum test and appears 
 
XIX] LACCASE. 313 
 
 to a certain extent to rely upon this method of recognition. 
 This is unfortunate, as most investigators do not find it give 
 entirely satisfactory results. He says that an alcoholic tincture 
 of gum guaiacum becomes blue in the presence of air and a little 
 laccase ; if much of the latter is present, it turns from blue to 
 green and subsequently to yellow. In most cases however 
 he has confirmed his results by isolating the enzyme and 
 proving its presence by its action. This is really the only 
 satisfactory method of demonstrating its existence. By the two 
 methods conjointly he has found laccase in the roots of the 
 beet, carrot and turnip; in the tubers of the potato and the 
 Jerusalem artichoke ; in the tuberous roots of Dahlia ; in 
 certain rhizomes ; in the fruits of the apple, pear, quince and 
 chestnut; in the vegetative parts of lucerne, clover, rye-grass, 
 and asparagus; and in the flowers of Gardenia. It may be 
 prepared from these sources by extraction with water and pre- 
 cipitation of the extract with alcohol. If the tissue is green, 
 the extract may be saturated with chloroform and allowed to 
 stand for 24 hours to free it from the colouring matter, after 
 which the precipitation by alcohol may be carried out. 
 
 Rey-Pailharde has found laccase in germinating seeds, 
 especially of plants of the Leguminosw. 
 
 The activity of laccase appears to be associated in some 
 way with the presence of manganese. Its ash always con- 
 tains traces of an oxide of this metal, sometimes as much 
 as 2 per cent. Bertrand states that the activity of a pre- 
 paration of the enzyme is proportional to the amount of 
 manganese which is present. 
 
 When prepared from Lucerne it is poor in this constituent, 
 and the efifect of the addition of a salt of the metal can be 
 easily studied. Bertrand describes a typical experiment on 
 this point. He gathered several kilograms of lucerne at the 
 time of flowering and bruised them in a mortar, pressing out 
 the sap, which was then saturated with chloroform and allowed 
 to stand in the dark for 24 hours. The juice was next filtered 
 and 2^ volumes of alcohol added to precipitate the laccase. 
 The precipitate was taken up with a little water, the solution 
 filtered and the laccase again thrown down by large excess of 
 
314 OXIDASES, OR OXIDISING ENZYMES. [CH. 
 
 alcohol. The final precipitate was collected and dried in vacuo. 
 It contained a mere trace of manganese. 
 
 To 50c.c. of a solution of hydroquinone "l gm. of this 
 precipitate was added, and the whole was agitated for 24 hours 
 in contact with air. There was then only a red coloration 
 produced. To a further quantity of 50 c.c. of the hydroquinone 
 solution •! gm. of the precipitated laccase and 1 mgr. of man- 
 ganese in the form of the sulphate were added together, and in 
 less than 2 hours crystals of quinhydrone were formed. In the 
 latter case there was an evident oxidation, much more extensive 
 than after 24 hours' agitation in the absence of the manganese. 
 
 In an experiment so arranged that the absorption of oxygen 
 could be measured it was found after 6 hours' agitation with air 
 at 15° C. that with laccase alone 2 c.c. oxygen were taken up ; 
 with a salt of manganese alone '3 c.c. were absorbed ; but with 
 both present together 6"3 c.c. of oxygen were fixed. 
 
 The manganese is thus seen to play a very active part in 
 the ordinary action of the enzyme. No other metal was found 
 to be capable of replacing it. 
 
 Manganese combined with various acid radicals was found 
 in a further series of experiments to have a certain power of 
 causing the oxidation of hydroquinone, the protoxide appearing 
 to act as a carrier of the oxygen. Comparing the action of these 
 salts of manganese with the conjoint action of manganese and 
 laccase, Bertrand advances the theory that the oxidases may 
 be conceived to be special combinations of manganese with 
 certain proteid bodies containing acid radicals, the latter vary- 
 ing with the particular enzyme. In such combinations the 
 acid radical has just the necessary affinity to keep the metal 
 in solution. The work of conveying the oxygen is in Bertrand's 
 opinion discharged by the manganese, while the proteid matter 
 gives to the oxidase its other characters, such as are made 
 evident by the action of heat, and the various reagents used to 
 identify it. 
 
 Whether this hypothesis be accepted or not, the experiments 
 show that laccase is at any rate much assisted in its working by 
 the presence of manganese if its activity is not entirely de- 
 pendant upon its association with that metal in some form. 
 
^IX] LACCASE. 315 
 
 Besides the plants already mentioned laccase appears to 
 exist in a considerable number of Fungi. In these plants the 
 phenomena of oxidation are very prominent, and in consequence 
 of this fact Bourquelot and Bertrand instituted in 1896 an 
 investigation of them with a view to ascertaining whether 
 laccase or some similar enzyme plays a part in their metabolism. 
 As in other cases, at the outset these observers laid consider- 
 able stress on the guaiacum reaction, and they found that the 
 liquid that can be expressed from many fungi very rapidly 
 oxidises the tincture with the formation of a blue colour, but 
 that it does not bring about this change if it is first boiled. 
 
 The reactions of the expressed juice with other bodies than 
 tincture of guaiacum leave no doubt that it contains the same 
 principle as the sap of the lacquer tree. It causes the brown- 
 ing of the laccol prepared from the latex of Rhus ; it yields 
 crystals of purpurogalline when allowed to act upon pyrogallol, 
 produces quinone and quinhydrone from hydroquinone, and 
 gives a very distinct brown colour with gallic acid. 
 
 The fungus which yields laccase most readily is Russula 
 faetens Pers., one of the Basidiomycetes, which is fairly common 
 in woods during the summer. 125 grams of this fungus 
 extracted with its own weight of chloroform water yielded 60 c.c. 
 of a liquid which was at first pale yellow in colour, but which 
 gradually reddened on exposure to air. When made to act on 
 gallic acid in a closed flask which was constantly shaken it was 
 found that the oxygen was gradually absorbed, 15 c.c. dis- 
 appearing during the first hour of action. It gave also the 
 reactions just described with laccol, pyrogallol, &c. 
 
 When the extract so prepared was boiled, it gradually lost 
 its enzymic powers. Bourquelot and Bertrand say however that 
 it is more resistant to heat than most enzymes, and that to 
 ensure complete destruction the boiling should be maintained 
 for a short time. 
 
 When the extract of Russula is poured into an excess of 
 alcohol it yields only a small amount of precipitate, but this 
 when separated off gives up the enzyme to cold distilled water. 
 The precipitation of the laccase is not complete however when 
 the extract is so treated. 
 
316 OXIDASES, OR OXIDISING ENZYMES. [CH. 
 
 A very large number of species of Fungi have been ex- 
 amined, chiefly belonging to the Basidiomycetes, more than half 
 of which have been found to contain laccase, capable of acting 
 on the aromatic bodies mentioned. Of these the genera Rtissula, 
 Lactarius, Boletus, and Psalliota are the most noteworthy. 
 
 The Gasteromycetes as a rule contain little, if any, and the 
 Ascomycetes and Myxomycetes so far as they have been ex- 
 amined are free or nearly free from the enzyme. 
 
 Besides working at the effect of laccase on the aromatic 
 bodies as described above, Bourquelot and Bertrand investigated 
 the nature of the changes of colour which supervene when 
 many of the fleshy fungi are cut and the damaged surfaces 
 exposed to the air. The tissue of Boletus changes almost 
 instantaneously under such conditions, assuming a blue 
 colour, the depth of tint and rapidity of appearance varying 
 somewhat in different species. Lacterius becomes violet when 
 wounded, while Russula turns first red and finally black. 
 
 There have been several theories as to the cause of this 
 change of colour. Schcenbein noticed the phenomenon as long 
 ago as 1856 and he attributed it to the action of ozone upon a 
 particular chromogen in the fungus, saying that the latter also 
 contains a substance capable of titinsforming the oxygen of the 
 air into ozone. In 1872 Ludwig made some investigations into 
 the subject and confirmed Schcenbein as to the existence of a 
 special chromogen in the tissue. 
 
 In the light of the recent woirk on the oxidases Bourquelot 
 and Bertrand were led to the view that one of the latter probably 
 is concerned in the alteration of the chromogens. According to 
 Schcenbein there was evidently something concerned besides the 
 chromogen, and in his opinion the work effected by the par- 
 ticular constituent in question was the transformation of oxygen 
 into ozone. Whatever it may have been it cooperated with the 
 oxygen of the air in causing the oxidation of the chromogen. 
 As this is apparently the part played by laccase in the formation 
 of the lacquer varnish, it seems probable that Schoenbein's 
 hypothetical oxygen transformer was really an oxidising enzyme. 
 
 Working on this hypothesis Bourquelot and Bertrand car- 
 ried out the following experiment. A definite weight of Boletus 
 
XIX] LACCASE. 317 
 
 cyanescens Bull, was extracted with boiling alcohol of 95 per 
 cent, concentration, the fungus being cut up as far as possible 
 out of contact with air. The extraction was continued for a 
 quarter of an hour, after which the liquid was cooled and filtered. 
 The alcoholic extract so prepared was faintly yellow in colour, 
 and it contained the substance which normally turns blue on 
 exposure. So prepared it retained its colour for a considerable 
 time, even when diluted with water and allowed to stand in 
 contact with air. 
 
 The investigators added to such an extract, diluted with its 
 own volume of water, a small quantity of the extract of Russula 
 prepared as described above. In half-a-minute a purple colora- 
 tion appeared which passed rapidly into blue. The same effect 
 followed on the addition of a little laccase prepared from the 
 latex of the lacquer tree. If the enzyme was added slowly 
 without agitation the tint was seen to be assumed gradually, the 
 upper layers of the liquid in contact with the air being coloured 
 first and the tint spreading thence throughout the whole. 
 
 Hence Bourquelot and Bertrand infer that the oxidase which 
 can effect these changes is identical with the laccase of Rhus and 
 other plants, and that in addition to acting on aromatic bodies 
 such as hydroquinone and pyrogallol it also assists to oxidise 
 the chromogens of certain fungi, especially those which yield 
 a blue or a red colouring matter. The laccase exists in the 
 juice of the fungi side by side with the chromogen', but when 
 the juice is boiled before exposure to the air has taken place 
 the laccase is destroyed and the chromogen in consequence 
 remains unchanged. 
 
 An enzyme similar in many respects to laccase has been 
 described by Pi^ri and Portier as existing in the gills, labial 
 palps, and blood of certain molluscs. 
 
 Tyjvsinase. 
 
 In other Fungi there are different chromogens which do not 
 turn blue on exposure to air but become red and finally black. 
 Of these Russula nigricans Bull, is perhaps the most con- 
 spicuous example. The substance which gives rise to the 
 
318 OXIDASES, OK OXIDISING ENZYMES. [CH. 
 
 black colour is almost insoluble in alcohol, but after the fungus 
 has been boiled with this reagent it can be extracted from the 
 residue by subsequent maceration with boiling water. When 
 such an extract is treated with a little fresh cold water extract 
 of the fungus, or a piece of the tissue is added to it the liquid 
 turns red and after a time black. If the chromogen is extracted 
 from the fungus by boiling water and rapidly pressed and the 
 exuded liquor filtered and concentrated to a small bulk, it 
 deposits colourless needle-shaped crystals, usually collected 
 together into spheres. They are not soluble in alcohol nor 
 readily in cold water but they dissolve freely in hot water. 
 They have been identified by Bertrand with tyrosin. 
 
 Bertrand has observed the same general course of behaviour 
 with the expressed juice of the roots of the beet, the tuberous 
 roots of the dahlia, and the tubers of the potato. In these cases 
 also he has identified tyrosin in the tissues. 
 
 The similarity of behaviour to that observed in the cases of 
 Boletus, Lactarius, &c. points to a similar cause of the change 
 of colour. Laccase however has no power to set up the black- 
 ening. Nor will simple oxidising agents bring it about. Bertrand 
 asserts that it is an oxidation process due to the presence of a 
 special oxidase, and he has named the enzyme in question 
 tyrosinase. 
 
 If a little of the cold water extract of Bussula nigricans is 
 added to a solution of tyrosin, the mixture becomes at first red, 
 and subsequently assumes an inky blackness, while finally a black 
 amorphous precipitate settles out. If this is carried out in a 
 glass vessel without agitation the colour first appears at the 
 surface of the liquid. If it is conducted in a closed vessel from 
 which air is excluded the change of colour does not take place. 
 Nor is the change induced if the extract of the fungus is boiled 
 before being added to the solution of tyrosin. In a closed 
 vessel in the presence of air, the absorption of oxygen can be 
 measured coincidently with the blackening of the liquid. 
 
 Tyrosinase can be extracted not only from Bussula, but 
 from the dahlia and the beetroot. It is immaterial which of the 
 three serves as the source of the oxidase as the effect upon 
 the tyrosin is the same in all cases. Bussula appears to 
 
XIX] TYROSINASE. 319 
 
 contain it in greatest quantity. The same mode of extraction 
 can be employed with either material. 
 
 In some species of Russula the two oxidases so far discussed 
 exist side by side. Bertrand has separated them by the 
 following treatment. One and a half kilogrammes of freshly 
 gathered Russula delica Fries, was reduced to pulp and macer- 
 ated for half-an-hour with its own weight of chloroform-water 
 at the ordinary temperature. On pressing it, about 2 litres of 
 a mucilaginous fluid was obtained, to which 3 htres of 95 per 
 cent, alcohol were added. A precipitate fell which was fil- 
 tered off. The filtrate was concentrated to half a litre by 
 distillation at 50° C. in vacuo, and when so obtained was found 
 to be capable of acting with considerable energy on pyrogallol 
 and hydroquinone, but to have no effect on tyrosin; it con- 
 tained therefore only laccase. 
 
 The precipitate was washed with 200 c.c. of chloroform 
 water and when it was well swollen up, forming a semi-solution, 
 it was precipitated by addition of 400 c.c. of alcohol and pressed 
 dry. It was further purified by a repetition of this treatment. 
 Dried at 35° C. it weighed about 7 grms. This precipitate 
 yielded to cold water after some hours' maceration, a principle 
 which oxidised tyrosin rapidly, but had hardly any perceptible 
 action on either hydroquinone or pyrogallol. 
 
 Tyrosinase is destroyed at a much lower temperature than 
 laccase: it is injured at about 50° C. and perishes rapidly at higher 
 points. It is possible to prepare laccase alone from a mixture 
 of the two, by heating the liquid containing them to 70° C. It 
 then oxidises hydroquinone, but is without action on tyrosin. 
 
 Bourquelot has recognised tyrosinase in many genera of 
 Fungi, among which may be mentioned Boletus, Russula, Lacta- 
 rius, Paadllus, Goprinus, PsalUota, Hebeloma, PhoUota, Collyhia, 
 Glitocyhe, Tricholoma and Amanita ; in all these it is associated 
 with laccase, but in the case of Amanita, the latter enzyme is 
 present only in small quantities. 
 
 Besides oxidising tyrosin, Bourquelot has found tyrosinase 
 to act on all the cresols, resorcinol, guaiacol, metatoluidine, 
 xylidine, ortho-, meta-, and para-xylenol, thymol, carvacrol, and 
 a and ;S naphthol. 
 
320 OXIDASES, OR OXIDISING ENZYMES. [CH. 
 
 He has noted a further peculiarity in its behaviour in bhat 
 it is effective when dissolved in a mixture of water and either 
 ethyl or methyl alcohol, provided that not more than 50 per 
 cent, of the spirit is present. The alcohols themselves are not 
 afi'ected by it. 
 
 (Enoocydase. 
 
 Another of these oxidising enzymes has been discovered to 
 play a prominent part in causing a particular disorder in certain 
 wines to which the name "casse" or " cassure " has been applied. 
 According to Bouffard a wine affected in this way loses its 
 characteristic colour, and after 3 or 4 hours it contains a red- 
 brown precipitate. If the wine is at rest the decoloration 
 begins at the surface, where a thin pellicle of colouring matter 
 forms, and the disturbance gradually spreads to layers deeper 
 and deeper in the liquid, until at last the walls of the vessel 
 are covered by adherent matter, and the liquid is almost de- 
 colorised, assuming a moderately characteristic yellow tint. 
 The deposits are formed of the colouring matter of the wine, 
 and are insoluble in solutions of tartaric acid, even if concen- 
 trated. The changes are not attended by any evolution of gas. 
 Bouffard says that such vrines can be preserved from the dis- 
 order by heating them to 60° C. or by the addition of traces of 
 sulphurous acid. The change is not due to bacterial action, for 
 it is not hindered by filtration through porcelain, nor by the 
 addition of reagents which are fatal to microbes, such as salicylic 
 acid or bichloride of mercury. 
 
 Gouirand has shown that this change is due to some principle 
 which exists in the wine itself. He took some samples of 
 affected wine and after filtering a quantity through porcelain, 
 a large addition of alcohol threw down a precipitate of a floccu- 
 lent character. When this was collected and washed, a small 
 quantity of it added to sterilised sound wines very speedily 
 produced the disorder. 
 
 This substance is decomposed by heating. In some of 
 Gouirand's experiments he treated samples of sound wines 
 with a small quantity of it, and dividing them into two parts, 
 
XIX] CENOXYDASE. 321 
 
 he heated half to 80° C. In periods varying from 12 to 72 hours 
 the disorder was pronounced in the unheated samples, while 
 the controls remained clear and limpid indefinitely. Warming 
 the controls only to 60° C. gave variable results ; in some it in- 
 hibited the action, in others it only retarded its progress. The 
 substance was not affected by heating to 50° C. 
 
 When healthy wines were precipitated by alcohol in the 
 same way as the unsound ones, the precipitate had no power of 
 setting up the disorder when added to other samples. 
 
 Martinand has ascertained that this substance is present in 
 ripe grapes. An extract of these gives all the reactions of 
 laccase, oxidising hydroquinone, pyrogallol, &c., but it loses the 
 power of producing these changes if heated to 100° C. If 
 however there is added to the extract, after cooling, a little of 
 the precipitate yielded- when the juice of fresh grapes is treated 
 with a large excess of alcohol, it regains the power. There is thus 
 present in the grapes themselves as in the wine prepared from 
 them, a certain amount of this oxidising substance, which from 
 its behaviour must be classed with laccase and tyrosinase, as 
 an oxidising enzyme. 
 
 The name cenoxydase has been given to this body. It ap- 
 pears to resemble laccase very closely but it is not certain 
 that it is identical with it. 
 
 Martinand has proved it to be present in other fruits 
 than grapes ; plums, pears and apples especially may be men- 
 tioned. It appears to develop with the ripening of the fruit, 
 unripe grapes containing, very little. A good deal seems to be 
 lost in the preliminary processes of wine-making, wine itself 
 containing relatively little, when compared with the freshly 
 expressed grape-juice. 
 
 Martinand finds that the cenoxydase can be removed from 
 wine by shaking it with ether, which takes from it a body 
 having some of the properties of tannin ; this becomes olive- 
 green or yellowish-brown on the addition of ferric chloride, is 
 turned red by alkalis, and gives a white precipitate with 
 albumin but not with gelatin. After the wine has undergone 
 oxidation, most samples do not give up this body to ether, and 
 many others yield only very small quantities of it. 
 
 G. F. 21 
 
322 OXIDASES, OR OXIDISraO ENZYMES. [CH. 
 
 Wine treated with ether in this way, and kept neutral, is not 
 subject to self-oxidation. We may therefore infer that the 
 enzyme is possibly associated in the wine with this body 
 which is soluble in ether. 
 
 Martinand finds that the oxidase is destroyed when its 
 solution is heated to 72° and kept at that temperature for four 
 minutes. Exposure to 55° C. for IJ hours is also fatal to it. 
 Intermediate temperatures bring about the same destruction 
 after intermediate times of exposure. 
 
 Bouffard has observed that the temperature of destruction 
 varies a good deal under different circumstances. He has found 
 that wines beginning to be attacked with the disorder have 
 been completely preserved by being heated to 60° C, and that 
 warming them only to 55° C. materially helps them to resist it 
 He has further extracted normal wine by the alcohol method 
 and side by side with it samples of the same wine after being 
 heated to 60° C. The precipitate in the latter case had no 
 oxidising power, while that in the former was very active. 
 Further investigation showed him that the nature of the 
 medium exercised a great influence on the destruction. When 
 the enzyme was heated in an aqueous solution of neutral re- 
 action it withstood all temperatures below 72"5°C. but when 
 10 per cent, of alcohol or '5 per cent, of tartaric acid was present 
 destruction was complete at 52"5° C. If double these percent- 
 ages of alcohol or acid were present, the temperature necessary 
 for destruction was reduced 5° C. He agrees with Martinand 
 however in saying that it can be destroyed by prolonged heating 
 in neutral media at 60° C. 
 
 Dealing with the action of various reagents upon oenoxydase, 
 Bouffard has ascertained that it is destroyed by the action of 
 very dilute sulphurous acid, the necessary amount being '02 
 grm. per litre of the solution of the enzyme. 
 
 Cazeneuve has extracted the enzyme from unsound Beau- 
 jolais and examined many of its properties. He precipi- 
 tated the wine by excess of strong alcohol and found the 
 deposit was of a gummy consistency. He took up the 
 gummy precipitate with water and reprecipitated it with 
 alcohol, collected the deposit rapidly and dried it in vacuo. 
 
XIX] CENOXYDASE. 323 
 
 He found the precipitate chiefly gum, impregnated with 
 cenoxydase. 
 
 In most respects Cazeneuve's results agree with those ah-eady 
 quoted, but he finds further that it acts slightly on alcohols and 
 ethers and on the essences which give wines their peculiar 
 bouquet. In its action on the wine he observes that it causes 
 a disengagement of carbonic dioxide, and that after its action 
 there is a diminution of the quantity of alcohol and acid. 
 
 He attributes the noticeable effects produced to the action 
 of the enzyme on the tannins. As stated above Martinand has 
 shown that if these are removed by ether the disorder of the 
 wiue does not occur. Whether this is due to the removal of 
 the cenoxydase with the tannin or to the abstraction of the 
 latter only seems uncertain. 
 
 Cazeneuve further establishes a fact which indicates clearly 
 that the disorder is due to the enzyme. He has submitted 
 sound wine to the influence of a current of oxygen for some 
 time and also to the action of ozone and he finds that neither 
 process causes " la casse." 
 
 The enzyme can be preserved unchanged for some consider- 
 able time if dissolved in weak alcohol or in wine which does 
 not contain more than 9 per cent, of spirit. It is however rapidly 
 altered by strong alcohol. 
 
 Laborde has suggested a different origin for cenoxydase. 
 He finds the fungus Botrytis cinerea grows freely on grapes 
 and on sterilised wine " must," and an investigation of its life- 
 history has shown him that it normally secretes the enzyme. 
 In his experiments he employed a culture fluid in which this 
 Botrytis had been growing freely and he compared the actioa 
 of this liquid before and after boiling it. In the first case he 
 mixed a certain volume of the culture fluid with an equal 
 quantity of a perfectly sound wine and kept it in contact with 
 the air for 4 hours at the ordinary temperature. At the expira- 
 tion of that time all the colouring matter had been precipitated. 
 When he boiled the culture medium before adding it to the 
 wine no such change took place. 
 
 Laborde found that the oxidising power of the enzyme as it 
 existed in the mixture of the wine and the culture medium was 
 
 21—2 
 
324 OXIDASES, OR OXIDISING ENZYMES. [CH. 
 
 destroyed by heating the latter to 70° C. If no wine was 
 present it would resist even a higher temperature than this. 
 He has given the following table to show the effect of gradually 
 heating the culture-medium alone. 
 
 60° C. destroys about half of the oxydasic power. 
 
 65° C. „ „ two-thirds 
 
 70° C. „ ., four-fifths 
 
 85° C. „ the whole 
 
 The oxidase is slowly destroyed by absorbing oxygen, losing 
 about half its power in two days and nearly all in twelve days. 
 The destruction is greater in proportion during early than late 
 periods of oxidation. 
 
 The fungus contains most oxidase when it is in full fructifi- 
 cation. 
 
 It was mentioned above that Martinand had found oenoxy- 
 dase in the juice* of apples, pears and plums. Either the 
 same en2yme or a similar one has been described by Lindet as 
 causing oxidation of the tannin in the cider-apple. If slices of 
 apple, or a mass of the pulp, or sterilised sponges soaked in the 
 expressed juice are placed under a bell-jar over mercury, the 
 material rapidly reddens, and there is a simultaneous absorption 
 of oxygen and an evolution of carbonic dioxide. The phenomena 
 are the same if the juice in which the sterilised sponges are 
 soaked has been filtered through porcelain, or if antiseptics are 
 added, so that it is evident that the changes are not due to the 
 presence of micro-organisms. If boiled juice is used, it remains 
 uncoloured and there is no exchange of the gases mentioned. 
 
 The juice may be precipitated by alcohol and the precipitate 
 collected and washed in the usual way and it is then found to 
 be capable of setting up similar changes in boiled juice. 
 
 It is of course a common experience that the behaviour of 
 the pulp of a raw apple on exposure to air is very different from 
 that of a cooked one. The latter remains uncoloured, while the 
 surface of the raw pulp soon turns a reddish-brown, particularly 
 if it is unripe. 
 
 Lindet holds that the enzyme attaches itself to the tannin, 
 and explains the change of colour seen on wounding the fruit 
 
XIX] (ENOXYDASE. 325 
 
 by the suggestion that in the intact pulp, the tannin and the 
 enzyme are situated in different cells, being brought into con- 
 tact in consequence of the wound. This suggestion seems 
 however unnecessary, as the oxidases have been shown to work 
 upon the aromatic bodies they attack only when they are in the 
 presence of oxygen. The access of the latter is only possible 
 when the surface of the pulp is exposed. 
 
 Other vegetable oocidases. 
 
 The various flavours and the odours which constitute the 
 "bouquet" of different wines have been generally associated 
 with peculiarities of the fermentations induced by the dififerent 
 yeasts employed. Kecently Tolomei has brought forward 
 reasons for thinking that some of them may be due to definite 
 oxidases which can be extracted from the yeasts. 
 
 He first demonstrated the existence of oxidases in yeast by 
 cultivating in sterilised wine must, a crop of Saccharomyces 
 ellipsoideus that had originally developed in muscatel grape 
 juice. After a few days there was a considerable growth of the 
 organism and he separated it from the must and exposed it to 
 the air. After a time he extracted it with chloroform-water, 
 and found that the liquid then contained an oxidase which gave 
 Bertrand's reactions for laccase. He obtained similar results 
 with S. cerevisiae, and S. apiculatus. In the case of the former, 
 a young beer-yeast was suspended in a solution of glucose, and 
 a little alcohol was added ; the whole was then kept at 0° C. for 
 3 days, and was finally filtered through porcelain to free it from 
 yeast-cells. It was found to absorb oxygen from the air and to 
 give off carbon dioxide, and to form sulphuretted hydrogen 
 when in contact with sulphur. If heated to 72° 0. it lost all 
 these properties. When alcohol was added to the liquor a 
 precipitate fell which contained the oxidase, and when a little 
 of this precipitate was added to some of the original liquor that 
 had been sterilised by heat, it restored to it the properties 
 which had been lost during the sterilisation. 
 
 Tolomei showed further that the bouquet of muscatel wine 
 was caused by the oxidase of S. ellipsoideus. He extracted some 
 
326 OXIDASES, OR OXIDISING ENZYMES. [CH. 
 
 of the enzyme from this muscatel yeast, and added it to an 
 ordinary white wine, at the same time exposing it to the air. 
 The wine acquired a muscatel bouquet which it did not pre- 
 viously possess. 
 
 Jacquemin attributes the development of the special 
 bouquets of wines to the presence of certain glucosides in 
 the grapes. He says that these are present also in the leaves 
 of the vines, and that if these organs or extracts of them 
 containing the glucosides are added to the must, the ferment- 
 ation being subsequently carried out with pure yeasts, the 
 various flavours and bouquets are acquired by the fermenting 
 liquids. He attributes the transmission of these features 
 therefore to the secretion of glucoside-splitting enzymes and 
 not oxidases by the yeasts. 
 
 Buchner also says that beer-yeast contains an oxidase, to 
 the action of which he attributes the fact that an extract of 
 this fungus turns brown after a prolonged exposure to the air. 
 
 Effront has observed a considerable absorption of oxygen 
 by yeast when it was finely fragmented and subsequently 
 exposed to the air. The absorption was accompanied by a 
 sensible increase of temperature. Effiront suggests that the 
 phenomenon is due to the action of an oxidase. 
 
 Br&iudat has recently found an oxidase in the leaves of 
 Isatis alpina and other indigo-yielding plants. As already men- 
 tioned in a preceding chapter the formation of indigo involves 
 the decomposition of a glucoside, indican, which is effected by 
 a ferment resembling emulsin. The products of this decom- 
 position are indigo-white or leudndigo, and indighicin, which is 
 a sugar. The indigo- white is converted into indigo-blue by an 
 oxidase which is present in the leaves. Br^audat says that it 
 acts most advantageously in the presence of a weak alkali such 
 as lime-water. The alkali alone does not produce the effect. 
 
 Tolomei has foufid an oxidase in ripe olives, which appears 
 to oxidise the oil, giving rise to oleic, acetic, and sebacic acids. 
 Bouffard and Semichon state that purple grapes contain an 
 oxidase which in the presence of a current of air oxidises and 
 precipitates the colouring matter, so that white wine can be 
 prepared from the juice. Lepinois has found a similar body in 
 
XIX] ANIMAL OXIDASES. 327 
 
 the expressed juice of aconite and belladonna which destroys 
 the green colouring matter. Boutroux says that the colour of 
 brown bread is due to another oxidase which is in the bran. 
 
 Within the last year oxidases have been discovered in malt 
 extract (Grliss) in the leaves of Gorchorus (Kiouri), Helleboms 
 (Vadam), Digitalis (Brissemonet and Joanne), Vitis (Oornu), in 
 the latex of Schinus molle (Sarthou), in the root of Valeriana 
 officinalis (Carles), and in the Coli bacillus (Koux). Woods 
 attributes the changes in the chlorophyll of leaves to the 
 same cause. 
 
 Animal oxidases. 
 
 Oxidative processes have long been known to take place in 
 blood when shed and exposed to the air. Claud Bernard first 
 pointed out that under these conditions sugar disappeared, and 
 his results have been confirmed by many subsequent observers. 
 Only Avithin recent years, however, has it been suggested that 
 this disappearance of sugar is due to the action of an enzyme, 
 but this view is now put forward by several observers. 
 
 Lepine and Barral in 1890, in the course of an investigation 
 into the changes taking place in sugar in the blood, found that 
 glycolysis could be detected after blood had been shed, and that 
 it was more rapid in proportion as the temperature was raised 
 till it reached 54° C, at which point it suddenly stopped. The 
 physical condition of the fluid was not appreciably different 
 at this point from what it was at 52° C. when glycolysis was 
 very evident. Lepine and Barral found farther that the blood 
 drawn fi:om the portal vein lost more sugar than that taken 
 fi:om the splenic vein under identical conditions. 
 
 Arthus showed a httle later that the presence of actually 
 living elements in the blood were not essential to the process. 
 He collected blood aseptically into sterilised vessels and de- 
 fibrinated it. Maintaining it then at 10° C. for several days, 
 with antiseptic precautions, it continued to lose sugar. In 
 other experiments, he showed that glycolysis took place in 
 serum, in oxalated plasma free from corpuscles, and in blood 
 diluted with several volumes of water. He found that freshly- 
 
328 OXIDASES, OE OXIDISING ENZYMES. [CH. 
 
 drawn blood showed comparatively little glycolytic power, but 
 that it was developed on standing for some time. 
 
 These experiments are strikingly suggestive of the presence 
 of an oxidase ; the process shows a minimum point of activity 
 at 0°C., an optimum at 40° — 50° C, and a maximum one at 
 64° C. Its gradual development is remarkably like that of the 
 formation of the pancreatic digestive ferments. 
 
 The action was also examined with some completeness 
 in 1892 by Seegen, who found that the disappearance of the 
 sugar was not influenced by the presence of chloroform. This 
 reagent prevents the action of living cells and micro-organisms, 
 but does not inhibit the work of enzymes. Seegen found that 
 the exclusion of bacteria by other means did not prevent the 
 glycolysis, and argued consequently in favour of the presence 
 of a sugar-destroying enzyme. He demonstrated the same 
 influence of temperature as had been found by Lepine and 
 Barral and by Arthus. 
 
 Both Seegen and Arthus suggested that the enzyme was 
 formed in consequence of post mortem changes taking place in 
 the blood. Arthus held the source to be the white corpuscles 
 or leucocytes. Lepine and Barral found they could extract it 
 from the corpuscles in greater quantity than from the serum. 
 
 Further experiments made by many observers have shown 
 that the power of destroying sugar is not confined to the blood. 
 Lepine found that the removal of the pancreas of the dog was 
 followed by an intense diabetes, the ratio of sugar to urea in 
 the urine increasing enormously. He at once associated the 
 pancreas with the secretion of an oxidase. In its absence, sugar, 
 which would in the general course of events be destroyed in 
 the blood, passed out of the system in the urine. This view 
 was supported by the observation afready quoted, that the 
 destruction of sugar in the portal vein is much greater than 
 in the splenic. Lepine found further that the chyle, which 
 must receive some of the pancreatic secretion through the 
 intestinal wall, also contained the glycolytic property. The 
 injection of chyle into the jugular vein of an animal caused 
 a marked reduction in the sugar in the living blood. The 
 addition of chyle to a 1 per cent, solution of glucose kept 
 
XIX] ANIMAL OXIDASES. 329 
 
 in vitro at 38° C. caused a decided reduction of the amount 
 of the sugar. 
 
 Lepine and Barral in a subsequent paper stated that, in 
 the cases of both blood and chyle, the glycolytic power was 
 increased by a rise of temperature and impeded by the presence 
 of carbon dioxide. 
 
 Lepine subsequently carried out a series of researches on 
 'the pancreas of the dog, which confirmed his view that the 
 secretion of a glycolytic oxidsise is one of the functions of that 
 organ. He ground up the pancreas, with aseptic precautions, 
 immediately after its removal fi-om the body, and macerated it 
 for two to three hours at 38° C. in water containing '2 per cent, 
 of a mineral acid, and then neutralised the extract with sodium 
 hydrate. To 100 c.c. of the resulting liquid he added half a 
 gramme of glucose and digested it for an hour at 38° C. In a 
 series of such experiments he found there was a disappearance 
 of sugar, ranging from 10 to 50 per cent. A fresh pancreas 
 similarly extracted with water instead of dilute acid, yielded an 
 extract with very little glycolytic power. Lepine inferred that 
 a glycolytic enzyme could be prepared from the tissue he 
 used, just as similar treatment yields trypsin from the same 
 gland. 
 
 He supported his hypothesis still further by an experiment 
 in which he compared the glycolytic power of the blood leaving 
 the pancreas during active secretion with that possessed by 
 it when the gland was at rest. He found that during the 
 secretion caused by stimulation of the vagus, blood drawn from 
 the pancreatic vein possessed little glycolytic power, but that 
 the latter became considerable in the blood from the same vein 
 during the hours immediately following the cessation of the 
 secretion. 
 
 The work of Abelous and Biamfe, carried out in 1894, has 
 advanced our knowledge of this enzyme still further. These 
 investigators examined the action of the blood on salicyl- 
 aldehyde. This body was not oxidised to salicylic acid by 
 the air, nor by distilled water, nor by normal saline solution 
 (a solution of sodium chloride containing -6 per cent, of the salt). 
 But when defibrinated blood, or blood-serum was added to the 
 
330 OXIDASES, OE OXIDISING ENZYMES. [CH. 
 
 aldehyde and the mixture kept at a temperature of 37° C. 
 salicylic acid was formed. The oxidation was found to vary 
 in amount with the blood of different animals. 
 
 Abelous and Biam^s, following up Lepine's work on the 
 pancreas, looked for the presence of glycoljrtic power in other 
 tissues. They found that it was manifested also by the testes, 
 the thyroid glands, the liver, kidney, lungs, and spleen. 
 
 Spitzer has still more recently confirmed the statements of 
 previous observers, but he originally opposed the view that the 
 oxidation is due to the action of an enzyme, comparing it pre- 
 ferably with the oxidation produced by hydrogen-peroxide and 
 other oxidising agents. 
 
 In a paper which appeared in 1897 he published the 
 result of some researches on the oxidative powers of various 
 tissues, estimating their capabilities by measuring the quantity 
 of oxygen they could liberate from hydrogen-peroxide. In 
 some cases he also ascertained the quantity of salicyl-aldehyde 
 which they could convert into salicylic acid. He found the 
 tissues could be arranged in this respect in an order differing 
 but little from that published by Abelous and Biam^. Spitzer 
 has stated more recently that he has found them capable of 
 converting arsenious into arsenic acid. 
 
 Spitzer made a very important advance by ascertaining that 
 these different tissues owed their oxidising powers to nucleo- 
 proteid substances which they contained. He was able to 
 prepare nucleo-proteids from every tissue which he found capable 
 of inducing glycolysis, and he ascertained that the oxidative 
 energy of a tissue was proportional to the amount of nucleo- 
 proteid which it gave up to an appropriate solvent. He found 
 farther that these compounds showed the same peculiar relation 
 to temperature as the original organs exhibited, and that they 
 were affected in precisely the same way by various antiseptics 
 and poisons. 
 
 Seegen attributes the glycolytic power of the blood to a 
 similar substance. 
 
 Jaquet, and Salkowski and Katsusaburo Yamagiwa have 
 confirmed the results of the investigators quoted and have 
 concluded from their own researches that the action is due to a 
 
XIX] ANIMAL OXIDASES. 331 
 
 soluble enzyme which is destroyed by boiling and by prolonged 
 contact with alcohol. 
 
 Hammarsten has stated that the gastric mucous membrane 
 contains another enzyme belonging to this group. It has the 
 property of converting lactose (milk-sugar) into lactic acid. 
 
 Another oxidase has recently been described by Fieri and 
 Portier as existing in the gills, labial palps, and blood of several 
 of the acephalous molluscs, particularly Artemis exoleta. My a 
 arenaria, Tabes pullastra, Ostrea, edulis, and Pecten jacobceus, 
 besides one or two other species. It has many poiats of 
 resemblance to laccase, especially with regard to its action on 
 hydroquinone. The authors say that if one of the gills or a 
 piece of one of the labial palps of any of these molluscs is 
 warmed to 50° — 60° 0. in a solution of hydroquinone of 1 per 
 cent, concentration, the odour of quiuone is soon evolved, and if 
 the liquid is subsequently concentrated by evaporation, crystals 
 of quinhydrone are deposited over the surface of the tissue. 
 
 The oxidase can be extracted from the gills or the labial 
 palps of the animals by chloroform-water, or by solutions of 
 sodium fluoride or of salicylic acid, and can be precipitated from 
 its solvents by excess of alcohol. It is active in either neutral 
 or faintly acid media. Like laccase it does not decompose tyrosin. 
 
 Some observations of Abelous and Gerard call for notice 
 here, as indicating possibly the existence of enzymes possessing 
 the power of obtaining oxygen from somewhat stable compounds 
 instead of utilising the free oxygen of the air. Many observers, 
 among whom may be mentioned Gautier, Bokomy, Ehrlich, and 
 Binz have shown that various animal tissues have reducing 
 properties, being able to convert alkaline nitrates into nitrites. 
 Abelous and Gerard have examined various organs of the horse 
 from this point of view, and find many of them possess this 
 power but to different extents. They arrange the tissues in 
 the following order; Uver, kidney, supra-renal capsules, lung, 
 testicles, intestines, ovary, sub-maxillary glands, pancreas, 
 spleen, striated muscle, brain. An extract of these organs, 
 prepared with either water or glycerin, in the presence of 
 chloroform, and filtered till free from all traces of cells, when 
 mixed with a solution of nitrates and allowed to stand for some 
 
332 OXIDASES, OB OXIDISING ENZYSIES. [CH. XIX 
 
 time reduces them to nitrites. The extract loses the power on 
 being boiled. The action is not exerted at 0" C. but above that 
 poiat rises gradually and reaches a maximum at about 40° C. ; 
 from this point it diminishes being very little at 60° C. and 
 ceasing a little below 70° C. Hydrogen increases and carbon 
 dioxide diminishes its intensity. They attribute the action 
 consequently to an enzyme ' which they say works most ad- 
 vantageously in a faintly alkaline medium. The enzyme can 
 be precipitated from the extracts by alcohol, but contact 
 with the spirit soon weakens it. The nitrates most easily 
 reduced are those of potassium and ammonium. Besides 
 attacking nitrates it reduces butyric acid to butyric aldehyde 
 and bleaches methylene blue. 
 
333 
 
 CHAPTER XX. 
 
 ALCOHOLIC FERMENTATION. 
 
 Alcoholic fermentation is the one which has been longest 
 known and most closely studied. In an earlier chapter we 
 have briefly summarised the steps by which our present know- 
 ledge of it has been obtained, and we have seen how closely it 
 has been associated during the present century with the various 
 problems presented by the biology of yeast. 
 
 The first observations upon its true nature were those made 
 by Becher in 1682, in the course of which he ascertained the 
 fundamental facts that it was only possible in saccharine fluids, 
 and that the alcohol proceeded from a decomposition of some 
 constituent present in them. 
 
 More than a hundred years passed before any very im- 
 portant addition was made to our knowledge of the chemical 
 changes involved in the process. 
 
 The researches of Lavoisier mark a definite epoch in the 
 gradual advance of our information, for they first showed 
 the relation existing between the sugar and the products 
 found after the fermentation of the latter had taken place. 
 Lavoisier was the first observer who studied the process quanti- 
 tatively, and applied new methods of analysis in his research. 
 By determining the percentages of carbon, hydrogen, and oxygen 
 in the sugar, and in the products resulting from the fermen- 
 tation, he arrived at the conclusion that the operation consisted 
 of the separation of the sugar into two parts, one of which 
 became oxygenated at the expense of the other to form carbon 
 dioxide, while the second became converted into alcohol after 
 
334 . ALCOHOLIC FERMENTATION. [CH. 
 
 parting with a portion of its oxygen. He said further that if it 
 were possible to recombine these two substances, alcohol and 
 carbon dioxide, sugar would again be formed. 
 
 Though the methods of analysis used by Lavoisier were 
 imperfect and his figures were inaccurate in consequence, we 
 know that his general conclusions were sound. About the 
 year 1815 analyses by Gay-Lussac, Th^nard and de Saussure 
 fixed definitely the composition of sugar and alcohol. These 
 more accurate analyses confirmed Lavoisier's position, but re- 
 vealed a discrepancy which for a long time remained unexplained. 
 Computation of the composition of cane-sugar based upon the 
 weights of carbon dioxide and alcohol formed during its fermen- 
 tation, pointed to its having the formula CsHuOb (taking the 
 modem values of the atomic weights) the decomposition being 
 capable of expression by the equation 
 
 CeH^Oe = 2QHeO + 2CO,. 
 
 The analyses made by Gay-Lussac and Th^nard of cane-sugar 
 itself, demanded the formula CisHjjOu. These authors were 
 unable to account for the discrepancy except on the assumption 
 that the analyses of the sugar were not quite accurate. Dumas 
 and Boullay suggested as a more probable explanation, that 
 the fermentation was accompanied by the absorption of water 
 before or at the same time as the splitting of the sugar. Thus 
 the equation representing the reaction would become 
 
 CJIjOn + HP = 40^60 -1- 4C0,. 
 
 Very shortly afterwards the discovery was made by Dubrun- 
 faut that before cane-sugar could ferment it became transformed 
 into another sugar that was not crystallisable, and Biot showed 
 that under the action of weak acids it became decomposed into 
 two other sugars, now known as glucose and finictose. 
 
 Berthelot was the first to show that besides inorganic 
 reagents an enzyme possesses the power of bringing about this 
 transformation and that the living yeast-cell forms this enzyme, 
 already described in a preceding chapter under the name of 
 invertase. 
 
 The fermentation of cane-sugar has thus been proved to 
 take place in two stages; in the first it is split up with 
 
XX] ALCOHOLIC FERMENTATION. 336 
 
 hydrolysis into the two hexoses glucose and fructose, according 
 to the equation CiaHjaOu + H2O = CeHuOn + CeHiaOe ; in the 
 second both of these are decomposed, forming alcohol and 
 carbon dioxide as suggested by Gay-Lussac. 
 
 Another fact came to light sometime after the publication 
 of the results of Dumas, Dubrunfaut, and Biot, the significance 
 of which for some time met with but slight recognition. In 
 1847 Schmidt of Dorpat found the presence of succinic acid in 
 small amount in fermenting liquids. It remained however for 
 Pasteur to complete the discovery of which this fact was part. 
 To his careful researches we are indebted for our knowledge 
 that from 4 to 5 per cent, of the cane-sugar used in a fermen- 
 tation does not undergo the ordinary decomposition, but splits 
 up into glycerine, succinic acid and carbon dioxide. 
 
 Pasteur has suggested the following equation as representing 
 the change. 
 
 esOsHiA + 6OH2O = 24C4H,04 -1- I44C3H8O3 + 60COj 
 succinic acid glycerine. 
 
 Monoyer has represented the reaction rather differently 
 thus : — 
 
 8CeH,,0e + 6H,0 = 2C4He04 + 12C3H8O3 -I- 4C0, +0, 
 
 According to this hypothesis the change is associated with 
 the liberation of a small quantity of oxygen, a fact the im- 
 portance of which will appear later. 
 
 Bechamp and later Duclaux have found that acetic acid 
 also is formed in small quantities during alcoholic fermentation. 
 Duclaux states that its presence is constant, but that the 
 amount never exceeds "05 per cent, of the weight of sugar, if 
 the fermentation is stopped as soon as all the latter has been 
 decomposed. Bechamp says that under appropriate conditions 
 this quantity can be largely exceeded. 
 
 Other observers have noticed variable but always minute 
 quantities of aldehyde as accompaniments of fermentation. 
 
 The ordinary alcohol produced is often found to be mixed 
 with higher alcohols, propylic, isobutylic, amylic, caproic, oenan- 
 thylic, and caprylic all having been identified, amylic alcohol 
 sometimes occurring in some quantity. 
 
336 ALCOHOLIC FERMENTATION. [CH. 
 
 The amounts of all these less prominent constituents seem 
 to vary a good deal under different conditions of fermentation. 
 More succinic acid is produced when the operation is slow 
 than when it is rapid. The different kinds of yeast show 
 considerable differences among themselves with regard to these 
 various products. Indeed the latter seem to be more closely 
 related to the biological work of the cells than the ethylic 
 alcohol, the appearance of which is the most conspicuous feature 
 of the process. The relation of the alcohol to the ordinary 
 metabolism still remains a subject of discussion. 
 
 We have seen that for a long time alcoholic fermentation 
 was associated only with cane-sugar. Much light has been 
 thrown upon the composition of various sugars in recent years, 
 especially by the researches of Emil Fischer. Without entering 
 into details, which would be beyond the scope of the present 
 work, we may divide the sugars that are of general occurrence 
 into two main groups, the simplest of which have the empirical 
 formula C„Hj„0„, the value of n in those known up to the 
 present ranging from 2 to 9. The members of the other group 
 are more complex, being theoretically composed by the com- 
 bination of either 2 or 3 molecules of a hexose with elimination 
 of one or two molecules of water. Thus we have cane-sugar 
 C12H22O11 corresponding to 2(C6H]206) — HaO, and raffinose, 
 CisHsjOis represented by 3(C5Hi206) — 2H2O. This group is 
 generally known as the polysaccharides. 
 
 The sugars which are fermentable appear to belong entirely 
 to the first group and to be represented especially by the hexoses 
 CeHijOs- Some of the others are however capable of ferment- 
 ation, but only those in which the value of « is divisible by 3. 
 
 Certain of the polysaccharides appear at first sight to be 
 fermentable, particularly cane-sugar and maltose. It has been 
 found however by Fischer that they are not directly attacked 
 by the alcohol-producing principle, but undergo a preliminary 
 hydrolysis with the consequent formation of hexoses. We have 
 seen that this fact was established for cane-sugar by Dubrunfaut 
 and later by Berthelot. This sugar by the action of invertase 
 yields glucose and fructose. Similarly each molecule of maltose 
 is converted by hydrolysis into two molecules of glucose; lactose 
 
XX] ALCOHOLIC FERMENTATION. 337 
 
 or milk-sugar is resolved into glucose and galactose. The other 
 polysaccharides behave similarly. 
 
 The different hexoses also show greater or less facility of 
 conversion into alcohol and carbon dioxide. Glucose is most 
 readily attacked, fructose less so, and galactose undergoes 
 fermentation only with great difficulty. 
 
 The progress of a fermentation has been found to depend 
 also, and in very large measure, on the species of yeast which is 
 employed. A great development of our knowledge in this 
 direction has been made during the last twenty years, which 
 has been due very largely to the labours of Hansen. 
 
 It is difficult to overestimate the importance of his re- 
 searches into the biology of yeast. Indeed it may be said that 
 since Pasteur no one has done so much to elucidate the 
 problems of alcoholic fermentation. Hansen was one of the 
 pioneers who elaborated exact methods for the cultivation of 
 micro-organisms in a state of purity or isolation. By the 
 methods which he adopted he made it clear that the Saccharo- 
 mycetes really form a separate and distinct group of alcohol- 
 producing fungi, and he brought out new points of view with 
 regard to species and races. It is not in the province of this 
 work to discuss his results in detail, as they turn more on the 
 biological peculiarities of the yeast-plant than on the wider 
 question of the chemical and physical peculiarities of the 
 changes which it induces. As an illustration of the scope of his 
 researches we may however mention the investigations he made 
 upon the conditions of the formation of spores and films, and 
 upon the morphology of these developmental structures and 
 their course of life, taking especially the yeast known as 
 Saccharomyces apiculatus as a representative of the group. 
 His experiments upon the alterations induced by differences of 
 conditions of life, such as exposure to the cold of winter, and 
 upon variations following such primary alteration, have thrown 
 a flood of light upon this section of the subject. 
 
 Hansen's researches have had even greater effect oa the 
 industrial methods of brewing and of the manufacture of wine. 
 It is hardly too much to say that he and his pupils have almost 
 revolutionised the methods in use. 
 
 G. F. 22 
 
338 ALCOHOLIC FERMENTATION. [CH. 
 
 Hansen's work has not however been all that has been 
 carried out in this field of research. Other investigators have 
 described other species, while other fungi than yeasts, especially 
 species of Mucor, have been found capable of setting up fermen- 
 tation. A copious literature has sprung up on the subject, 
 which cannot however be considered here in any detail. 
 
 The course of action on mixtures of fermentable sugars in 
 the presence of different yeasts is often very different. Most of 
 the latter contain hydrolytic enzymes which efifect the hydrolysis 
 of the polysaccharides cane-sugar and maltose. If either of 
 these inverting enzymes is not present, however, the yeast 
 cannot ferment the corresponding sugar. The yeasts for ex- 
 ample which do not form invertase have no action on cane- 
 sugar. Hansen's six true species of Saccharomyces all hydrolyse 
 both cane-sugar and maltose, and subsequently cause their 
 products to undergo alcoholic fermentation. S. Marxianus 
 differs in not attacking maltose, while S. membrancBfadens does 
 not ferment either of them. Other organisms have similar 
 idiosyncracies. 
 
 As the fermentation of sugars by yeast is thus seen to be a 
 function of the living cell, everything that affects the life of 
 the latter has a certain influence on its fermentative power. 
 Substances which serve as food for the yeast promote fermen- 
 tation; acids and bases are deleterious to the cell, their 
 influence being the more marked as their concentration is 
 increased. The proportion of acids which are effective in this 
 direction have been given differently by various observers. 
 Hayduck found that •! °/o of sulphuric acid reduced the energy 
 of fermentation, while -7 "/o inhibited it altogether. Hydro- 
 chloric acid was found to be less deleterious, but fermentation 
 was unfavourably influenced by '2 "/». The development of the 
 yeast-plant was promoted by '02 "/o of sulphuric acid or by 
 ■1 — "5 "/o of lactic acid, but unfavourably influenced by '07 °/o of 
 the former or l'5"/o of the latter. In the presence of •2°/^ of 
 sulphuric acid or 4°/o of lactic acid the yeast-cells did not 
 undergo multiplication. Bokomy found the influence of sul- 
 phuric acid was deleterious, and' fermentation was impeded 
 when as little as one part in 20000 was present, while four 
 
XX] ALCOHOLIC FERMENTATION. 339 
 
 times that amount stopped it entirely. The writer found that 
 the development of the yeast-plant was markedly impeded by 
 the presence of "S'/o of lactic acid. 
 
 Among deleterious bodies Bokorny mentions corrosive sub- 
 limate, potassic permanganate, free chlorine, and iodine. Phos- 
 phorus also impedes fermentation. Bokorny prepared a culture 
 medium containing one part of it in 20000, besides cane-sugar 
 and mineral salts. A feeble fermentation was excited in this 
 liquid 6 hours after the addition of yeast. 
 
 Bromine is less harmful than chloride or iodine. One part in 
 10000 parts of a fermenting solution does not cause inhibition ; 
 solutions of potassic chlorate or potassic iodate of 1 per cent, 
 concentration are also without deterrent effect. Very dilute 
 solutions of potassic cyanide or strychnine nitrate (1 : 50000) 
 do not cause complete inhibition, nor does quinine acetate in a 
 concentration of "1 per cent. 
 
 Other carbohydrates than sugars have been stated by 
 various writers to be capable of undergoing alcoholic fermen- 
 tation. L^vy has produced alcohol by fermenting artichoke 
 tubers with yeast ; Buchner has shown that the glycogen of the 
 yeast can give rise to alcohol. 
 
 Pasteur found that when a considerable quantity of yeast 
 was allowed to act upon a very small amount of sugar the 
 fermentation proceeded for some time after the sugar had all 
 disappeared. The later stages of the process were marked 
 consequently by the fermentation of other carbohydrates which 
 the yeast contained. He showed this to be the case by ex- 
 amining the liquid for sugar as the fermentation proceeded. 
 Eventually three times as much carbon dioxide was evolved as 
 could have resulted from the sugar employed. The alcohol 
 formed corresponded in amount to the volume of the carbon 
 dioxide. 
 
 Pasteur showed further that when fresh yeast was mixed with 
 water and kept for a time at 25° C, carbon dioxide was very 
 soon evolved and on distillation of the liquid after a not very 
 prolonged exposure, alcohol was found to be present. Under the 
 conditions of the experiment this must have proceeded from the 
 fermentation of the other carbohydrates of the yeast employed. 
 
 22—2 
 
340 KOUMISS. [CH. 
 
 These experiments are found upon examination however ta 
 be strictly comparable to those in which the polysaccharides 
 take part. The material is not directly fermentable, but gives 
 rise first to sugars of the hexose type. The carbohydrate which 
 the artichoke tuber contains in greatest amount is inulin, and 
 we have seen in a preceding chapter that the zymogen of inulase 
 is also present in its cells. Inulase is produced from the latter 
 and converts the inulin into fructose, which is easily fermentable 
 by the yeast employed. The method adopted in practice is to 
 treat the washed and sliced tubers with 4 times their weight of 
 water containing 2 per cent, of potassium bitartrate. The 
 liquid is allowed to stand for four or five hours at a temperature 
 of about 16° C. and is then decanted off", and the extraction 
 repeated with a further quantity of the same solution. The 
 resulting extract, containing the fructose resulting from the 
 hydrolysis of the inulin, has a specific gravity of about 1030. It 
 is sterilised by heating it to the boiling point three times on 
 successive days and the yeast is then added, after which fer- 
 mentation proceeds rapidly. The latter process is conducted at 
 a temperature of 20 — 25° C. and is continued for 3 days, the 
 alcohol being subsequently separated by distillation. The inulin 
 is converted into fructose during the preliminary extraction with 
 the dilute solution of potassium bitartrate. Similarly in 
 Pasteur's experiments there can be no doubt the fermentation, 
 supervened upon a preliminary hydrolysis. 
 
 The fermentation of milk-sugar or lactose underlies the 
 production of the beverages kno\vn as Koumiss and Kephir, 
 which are manufactured and used largely in Russia and in 
 central Asia. Lactose is a polysaccharide having the same 
 empirical formula as cane-sugar and like it is decomposed by 
 an enzyme of the same type as invertase, as has already been 
 mentioned. The products of its hydrolysis are glucose and 
 galactose, both of which can give rise to alcohol though the 
 last named is only fermentable with difficulty. 
 
 Koumiss is chiefly prepared in the steppes of South Western 
 Siberia and the countries adjoining. The milk used in its 
 manufacture is yielded by mares, and there are variations in its 
 mode of preparation in different parts. It is apparently due to- 
 
XX] KEPHIR. 341 
 
 the action of several organisms, one of which is a yeast, while 
 others ate lactic-acid-producing bacteria. The organisms are 
 not isolated for the purpose of the fermentation, but a certain 
 quantity of old koumiss is mixed with the fresh milk, usually 
 one part of the former to ten parts of the latter. The fermen- 
 tation is conducted in small casks or vats which are fitted with 
 a stirring apparatus. It is found advisable to employ the latter 
 for about 5 minutes at the commencement of the operation so 
 that the whole mass of liquid may be well aerated. When the 
 fermentation is nearly finished the liquid is transferred to 
 strong bottles which are closely corked and the corks wired 
 tightly down. The continuation of the fermentation makes 
 the koumiss an effervescent beverage. The change which takes 
 place in the milk is the production of alcohol, carbon dioxide, 
 and lactic acid at the expense of the milk-sugar. The latter 
 exists in the milk to the extent of 5*5 per cent. In the koumiss 
 there is usually about 1'3 per cent, of sugar, 1'6 per cent, of 
 alcohol, and nearly 1 per cent, of both lactic acid and carbon- 
 dioxide. 
 
 There appears to be a slight diminution in the proteids of 
 the milk, amounting to about "1 per cent., but the nature of 
 the changes they undergo has not been ascertained. There is 
 reason to think the casein is converted into acid-albumin and 
 peptone, as it does not curdle under the influence of the lactic 
 acid as normal milk would do. The agenj; effecting this is 
 probably a proteolytic ferment, such as we have seen to be 
 secreted by both yeast and bacteria. 
 
 Kephir is another effervescent beverage, prepared from cow's 
 milk, and very much used in the mountains of the Caucasus. The ' 
 nature of the organism employed in the fermentation is not well 
 known, but it appears to consist of a yeast which is associated 
 symbiotically with one or more bacteria. It exists in the 
 form of gelatinous lumps which are mainly comp6sed of a modi- 
 fication of cellulose, probably forming the sheaths or zoogloea of 
 the bacteria, and which entangle the yeast mechanically. The 
 method of preparation of the kephir is much the same as that 
 of koumiss. The milk is kept at a temperature of 18 — 19° C. 
 and a quantity of the organism is added to it in the fermenting 
 
342 THE GINGER-BEER PLANT. [CH. 
 
 vat, and the liquid kept in agitation. The fermentation is 
 usually well advanced in ahout 24 hours, when the liquid is 
 bottled and the fermentation completed in the bottles. 
 
 Mix has described a similar organism which is in use in 
 North America, which can ferment lactose and glucose. 
 
 The products of the kephir fermentation differ somewhat 
 from those of koumiss. The sugar of the milk falls from 4 to 
 about 2 per cent. ; the fatty matters are diminished in about 
 the same proportion, and the proteids are reduced to about 
 three-fourths of their original quantity. The alcohol generally 
 reaches a concentration of nearly one per cent., and the lactic 
 acid is formed in slightly larger amount, the ratio between 
 them being 8 : 9. Koumiss we have seen contains sensibly less 
 lactic acid than alcohol. The proteids do not appear to be 
 peptonised by the kephir organism, though some change takes 
 place in the casein in consequence of which it is very incom- 
 pletely precipitated by the lactic acid. 
 
 In both these processes we have almost certainly to recognize 
 two different fermentations proceeding side by side, the one 
 due to the yeast, and resulting in the formation of the alcohol ; 
 the other due to the bacteria, and giving rise to the lactic 
 acid. 
 
 A third instance of an alcoholic fermentation combined 
 with other chemical changes is afforded by the curious or- 
 ganism which is used in many rural districts of England for 
 the manufacture of an effervescent beverage known as ginger- 
 beer. In several respects it presents many resemblances to 
 the kephir of the Caucasus. Though it has been in use for 
 many years it was only in 1887 that attention was called to 
 its peculiar composition. A complete investigation into the 
 nature of the organisms composing it was carried out by 
 Marshall Ward in 1891. 
 
 The ginger-beer plant resembles the kephir organism in its 
 outward appearance, being found in white semi-translucent 
 masses of irregular shape and almost gelatinous consistency. 
 In size they vary from the dimensions of a pin's head to a 
 diameter of nearly an inch. In a fermenting liquid they grow 
 very freely, and become much softer and more slimy than they 
 are in the dry condition. 
 
XX] THE GINGEE-BEER PLANT. 343 
 
 The appearance of a fermentation is thus described by 
 Marshall Ward : — " The most striking characteristics of the 
 above-described lumps of ginger-beer plant however only 
 become evident when they are placed in saccharine solutions 
 and they are perhaps best shown roughly as follows. A soda- 
 water bottle is filled three parts full of Pasteur's fluid, or any 
 other similar solution of sugar in water, and a lump of ginger 
 added. Into this mixture are placed a few lumps of the ginger- 
 beer plant ; the bottle is then well corked and laid in a warm 
 place, and observed from time to time.' In from 24 to 28 hours 
 depending on the season, temperature, &c., the liquid is 
 observed to become more and more turbid, and bubbles of gas 
 begin to ascend, the fermentation soon goes on rapidly, and 
 unless the cork is well secured by string or wire, it will be 
 blown out. This primary turbidity is found to be due almost 
 entirely to innumerable yeast-cells, and further examination 
 proves that these yeast-cells are shed from the lumps of ginger- 
 beer plant (which rise and fall with varying buoyancy in the 
 liquid) and then multiply in the medium, and soon form a 
 greyish deposit at the bottom. The buoyant dancing of the 
 lumps is seen to be determined by the copious evolution of gas 
 bubbles from their surfaces.... The liquid... is not only sur- 
 charged with carbon dioxide, but is evidently more or less 
 viscous, with a viscosity which is different from any property 
 directly imparted to it by the sugar and other materials added.... 
 As time goes on this viscosity increases and it sometimes 
 happens that the liquid becomes so thick that the gas-bubbles 
 rise comparatively slowly. The viscosity is not due to the 
 mere presence of yeast-cells, because they fall to the bottom ; 
 it is due to the presence of innumerable swollen or slimy vermi- 
 form bodies distributed through the mass of the liquor. Myriads 
 of rod-shaped bodies (Bacteria) are also observable.. . .The ginger 
 beer is distinctly acid aa well as viscous; the colour of the 
 liquid is paler than that of the original solution." 
 
 A very detailed and elaborate investigation into the compo- 
 sition of the lumps of the " Plant " showed it to be composed 
 of several organisms, of which two specific forms constitute the 
 ginger-beer plant proper, while the rest are merely due to 
 admixture of other forms from outside. The two in question 
 
344 THE GINGER-BEER PLANT. [CH. 
 
 are a Yeast, Saccharomyces pyriformis, and a Schizomycete 
 which has been named Bacterium vermifurme. Marshall Ward 
 found it possible indeed under proper conditions to reconstruct 
 the "Plant" from pure cultures of these separate organisms 
 originally isolated from a fermenting liquid. 
 
 The bacterium is a peculiarly vermiform organism con- 
 sisting of filamentous or rod-like, often homogeneous, bodies 
 much coiled and twisted together, which are sometimes found 
 to be broken up into short rodlets or even cocci, the cells being 
 thus arranged in chains. The separate filaments are invested 
 with a pellucid swollen gelatinous sheath, wliich gives to the 
 complete organism its peculiar consistency. In the midst of 
 this gelatinous matrix of coiled filaments the yeast-cells appear 
 to be mechanically entangled. The whole forms a symbiotic 
 plant, which has a superficial resemblance to the thallus of a 
 lichen, in which a yeast has replaced the alga. 
 
 The ginger-beer plant is capable of causing fermentation in 
 solutions of grape-sugar and of cane-sugar, but it acts most 
 readily on the latter. It differs from kephir in not being 
 able to ferment sugar-of-milk (lactose). 
 
 In addition to the carbon dioxide evolved, the solution at 
 the end of a fermentation contains alcohol and acetic acid, with 
 relatively large quantities of another acid which has not been 
 completely examined, but which appears to resemble lactic acid. 
 The alcohol and acetic acid are formed during the earlier stages 
 of the fermentation, the other acid appearing somewhat later. 
 
 Another organism of a similar nature has recently been 
 investigated by the writer in collaboration with Marshall 
 Ward. It occurs as a growth parasitic upon the sugar- 
 cane, and was originally procured from Madagascar. It consists 
 of gelatinous masses closely resembling the ginger-beer plant, 
 and like it, is composed of a yeast and a bacterium, the 
 latter again being very much like the bacterium last described. 
 It ferments cane-sugar, maltose, glucose, and fructose, but not 
 lactose. The products of the fermentation are alcohol, carbon 
 dioxide, and certain acids, of which the chief are acetic and 
 succinic. When cultivated in solution of cane-sugar it produces 
 a quantity of viscous material, which apparently consists in 
 
XX] KOJi. 345 
 
 great part of the extremely diffluent sheaths of the bacterium. 
 This substance can be obtained in large quantities from pure 
 cultures of the latter, and it has been found to consist of two 
 modifications of cellulose, resembling the dextrans, or hemi- 
 celluloses of various writers. The yeast produces little besides 
 alcohol and carbon dioxide, and small quantities of succinic 
 acid. The bacterium gives rise to the volatile acetic acid, but 
 forms the latter directly from the sugar and not indirectly through 
 the medium of the alcohol, which, unlike the ordinary acetic 
 microbe, it is unable to decompose. The bacterium is peculiar 
 also in being one of the very few which secrete invertase. 
 
 Besides the yeasts and such symbiotic fungi as have been 
 described, other fungi have the power of exciting alcoholic fer- 
 mentation. In the preparation of a spirituous beverage much 
 in use in the far East a material is employed by the Chinese 
 to which they give the name of Koji. This is prepared by 
 inoculating freshly steamed rice with the spores of Eurotium 
 (Aspergillus) oryzew. The grain, husked and bleached, is 
 steamed for 12 hours till it is soft ; it is then cooled on straw 
 mats. As soon as the temperature has fallen to about 28 — 35° C. 
 a little is mixed with the spores of the fungus and stirred into . 
 the rest of the mass. The mats with their coating of rice are 
 then allowed to stand in a sort of cellar, frequently dug out of 
 the ground, for 18 — 24 hours, at the end of which time a good 
 mycelium has developed from the spores. The grains are next 
 worked up by the hands and exposed on trays in the warmest 
 part of the cellar. This process is repeated about 12 hours 
 later, and the mass is cooled and moistened with a little water. 
 A third working up of the mass of grains and fungus is can-ied 
 out after a further similar interval, and after 14 — 16 hours more 
 incubation the Koji is ready for use. The active principle in 
 the fermenting mass is derived from the fungus, which as we 
 have already seen contains diastasic, inverting, and other en- 
 zymes. It has also the power of forming alcohol at the expense 
 of the sugars which are produced by the action of these enzymes 
 on the amylaceous and saccharine materials with which it is 
 mixed in the processes of manufacture. It does not affect 
 lactose. " 
 
346 ARRACK. [CH. 
 
 Another fungus possessiug similar properties has been de- 
 scribed by Wehmer, who has named it Aspergillus wentii. It 
 closely resembles A. oryzece, but can only be obtained by 
 covering beans, which have been boiled and superficially sun- 
 dried, with the leaves of Hibiscus tileacus. The fungus invari- 
 ably makes its appearance on the leaves. It is characterised by 
 possessing a light chocolate colour. The method of collecting 
 the mould exactly resembles that employed by the Chinese in 
 the manufacture of arrack. 
 
 Arrack is prepared in Java from rice starch by the action of 
 a substance which the natives call " raggi." This is a material 
 in which many microbes and fungi abound, but the one to 
 which the alcoholic fermentation is due is not known with 
 certainty. "Raggi" has been examined lately by Went and 
 Prinsen Geerligs, who have made cultures of several of the 
 organisms it contains. One of these has been called Chlamy- 
 domucor oryzece ; it is a fungus which developes a much-branched 
 unseptated mycelium. It is aerobic, and secretes rennet and 
 diastase, but does not invert cane-sugar nor set up the alcoholic 
 fermentation of glucose. Another fungus present has been 
 named Monilia javanica. It has the power of fermenting 
 glucose, levulose, raffinose, maltose and cane-sugar, the latter 
 of which it inverts. A true Saccharomyces, which the authors 
 call S. vordemannii, appears to be the principal agent in the 
 production of the alcohol of the arrack. 
 
 When Mucor racemosus is cultivated in a solution of sugar, 
 instead of forming an ordinary mycelium, it is found in the 
 form of round or ovoid cells which resemble very closely those 
 of brewers' yeast, and which like the latter multiply rapidly by 
 budding. Other species of Mucor behave similarly. These fungi 
 have the power of fermenting glucose but apparently cannot 
 attack fructose. When cultivated in invert-sugar they conse- 
 quently can only ferment one moiety of it. Generally the 
 fermentation is much slower than that excited by the true 
 yeasts and is more easily stopped by the alcohol produced. 
 M. erectvs can produce as much as 8 per cent, of spirit in a 
 saccharine solution; M. racemosus only about 3 per cent. ; M. 
 stoloni/er only 1-3 per cent. 
 
XX] ALCOHOLIC FERMENTATION. 347 
 
 Mucor racemosus does not produce alcohol and carbon 
 dioxide in the same proportion as yeast, viz. 100 : 96'3, but in 
 relatively larger amount, the ratio being stated by Fitz as 
 123"1 : 100. The spirit contains a little aldehyde, and succinic 
 acid is an accompanying product. Whether or no glycerine is 
 formed is doubtful. 
 
 The different species of Mucor are not all able to ferment 
 cane-sugar, as some of them do not secrete invertase and cannot 
 therefore effect the preliminary hydrolysis. 
 
 Penicilium glaucum and Rhizopus nigricans also are said to 
 be capable of exciting alcoholic fermentation. 
 
 A curious bacillus was described in 1891 by Perdrix, who 
 isolated it from the water of the Seine. It has among other 
 properties the power of producing alcohol when cultivated in a 
 starchy medium. The starch is first hydrolysed to sugar and 
 the latter is partially fermented. A solution of 4"5 grms. of 
 starch in 200 c.c. of water fermented by it in the absence of air 
 was found to yield the following constituents at the end of the 
 fermentation : — 
 
 Sugar 
 
 3'52 grms. 
 
 Acetic acid '08 grm. 
 
 Ethyl alcohol 
 
 ■347 „ 
 
 Butyric acid "175 „ 
 
 Amyl alcohol 
 
 •082 „ 
 
 
 During the process the liquid gave off "407 grm. of carbon 
 dioxide and '022 grm. of hydrogen. 
 
 Perdrix named the microbe Bacillus amylozyme. 
 
 It will ferment the starches of wheat, maize, rye and barley. 
 When yeast is added to a mixture in which it has excited a 
 fermentation the two organisms will work symbiotically and 
 under these conditions 90 per cent, of the theoretically possible 
 quantity of alcohol may be formed. 
 
 Fitz has stated that glycerine as well as sugar is capable of 
 giving rise to alcohol, the organism producing it being a microbe 
 to which he has given the name Bacillus aethylicus, and which 
 can be cultivated from an infusion of hay. He cultivated it in 
 •1 per cent, solution of Leibig's extract to which 3 per cent, of 
 glycerine had been added. 200 grammes of glycerine yielded 
 25"8 grammes of alcohol. 
 
348 ALCOHOLIC FERMENTATION. [CH. 
 
 Fitz's organism has been cultivated also by Emtnerling, who 
 found it capable of forming alcohol from mannitol as well as 
 from glycerine. Emmerling has shown that Granulobacter 
 butylicus produces butylic alcohol from glucose. 
 
 Alcohol also appears as a bye-product in several other fer- 
 mentations which will be considered in a subsequent chapter. 
 
 The formation of alcohol by the vegetable cell is not, how- 
 ever, confined to the lowly organisms which we have so far 
 described. There are many facts which lead us to the view 
 that it is a property of living protoplasm, which is, however, 
 only exercised when the latter is exposed to abnormal condi- 
 tions, the chief of which is the absence of oxygen. The earliest 
 of the observations recorded on this point were those of B^rard 
 in 1821. These are very meagre and incomplete, but we learn 
 from them that he found that when certain fruits were shut up 
 in a closed vessel containing carbon dioxide or some inert gas, 
 there was a continuous production of carbon dioxide, which he 
 attributed to a kind of fermentation. 
 
 Lechartier and Bellamy were the first observers to record 
 carefully the course of events under these conditions. Their 
 observations were published in 1869 and 1872, and they throw 
 a good deal of light upon alcoholic fermentation in general. 
 They examined principally succulent fruits such as apples, pears, 
 cherries, and gooseberries: also potatoes, and grains of wheat. 
 Placing these in closed vessels from which tubes were led off to 
 receivers containing mercury, they found that they gradually 
 absorbed all the free oxygen around them, and at the same time 
 evolved carbon dioxide. The disengagement of the latter gas 
 continued after all the oxygen had disappeared, but became 
 gradually less as time went on. During the experiments, the 
 fruits were kept from contact with each other, and from the 
 walls of the vessels in which they were enclosed. After several 
 months the fruits were removed, mashed to a pulp, and the 
 mass distilled, when a considerable quantity of alcohol was 
 separated from them. Very careful microscopic examination 
 showed the absence of all micro-organisms. 
 
 One of their experiments may be quoted in detail. On 
 Nov. 12, two pears, weighing 157 grammes and 125 grammes 
 
XX] ALCOHOLIC FERMENTATION. 349 
 
 respectively, were suspended in a glass vessel so as not to be in 
 contact with its sides, and a quantity of calcic chloride was at 
 the same time introduced, so that only dry air should surround 
 the fruits. The vessel was then closely stoppered, and from 
 the stopper a leading tube was taken to a receiver containing 
 mercury. The apparatus was kept closed till the 19th of the 
 following July. During that time, 1762 c.c. of carbon dioxide 
 were evolved, and the fruits at the moment of opening were 
 found to contain 2'62 grammes of alcohol. The pears had kept 
 their natural colour ; the skin was wrinkled but not moist ; their 
 condition was that of normally withered fruits. On weighing 
 them it was found they had given off 1^4 grammes of water, 
 but still contained a quantity equal to nearly 70 per cent, of 
 their weight. Towards the end of the experiment the evolution 
 of carbon dioxide gradually diminished. From March 3 to 
 April 8, only 28 c.c. were disengaged and the formation ceased 
 entirely at the latter date. The authors point out that this fact 
 was incompatible with the view that the fermentation was due 
 to an alcohol-producing micro-organism ; moreover, microscopic 
 investigation of the pulp taken at different distances from the 
 centre of the fruit showed that no such bodies were present. 
 In some of their experiments spores or cells of yeast made their 
 way into the fruits, and in these cases the progress of the fer- 
 mentation was altogether different from the one described, the 
 evolution of carbon dioxide continuously increasing after the 
 fermentation was once initiated. 
 
 During the time when the spontaneous fermentation was 
 taking place, the fruit became sensibly modified, the cellular 
 tissue being wholly or in part dissociated, and becoming 
 syi'upy, while the seeds lost the power of germination. 
 
 The experiments of Lechartier and Bellamy were subse- 
 quently repeated by Pasteur and their results confirmed. 
 
 When fruits were gathered and allowed to ripen without 
 being deprived of oxygen, there was no formation of alcohol 
 accompanying the process. The alcoholic fermentation in the 
 cases described was thus undoubtedly set up by the peculiar 
 conditions of the experiments. 
 
 Investigations published byBrefeld in 1876, and by de Luca. 
 
350 ALCOHOLIC FERMENTATION. [CH. 
 
 in 1878, have shown that similar results are met with in 
 experiments with seeds, leaves, and branches. About the same 
 time Miintz found that when he cultivated plants in pots in an 
 atmosphere of nitrogen instead of carbon dioxide, alcohol was 
 produced in their Hviug cells. He carried out his investigations 
 on many different plants, including maize, beets, cabbages, and 
 chicory, all of which remained healthy so long as they were 
 under observation. He detected alcohol in their tissues by 
 the iodoform reaction. 
 
 The occurrence of alcohol in the more purely vegetative 
 parts of plants, observed more than 20 years ago by Brefeld 
 and by de Luca as already mentioned, has met with abundant 
 confirmation in recent years. 
 
 Berthelot determined its presence in 1893 in the leaves of 
 wheat. He took leaves fireshly gathered from the plants and 
 transferred them at once to a flask, and replaced the air by 
 hydrogen. He then exposed the flask to a temperature of 
 110° C. and collected the distillate, which he found to contain 
 alcohol. In an experiment conducted on a larger scale he 
 obtained sufficient alcohol to prepare ethylene from it. 
 
 Maz6 in 1899 claimed to show that the leaves of the vine 
 normally contain alcohol. He obtained between 50 and 100 milli- 
 grams of spirit from 35 grams of the fresh tissue. He found 
 also that alcohol is present in measurable quantities in the 
 germinating seeds of the pea when access of oxygen to them 
 is prevented. 
 
 Devaux has shown that alcohol can be formed in woody 
 stems and that its presence is probably attendant upon partial 
 asphyxiation. He found the air in the stems of such trees to 
 be relatively poor in oxygen, containing often not more than 
 half the normal amount. By raising the temperature of such 
 stems so as to quicken their respiration, the oxygen content of 
 their interior rapidly sank, becoming after 3 days at 35° C. as 
 little as 1 per cent, in several cases. The simultaneous output 
 of carbon dioxide was greatly increased. Investigation showed 
 a coincident formation of alcohol. 
 
 Devaux has shown that this internal asphyxia exists in 
 some trees at the normal temperature (12° — 20° C.) and that 
 
XX] ALCOHOLIC FERMENTATION, 351 
 
 the quantity of alcohol in the fresh wood of such trees amounts 
 sometimes to 1 c.c. of absolute spirit per kilogram of wood. 
 
 Some experiments carried out in 1896 by Gerber, have also 
 shown the power of vegetable protoplasm iu the higher plants 
 to set up alcoholic fermentation. This observer carried out a 
 long series of researches on the changes undergone by various 
 fruits in the course of their maturation, and paid special 
 attention to the respiratory changes which can be observed 
 during this period. Attention may be called especially to the 
 ripening of fruits which contain considerable quantities of 
 tannin. Among these may be noted especially the fruit of 
 the Chinese date-plum {Diospyros kaki), which when ripe con- 
 tains a very agreeable volatile aromatic perfame. When- this 
 fruit was allowed to ripen in a confined atmosphere, and the 
 pulp examined, it was found to yield ten per cent, of ethylic 
 alcohol, mixed with other alcohols among which amylic 
 was conspicuous. The pulp also contained acetic acid. The 
 aromatic principle was found to be a mixture of amyl and ethyl 
 acetates, with traces of oenanthylates and pelargonates. The 
 tannin disappeared during the ripening, but the alcohols formed 
 did not appear to be developed from it, but rather from the 
 sugars of the fruit. Gerber associates its formation with the 
 absence of oxygen during the later stages of the ripening. He 
 obtained similar results with Diospyros costata. When the 
 fruits of this plant were gathered and allowed to ripen at a 
 temperature of 30° 0., the ratio of oxygen absorbed to carbon 
 dioxide evolved was 1 : 312 ; when the temperature was 
 maintained at 15° C. it was 1 : 1"26. Alcohol was produced 
 in the former case but not in the latter. The fermentation 
 appeared to be due to the supply of oxygen not being sufiBcient 
 for the needs of the plant when its metabolism was exalted by 
 the Mgher temperature, though it was so with the less active 
 metabolism at 15° 0. The order of events in the ripening of 
 these fruits was the following. In the early stages, the respira- 
 tion showed that they exhaled less carbon dioxide than they 
 absorbed oxygen, the tannin gradually disappearing during this 
 period, and being in Gerber's opinion completely oxidised to 
 carbon dioxide and water. After the disappearance of the 
 
352 ALCOHOLIC FERMENTATION. [CH. 
 
 tannin, the pectose which was present in the walls of the cells 
 was converted into pectine, which caused the softened character 
 of the pulp. The swelUng characteristic of pectine partially 
 obstructed the intercellular passages in the pulp, thus inter- 
 fering very seriously with the transport of air to the cells of the 
 interior, and causing the supply of oxygen to be considerably 
 lessened. At the temperature of 15° C. sufficient oxygen was 
 able to pass along the narrowed intercellular passages to 
 minister to the needs of the cells, but at the higher point of 
 30° C. this was not so, and a sort of asphyxiation set in. This 
 set up the fermentation noticed at that temperature; at once 
 the output of carbon dioxide increased, and the ratio of the 
 two gases concerned rose from 1 : 1"26 to 1 : 3'12. The in- 
 creased amount of carbon dioxide was made up of that which 
 resulted from the fermentation of the sugar, added to the 
 quantity proceeding from the respiration of the cells, the 
 latter being the only factor when the lower ratio was observed. 
 This view of the cause of the increase was supported by the 
 observation that when air was admitted more freely to the cells 
 by subdividing the tissue into small pieces, the ratio at once 
 fell slightly, although as such subdivision did not completely 
 remedy the obstruction of the intercellulaS* passages, the differ- 
 ence was not very marked. 
 
 Gerber obtained similar results with bananas and melons, 
 observed under corresponding conditions. 
 
 He came to the conclusion reached by Lechartier and 
 Bellamy, and by Pasteur, that the cause of alcoholic fermenta- 
 tion in these fruits is to be looked for in the diminished supply 
 of oxygen to the cells, that it is indeed a struggle against 
 incipient asphyxiation. The alcohols which he obtained were 
 not free as in the experiments of Lechartier and Bellamy, but 
 were combined with other volatile bodies to form the various 
 etherial substances which constitute the perfumes of the finits. 
 
 Returning to the consideration of the part played by the 
 yeast-cells in ordinary fermentation, we find that it has been 
 a subject of considerable speculation, and that two hypotheses 
 have been put forward to explain its action. Pasteur came to the 
 conclusion that the fermentative power was connected with nutri- 
 
XX] ALCOHOLIC FERMENTATION. 353 
 
 tion in the absence of free oxygen, and that the decomposition 
 of the sugar under these conditions was the expression of the 
 effort of the organism to obtain oxygen for respiratory purposes. 
 We have seen that according to Monoyer, the formation of 
 succinic acid and glycerin, which Pasteur first discovered to 
 accompany the alcoholic fermentation, is accompanied by the 
 liberation of a small quantity of free oxygen. Pasteur strength- 
 ened himself in this opinion by experiments on the cultivation 
 of yeast in the absence of free oxygen, carried on side by 
 side with others in which the gas was freely supplied to the 
 organism. In these experiments he found that the relative 
 weights of yeast formed and sugar decomposed were very 
 different under the two conditions. When no oxygen was 
 supplied, fermentation was very slow, and not much yeast was 
 produced, but for each part of yeast formed, 60 to 80 parts of 
 sugar disappeared. When oxygen was admitted, the ferment- 
 ation was very rapid, the yeast grew luxuriantly, but for one 
 part formed, not more than 4 to 10 parts of sugar were 
 decomposed. The difference was not due to any weakening of 
 the energy of the yeast in the second case, for when some of 
 it was removed, and made to act on sugar in the absence of 
 oxygen, it behaved just as did that which was used in the first 
 experiment. Pasteur was thus led to hold that fermentation 
 is a phenomenon of life in. the absence of oxygen, and devoted 
 especially to the production of the latter from the carbohydrate 
 material of the cell itself, or that existing in its environment. 
 
 This view was not however allowed to pass without chal- 
 lenge. Schiitzenberger argues against it with some force, 
 pointing out that Pasteur's inference does not take into account 
 all the observed facts. In the presence of free oxygen we have 
 a very active fermentation set up, while the yeast is said to 
 possess, bulk for bulk, less fermentative power. Schiitzenberger 
 claims that the power to decompose sugar is not the same 
 thing as the power of respiration, and that the two should be 
 kept distinct in forming a hypothesis of the nature of the 
 action. The yeast-cell possesses both these powers, and so 
 long as it exercises both freely the fermentative power is not 
 directly related to the respiratory process. If the decomposition 
 G. p. 23 
 
354 ALCOHOLIC FERMENTATION. [CH. 
 
 of the sugar were only the result of the effort of the yeast-cells 
 to respire at the expense of part of the oxygen contained in 
 the sugar molecule, it would appear that fermentation should 
 either not have taken place at all in the presence of free 
 oxygen, or that it should have been much less than in the 
 other case, whereas the reverse is what is found. 
 
 Schiitzenberger supports his objection by experiments 
 carried out to ascertain how respiration is affected under 
 changed conditions. The results he obtained were briefly the 
 following : — 
 
 (1) In a watery liquid without sugar, but containing oxygen 
 in solution, the quantity of oxygen absorbed in imit time by a 
 gramme of yeast is constant, whatever proportion of oxygen is 
 present. 
 
 (2) In a saccharine liquid, containing also albuminous 
 matter, and with oxygen in solution, the same result is ob- 
 tained, except that the quantity of the gas absorbed in unit 
 time is greater. 
 
 (3) In two fermentations, carried on side by side for some 
 time, one only being continuously supplied with oxygen, the 
 latter produced most alcohol. Hence Schiitzenberger came to 
 the conclusion that the sugar is alimentary. 
 
 On a consideration of all the phenomena described by 
 these several observers together with the facts ascertained by 
 Lechartier and Bellamy and by Gerber it seems possible to 
 reconcile these two hypotheses. We may agree with Schiitzen- 
 berger that when the yeast-cell is cultivated under normal 
 conditions its peculiar metabolism demands a certain vigorous 
 decomposition of sugar to minister to its nutrition. Under 
 these conditions it is freely supplied with oxygen and with food 
 material. Its life is consequently very vigorous; the cells 
 grow and multiply and the coincident fermentation is extremely 
 active. But it does not seem at all a strained view to suppose 
 that its protoplasm is Kke that of the cells of the frnits and 
 other organs examined by the other observers. We have 
 seen that when these are deprived of access to free oxygen 
 the ratio of the gaseous interchange is rapidly altered, the 
 carbon dioxide being increased two or three-fold. The stimulus 
 
XX] ALCOHOLIC FERMENTATION. 355 
 
 of incipient asphyxiation leads in them to the setting up 
 of fermentation, evidence of which is afforded by the increase 
 of the carbon dioxide evolved; supplementary evidence is 
 given by the coincident appearance of alcohol. Similar diffi- 
 culty is placed in the way of the yeast when it is deprived 
 of oxygen. The commencing asphyxiation checks the multi- 
 plication of the yeast-cells; instead of the weight of newly 
 formed yeast being one-fourth to one-tenth that of the de- 
 composed sugar it falls to one-eightieth. At the same time 
 an intensification of the fermentation takes place, a so-called 
 respiratory process being developed in addition to the already 
 existing alimentary one. The difference between the yeast and 
 the fruits will thus be seen to be that the nutritive processes of 
 the former involve production of alcohol from sugar while those 
 of the latter do not. The increased fermentative activity of the 
 yeast when deprived of oxygen corresponds to the only ferment- 
 ative power of the cells of the fruit, which does not appear so 
 long as its environment is normal. That the fermentation by 
 yeast under incipient asphyxiation is intensified may appear 
 to be contrary to experiment, as the ordinary fermentation in 
 free oxygen seems most vigorous. But the quantity of the 
 yeast engaged is much greater under these conditions, as the 
 cells multiply very rapidly, and this must be taken into account 
 in making the comparison. Taking the ratio of yeast to sugar, 
 we have seen that in incipient asphyxiation the yeast can 
 decompose 80 times its weight of sugar, whereas in the presence 
 of oxygen it can only ferment 4 — 10 times its weight. 
 
 We see thus that the action of the yeast-cell is not always 
 the same. In the normal condition, it is exciting ferment- 
 ation in connection with its nutrition. This is a special case, 
 apparently confined to such organisms as the lower fungi. 
 A much more wide-spread action of protoplasm can also be 
 noted, when the production of alcohol is coincident with the 
 effort of the living cell to supply itself with the oxygen which 
 is not presented by its envfronment. 
 
 By many writers this latter process is spoken of as " intra- 
 molecular respfration." This is rather an unfortunate phrase, 
 for the process has nothing in common with the intimate 
 
 23—2 
 
356 ALCOHOLIC FERMEJTTATION. [CH. 
 
 respiratory process. The latter consists of the absorption of 
 oxygen by the protoplasm, and of a series of decompositions of 
 the substance of the latter, whereby continually simpler bodies 
 are produced, the ultimate terms reached being carbon dioxide 
 and water. The carbon dioxide liberated in respiration there- 
 fore proceeds immediately from the protoplasm. In the process 
 of fermentation this is not the case ; the decomposition appears 
 to be conducted outside the protoplasmic molecule and to be 
 set up with the view of liberating from some external body the 
 oxygen which the living substance needs for respiration. The 
 process is essentially a fermentative and not a respiratory one. 
 
 There is however another question of considerable importance 
 which must not be overlooked in this discussion. During its 
 life the yeast plant not only requires nutritive material for its 
 development and growth, but it must also be famished with 
 sufficient energy to carry out its vital processes. This is a 
 primary necessity for all living organisms. 
 
 In the case of the yeast-plant, we find that a temperature 
 somewhat higher than that of the surrounding air is eminently 
 favourable for its growth and multiplication, though the par- 
 ticular temperature desired is not the same for all races of the 
 plant. The maintenance of this slightly raised temperature 
 demands the expenditure of a certain additional amount of 
 energy in the form of heat, and this must be obtained by the 
 plant in the course of its metaboKc changes. 
 
 Normally the energy required by an aerobic plant which 
 contains no chlorophyll is furnished largely by its respiratory 
 processes. The same is true of such cells of green plants as do 
 not possess chlorophyll corpuscles, or are not exposed to light 
 of sufficient intensity to enable them to absorb radiant energy. 
 In the respiratory process, we find a continual breaking up of 
 complex bodies into simpler ones, in which decompositions the 
 protoplasm is immediately concerned. The living substance is 
 itself the seat of such decompositions and its own breaking 
 down and reconstruction are largely involved in them. The 
 decompositions appear to be provoked by the access of oxygen 
 to the protoplasm. It is well known that the addition of 
 oxygen to a compound increases its instability. The ultimate 
 
XX] ALCOHOLIC FERMENTATION. 357 
 
 expression of these respiratory changes is the production of 
 carbon dioxide and water, and the decompositions are un- 
 doubtedly accompanied by the liberation of energy. 
 
 When sufficient oxygen is supplied to yeast the gentle 
 respiratory' processes of the healthy cells are supplemented by 
 the decomposition of the sugar, which liberates energy without 
 a direct process of oxidation. If no oxygen is supplied or if the 
 quantity is an inadequate one, the decomposition of the sugar 
 alone must be the source to which the plant must look for the 
 energy it needs. We can understand that increased ferment- 
 ative activity will accordingly be manifested under the stimulus 
 of commencing asphyxiation. The yeast may be regarded 
 under these, conditions as decomposing more sugar either for 
 the purpose of obtaining oxygen for its respiratory processes, 
 or perhaps for that of liberating energy directly without the 
 intervention of respiration. The same explanations may be 
 applied in the case of the ripening fruits and the other parts 
 of plants already instanced. 
 
 In the case of anaerobic plants the decompositions which 
 are provoked may in like manner have for their object a gradual 
 supply of oxygen to the organism. They may on the other 
 hand set up the decompositions for the purpose of supplying 
 energy directly, without the intervention of any oxygen ob- 
 tained from the decomposing body. In either case the main 
 object sought seems to be the energy needed for the various 
 vital processes. 
 
 There is reason, as we have seen, to believe that the 
 fermentation of sugar with its coincident production of alcohol 
 is at any rate at the outset connected with the alimentation of 
 the yeast plant. How far this is the case with the higher 
 plants cannot be regarded as definitely ascertained. The re- 
 searches of Berthelot and of Maz6 already quoted show that 
 in the leaves of the vine at any rate alcohol is of frequent if not 
 constant occurrence under normal conditions. Its most probable 
 antecedent is sugar, but the exact nature of the decomposition 
 which occurs, and the definite meaning of the alcohol, have still 
 to be ascertained. Its occurrence however suggests a parallelism 
 between the nutritive processes of the vine and of the yeast plant. 
 
358 ZYMASE. [CH. 
 
 The method by which the yeast-cell effects the decomposi- 
 tion of the sugar has till qtiite recently remained undiscovered. 
 During the gradual development of our knowledge of enzymes, 
 the minds of many physiologists have been directed to the 
 question of the possible existence of one of these bodies, capable 
 of giving rise to alcoholic fermentation. The yeast-cell is known 
 to be the seat of formation of several enzymes. At different 
 times invertase, diastase, glucase, and trypsin have been dis- 
 TX)vered in it as we have already seen. Till 1896 all efforts 
 to prepare from yeast an alcohol-producing enzyme failed. 
 
 Claud Bernard was engaged during the last few months of 
 his Hfe in investigating the formation of alcohol in the juice of 
 grapes of different degrees of ripeness and decay. During some 
 of his experiments he found the quantity of spirit increase, 
 though no yeast was present, and he attributed this to the action 
 of a soluble ferment. He did not however isolate it. His ex- 
 periments so far as they went were published posthumously by 
 Berthelot, in July 1878. In 1896 however Buchner was success- 
 ful in extracting the enzyme. His process was the following : — 
 
 One kilogramme of purified pressed yeast was mixed with 
 an equal weight of fine quartz sand and with 250 grms. of 
 kieselguhr, a fine infusorial earth. The mixture was carefully 
 ground in a mortar till a moist plastic mass was obtained. The 
 microscope showed that most of the yeast-cells were disin- 
 tegrated or at least ruptured. 100 c.c. of distilled water was 
 then stirred into the pasty mass, and it was wrapped up in a 
 cloth and submitted to strong pressure in a hydraulic press. 
 The pressure was gradually raised till it reached 500 atmo- 
 spheres. The liquid thus squeezed out measured 300 c.c. The 
 cake was removed from the press and groimd up again with 
 another 100 c.c. of water and the pressure renewed, when a 
 fiirther 150 c.c. of liquid was obtained. In all therefore 250 c.c. 
 of the contents of the yeast-cells were yielded by the treatment. 
 The liquid was shaken up with 4 grammes of kieselguhr and 
 filtered through fine filter-paper. The resulting extract was a 
 clear, slightly opalescent liquid of a yellow colour, possessing a 
 yeasty aroma. It had a specific gravity of 1-046 at 17° C. 
 When a small quantity of this solution was mixed with an 
 
XX] ZYMASE. 359 
 
 equal volume of a 37 per cent, solution of cane-sugar, a regular 
 evolution of carbon dioxide commenced in about half-an-hour. 
 When the operation was carried on at a low temperature the 
 disengagement of the gas was very gentle, but at about 35° C. 
 it was so energetic as to cause the liquid to froth. 
 
 The preparation of such an extract as this presents consider- 
 able difficulty, the operation of grinding the yeast being very 
 tedious. Since Buchner's results were published it has however 
 been successfully carried out by Delbruck in Berlin, and by the 
 writer and by MacFadyen, Morris and Rowland in this country. 
 
 The fermentation is due to an enzyme in the yeast extract, 
 to which Buchner has given the name zymase. The name is not 
 perhaps well chosen, as the same term has been applied to the 
 whole group of soluble ferments, though it has been replaced in 
 this connection by the word " enzyme." 
 
 The decomposition of the sugar effected by zymase is its 
 disruption into alcohol and carbon dioxide. If a quantity of 
 the yeast extract is mixed with a solution of a fermentable 
 sugar and placed in a flask fitted with a manometer, the mercury 
 very soon rises in the distal Hmb of the latter, and the pressure 
 of the gas keeps it there for several days, bubbles of gas from 
 time to time escaping through the mercury column. At the 
 end of some days a measurable amount of alcohol can be 
 separated from the liquid by distillation. The action goes on 
 until all the sugar has been decomposed. 
 
 The extract is perfectly active when saturated with chloro- 
 form, which inhibits the action of the yeast cell itself Other 
 antiseptics may be used instead of chloroform. Buchner recom- 
 mends the use of potassium metarsenite in the proportion of 
 2 gms. per 100 c.c. of solution. 
 
 When chloroform is used there ensues a gradual precipita- 
 tion of the proteids of the yeast solution, which mechanically 
 carry down a large portion of the enzyme. If such a solution is 
 filtered and the precipitate suspended in a quantity of water, 
 equal in bulk to the original, volume before filtration, the 
 latter has much more fermentative power than the filtrate, 
 when sugar is added to both. 
 
 If the solution of the enzyme is heated gradually, a copious 
 
360 ZYMASE. [CH. 
 
 coagulum of proteid matter is formed at 35° — 40° C, and after 
 separation of this the extract is found to have lost its fer- 
 mentative power. 
 
 The extract is very difficult to preserve, as the fenzyme 
 decomposes very rapidly. It may be kept for 2 to 5 days if 
 the temperature be maintained at 0° C, but at the temperature 
 of the laboratory it is usually destroyed in half that time. If it 
 is kept in contact with sugar Buchner says it can be preserved 
 for a considerable time longer, retaining some ferment power 
 after two weeks. 
 
 In some cases the yeast extract has been found to be active 
 after filtration through a porcelain or kieselguhr filter. Dif- 
 ferent preparations however show differences in this respect. 
 
 The enzyme, like so many others, is capable of resisting 
 desiccation to a certain extent. The yeast extract may be evapo- 
 rated under diminished pressure till it consists of a thin paste, 
 and the latter may then be spread out in a thin layer and 
 further dried at 35° C. The solid residue generally represents 
 about 10 per cent, of the weight of the liquid. It can be 
 preserved unchanged for three weeks. If during that time it is 
 dissolved in 5 times its weight of water and the solution filtered, 
 the latter contains the ferment in an active condition. 
 
 In his earlier experiments Buchner was unable to precipitate 
 the zymase fi-om the extract of the yeast. More recently, 
 working in conjunction with Albert, he has succeeded in doing 
 so. He poured yeast extract into 12 times its volume of a 
 mixture of 10 volumes of absolute alcohol and 2 volumes of 
 ether, when a flocculent precipitate slowly settled out, which 
 contained nearly all the zymase, which could be filtered off' and 
 dried without losing much of its activity. If less alcohol was 
 used the precipitation was very incomplete. 
 
 The precipitate so obtained was found to be soluble in 
 glycerin or in water. Filtration of the solution in glycerin 
 did not impair its power of setting up fermentation, but the 
 watery solution was not so potent after passing through a 
 filter. 
 
 The sugars which are capable of fermentation by zymase 
 are those which the yeast itself can decompose. Buchner has 
 
XX] ZYMASE. 361 
 
 fermented cane-sugar, maltose, glucose and fructose, but not 
 lactose nor mannitol. It appears at present that the ferment- 
 ation is not accompanied by the formation of succinic acid and 
 glycerin which is characteristic of that brought about by yeast. 
 
 At the outset of his investigations Buchner found that the 
 enzyme could not be prepared from every sample of yeast with 
 which he worked. Many other observers, following his method 
 of preparation with scrupulous care, also failed to extract it. 
 
 The ordinary yeast of our breweries taken from the ferment- 
 ing wort at the close of the fermentation usually fails to yield 
 any enzyme. If however it is taken while in very active condi- 
 tion, when the fermentation is at its height and the yeast 
 frothing vigorously, a considerable quantity of zymase may be 
 extracted from it. Buchner in one of his early papers suggested 
 that the amount of the enzyme may vary in different yeasts and 
 from time to time in the same yeast. The secretion is appar- 
 ently intermittent, and only called forth by the conditions which 
 excite the cells to vigorous growth and multiplication. This 
 again calls to mind the behaviour of many gland cells, such as 
 those of the stomach, which do not begin to secrete their charac- 
 teristic enzyme till stimulated by the absorption or supply of 
 nutritive material. 
 
 Since Buchner's discovery attention has been directed again 
 to the production of alcohol in fruits and other parts of plants 
 when they are deprived of oxygen, Effront has recently claimed 
 to have demonstrated the secretion of zymase by the cells 
 concerned in setting up alcoholic fermentation in these organs. 
 His first experiments were carried out with cherries, which he 
 sterilised by washing them with a dilute solution of formic 
 aldehyde. They were then carefully dried and plunged into 
 flasks containing olive oil. After three days a considerable 
 evolution of carbon dioxide was observed, which continued for 
 about three weeks. At the end of that time the cherries were 
 crushed and the juice extracted from them by pressure. The 
 juice was freed from oil by treatment with ether, and was dried 
 at a low temperature in vacuo. The resulting powder was 
 macerated with water for 12 hours and then subjected to strong 
 pressure. The expressed liquid after filtration was found to 
 
362 ZYMASE. [CH. XX 
 
 excite alcoholic fermentation when added to cane-sugar ; evolu- 
 tion of carbon dioxide, production of alcohol, and diminution of 
 the specific gravity of the liquid being all observed. If the 
 extract was kept at 40° C. for an hour before being mixed with 
 the sugar it produced no change in the latter. 
 
 Effiront has prepared zymase from peas and from barley by 
 a similar method. 
 
 From these researches it appears certain that the production 
 of alcohol whether in the presence or absence of oxygen is 
 brought about by the activity of an enzyme. Its secretion by 
 the cells of yeast attends the ordinary nutritive processes as 
 well as the abnormal decompositions set up by incipient 
 asphyxiation. The latter condition induces its formation in 
 other parts of plants. The absence of oxygen stimulates the 
 protoplasm of the cells to secrete it, the ultimate effect of its 
 appearance being the liberation of energy as already stated. 
 
363 
 
 CHAPTER XXI. 
 
 THE FERMENTATIVE POWER OF PROTOPLASM. 
 
 In the introductory chapter of this volume we saw that the 
 metabolism of the living cell consisted of two different series of 
 events, the one constructive, embraciag the building up of 
 simple substances into complex ones and culminating in the 
 formation or repair of the living substance itself; the other 
 destructive, marked by the decomposition of complex sub- 
 stances, with the ultimate formation of others of much greater 
 simplicity. We have seen further that many of the latter 
 processes are carried out by means of enzymes, whose modes 
 of action we have since examined. But if we turn our at- 
 tention especially to intracellular changes we must recognize 
 that at present all such changes cannot be traced to enzymic 
 activity. Indeed a very important factor of the intracellular 
 mechanism is the breaking down of the protoplasm itself, or its 
 " self-decomposition " as Pfluger has expressed it. 
 
 The living substance must be regarded as the seat of a 
 multitude of chemical changes, some constructive and others 
 destructive in their nature. The incorporation into its sub- 
 stance of the ultimately assimilated proteids, fats, and carbo- 
 hydrates, goes on continuously, and no sooner is it constructed 
 than it undergoes a species of self-decomposition, giving rise 
 to many residues of various kinds, some of which are re- 
 incorporated with new material into its molecules, while others 
 are broken down still farther and ultimately eliminated. 
 
 Not only does the substance of the protoplasm itself undergo 
 these decompositions, but different materials in the cells are 
 involved in them, and the residues of these are absorbed into. 
 
364 THE FERMENTATIVE POWER OF PROTOPLASM. [CH. 
 
 and incorporated with, the living substance in the same way as 
 those which spring from its self-decomposition. We find that 
 in many cells there is stored a reserve of nutritive material, 
 not quite in a suitable state for immediate assimilation, but 
 ready to form a nutritive pabulum when split up in the manner 
 indicated. Nor are such materials of use only as supplying 
 nutritive material ; by their decomposition a supply of energy 
 is afforded to the protoplasm, of which it avails itself in carrying 
 out constructive processes. Under the condition of an in- 
 sufficient supply of oxygen also, the protoplasm frequently 
 effects decompositions of the same kind, as we shall see in 
 the present chapter. 
 
 These changes are very largely of the same nature as the 
 decompositions effected by means of enzymes, and we may 
 consequently speak of them as exhibiting a fermentative power 
 of protoplasm. 
 
 In discussing this power or property of the living substance, 
 we may confine it to cases in which protoplasm provokes 
 decomposition of various bodies for the purposes of preparing 
 nourishment for itself, of liberating energy for its various 
 processes, or of obtaining oxygen for respiratory purposes. 
 
 Such operations can be observed more or less easily in 
 almost all organisms, from the highest to the lowest, and in both 
 the animal and the vegetable body. 
 
 We have seen that the liver of mammals contains a 
 quantity of glycogen stored in its cells. Glycogen is also present 
 in the muscles of the trunk and limbs. During life there is a 
 constant demand for carbohydrate material by all the living 
 substance, and this is supplied partly by the consumption of 
 the stored glycogen. In an earlier chapter we have seen that the 
 glycogen of the liver is probably converted into sugar by an 
 enzyme formed in the substance of the cell-protoplasm. Many 
 physiologists hold the opinion however that this is not the only 
 agent in the transformation, but that some at all events is 
 directly hydrolysed by the protoplasm. The same appears to 
 be true of the muscles, for up to the present these have not 
 been shown to be capable of giving up diastase to an extracting 
 liquid to more than a trifling extent. 
 
XXI] THE FERMENTATIVE POWER OF PROTOPLASM. 365 
 
 The consumption of the fat stored in the various adipose 
 tissues appears to be due to a similar cause. The fat ministers 
 to the nutrition not only of the cells in which it is deposited but 
 also of the rest of the body. It must for this purpose be re- 
 moved from the seat of storage, and this removal almost certainly 
 involves its decomposition with a view to facility of transport. 
 The enzyme lipase however is by no means universally present 
 in adipose tissue. 
 
 Other nutritive materials are also acted upon by proto- 
 plasm. In cases of starvation the substance of the body does 
 not diminish uniformly; the fat disappears most readily, and 
 the muscular tissue is drawn upon very seriously before there 
 is any wasting of the brain. Of the muscles those of the heart 
 do not suffer till some time later than those of the trunk and 
 limbs. The latter are practically digested with the view of 
 nourishing the other tissues, and the more important cardiac 
 muscles receive aliment derived from the consumption of those 
 which are more easily spared. The living substance itself must 
 therefore be capable of breaking up the proteid materials 
 of various parts, in the absence of similar substances normally 
 supplied by the food. 
 
 The vegetable organism affords more examples of this power 
 of protoplasm than the animal body. What is perhaps the ex- 
 ception in the latter case appears among the normal functions 
 of various vegetable cells. The power of the protoplasm to 
 effect the disruption of carbohydrates is seen in the trans- 
 formation and reconstruction of the transitory starch which is 
 constantly going on in various parts of the higher plants, 
 though no doubt in many cases diastase here takes part. The 
 same power may be noted in the changes found to take place 
 among the various sugars that the higher plants contain ; the 
 various acids which are normal constituents of the cell-sap of 
 succulent parenchyma are probably if not certainly produced 
 from sugars in the cells. Kohl has suggested that they are 
 formed much in the same way as lactic acid is formed in 
 certain fermentations carried out by bacteria. These acids are 
 not usually such simple ones as are formed by microbes; we 
 find malic, citric, tartaric, &c. instead of acetic, lactic, butyric, 
 
366 THE FERMENTATIVE POWER OF PROTOPLASM. [CH. 
 
 &c. This may however be due to the character of the meta- 
 bolism of the two classes of cells respectively, for the action is 
 intracellular in both cases. 
 
 The intracellular digestion of proteids in the absence of an 
 enzyme can be seen in the case of the tuber of the potato. 
 The outer portion of the tuber contains crystals of proteid of a 
 cubical form, the proteid being probably one of the vegetable 
 globulins. At the onset of germination of the tubers these 
 crystals are found to disappear, being made use of in the nutri- 
 tion of the young shoots. 
 
 The fermentative power of the protoplasm upon cellulose 
 can be observed in almost all growing cells which assume an 
 irregular shape. We have noticed in considering the develop- 
 ment of branches upon the hyphse of different species of Botrytis 
 that the accumulation of cytase at the points of branching leads 
 to a softening of the cell wall at those spots, and that then the 
 hydrostatic pressure in the hypha causes its gradual extension. 
 The shapes of such cells as form the stellate hairs upon the 
 leaves of Deutzias can be explained only by taking into account 
 the power of the protoplasm to soften the waU of the originally 
 nearly spherical cell at particular spots, so that in these areas 
 it becomes more easily extensible than over the remainder of 
 its surface. The hydrostatic pressure due to turgescence con- 
 sequently causes protrusions at the softened spots, and hence 
 the appearance of the mature hair. 
 
 Less conspicuous cases are of frequent occurrence in the 
 interior of the vegetable tissues. This cytolytic action of 
 protoplasm is evidently the same thing as the softening brought 
 about by cytase. 
 
 Till very recently the fermentations induced in ripe fruits 
 as demonstrated by Lechartier and Bellamy, by Pasteur, and 
 by Gerber, were held to be illustrations of this property of 
 protoplasm. Effront's researches point however to their being 
 due to the presence of zymase. 
 
 If we pass to cases observable among more lowly forms we 
 find evidence of the same property of living substance. The 
 leucocytes of the blood of vertebrates may be regarded in some 
 .sense as independent bodies, capable of carrying out their own 
 
XXl] LACTIC FERMENTATION. 367 
 
 processes of nutrition. Metschnikoff has observed that certain 
 of these corpuscles, which he calls phagocytes, are able to 
 absorb and digest bacteria which have made their way into the 
 blood stream. The bacteria themselves are capable of bringing 
 about an almost infinite variety of similar decompositions, many 
 of them causing specific fermentations. In a few cases they 
 have been shown to effect these changes by means of enzymes, 
 but this is very far from being universally the case. The direct 
 action of their protoplasm must consequently be recognized as 
 the exciting cause of these decompositions. Various processes 
 of intracellular digestion occurring in the hjrphse of Mucor and 
 other filamentous Fungi demand a similar explanation. 
 
 The influence of deficient oxygenation upon the metabolic 
 processes of plants throws a further light upon this property. 
 Many phenomena have been recorded which show how pro- 
 foundly the ordinary metabolism is modified when circumstances 
 prevent their obtaining oxygen freely. Boehm, and later de Luca, 
 have shown that if any part of a living plant is insufficiently 
 supplied with oxygen, hydrogen and sometimes marsh-gas are 
 evolved from it. Boussingault and Schulz have observed 
 similar phenomena. From plants containing mannite also 
 hydrogen is given off, while according to de Luca, acetic acid 
 is formed from malic acid in the fruits, flowers, and leaves of 
 the Privet. In the decomposition of proteid too, Boehm found 
 ammonia exhaled. 
 
 As already mentioned, we find many specific fermentations 
 set up by micro-organisms without any secretion of enzymes, 
 several of which call for a brief notice. Attention may first be 
 given to those which in their normal metabolism cause the 
 production of quantities of various acids. The phenomena of 
 putrefaction also may be mentioned in this connection. 
 
 Lactic fermentation. 
 
 The frequent appearance of lactic acid in solutions of sac- 
 charine matters has been noticed from very early times. Its 
 occurrence explains the souring of milk which is allowed to 
 remain exposed to the air, and it was from this phenomenon that 
 
368 LACTIC FERMENTATION. [CH. 
 
 the acid formed derived its name, lactic acid being for the first 
 time prepared from whey by Scheele in 1780. The acid is not 
 however formed exclusively from milk-sugar, but it can be 
 produced from glucoses and from almost all substances con- 
 vertible into them. Braconnot found it in a liquid obtained by 
 steeping rice in water; also in the juice of the sugar-beet, and 
 in infusions of peas, and seeds of other leguminous plants. The 
 polysaccharides offer more resistance to its formation than the 
 glucoses. Lactose, which is itself a member of this group, lends 
 itself much more readily to lactic than to alcoholic fermentation. 
 Bourquelot states that the formation of lactic acid is not preceded 
 by hydrolysis as is the production of alcohol. The reactions which 
 take place may be expressed by the equations CsHiaOs = 2C3HSO3, 
 and in the case of polysaccharides CiaHaaOu -I- H2O = 4C3H8O,, 
 the last term being the formula for lactic acid. 
 
 Besides the sugars mentioned, lactic acid may be produced 
 from sorbite, inosit, mannite, and dulcite ; also from malic acid, 
 carbon dioxide in this case being evolved, according to the 
 equation CjHeOs = C3H5O, + CO2. It occurs also in the butyric 
 fermentation of many carbohydrates. 
 
 Lactic fermentation arises from the activity of many species 
 of microbe, including the bacteria found in pus. Micrococcus 
 prodigiosiis, Erberth's bacillus, Bacterium coli commune, and 
 others. The most energetic form, the one which is most 
 frequently found in sour milk, is the organism observed and 
 described by Pasteur, Bacterium, acidi lactici. This microbe 
 appears under the microscope as rounded globules, or short 
 rods slightly constricted in the middle; they are generally 
 isolated, but sometimes form chains two or three cells in length. 
 They are capable of forming spores, which are found at their 
 extremities. These offer a considerable resistance to the 
 destructive action of heat. 
 
 The microbe is aerobic, and requires considerable quantities 
 of oxygen. It is killed by exclusion of this gas from a liquid 
 in which it is living. It flourishes best in a neutral medium, 
 and consequently in cultivating it it is advisable to add a 
 quantity of powdered chalk to the milk or saccharine solution. 
 This neutralises the lactic acid as fast as it is formed, and the 
 
XXl] BUTYRIC FERMENTATION. 369 
 
 fermentation can consequently proceed. An alkaline medium 
 is also unfavourable to its activity. If the fermentation is 
 allowed to go on without neutralisation of the acid, it is found 
 that the action of the organism is usually stopped when about 
 8 per cent, of lactic acid has been formed. 
 
 For the cultivation of the lactic microbe it is necessary for 
 the fluid to contain a certain amount of proteid material 
 besides the sugar. It grows most readily at a temperature of 
 about 35° C. 
 
 Lactic acid has often been detected in the contents of the 
 mammalian stomach during digestion, and considerable dis- 
 cussion has taken place as to its cause. Many observers have 
 argued in favour of its being a product of bacterial action there. 
 Others, among whom may be mentioned Hammarsten, have 
 attributed its presence to the secretion of a special enzyme by 
 the gastric mucous membrane. It is said that certain gastric 
 extracts have been treated with sodium hydrate solution of 
 such concentration as to destroy both pepsin and rennet. After 
 removal of the excess of soda, the liquid has been capable of 
 bringing about the conversion of milk-sugar into lactic acid. 
 The eiiZ3niie cannot however be said at present to have been 
 satisfactorily isolated. Maly has stated that when glucpse has 
 been fermented in the presence of the mucous membrane of the 
 stomach of the pig, a variety of lactic acid has appeared among 
 the products. The conditions of his experiments do not seem 
 however to have entirely excluded the possibility of the 
 presence of micro-organisms. Other observers have attributed 
 the same property to the gastric mucous membrane of herbi- 
 vora. 
 
 Butyric fermentation. 
 
 It is generally found that in the souring of milk there is a 
 formation of butyric acid, which appears to take place at the 
 expense of some of the lactic acid which is first formed. The 
 chief agent in its production is a special microbe, which has 
 been named by different observers Clostridium butyricum, Vibrio 
 butyricus, Amylobacter Clostridium, and Bacillus amylobacter. It 
 G. F. 24 
 
370 BUTYRIC FERMENTATION. [CH. 
 
 consists of slender cylindrical rods which are occasionally united 
 into short rows. Sometimes they are found in active move- 
 ment; at others they are gathered together into a zooglcea. 
 They are often very irregular in shape ; when they produce 
 their spores, the latter are solitary in each cell, and are generally 
 found at about the middle of its long diameter, causing the rod 
 to have a somewhat fusiform appearance. This organism is 
 very widespread, and may generally be found accompanying 
 the lactic acid microbe. It differs in its habit of life from the 
 latter, being anaerobic and easily killed by the presence of free 
 oxygen. 
 
 Butyric acid is produced by this organism, not only from 
 lactic acid and substances which can give rise to the latter, such 
 as sugar and starchy bodies, but also from higher acids, such 
 as tartaric, citric, malic and mucic acids. The decomposition 
 which it provokes in these substances is of a soniewhat complex 
 character, being usually marked by the disengagement not only 
 of carbon dioxide, but of free hydrogen as well. According to 
 Van Tieghem, it has also the power of splitting up certain 
 varieties of cellulose, or of pectic compounds associated with the 
 latter, particularly the middle lamella of the cells of many 
 tissues. It does not, however, attack starch. 
 
 The bacillus is constantly found in the alimentary canal of 
 animals, particularly such as are herbivorous. It no doubt 
 plays a very important part in the digestion of cellulose in the 
 mammalian body. De Bary suggests that this digestion occurs 
 in two stages; hydrolysis of the cellulose first takes place by 
 virtue of a cytolytic enzyme, resulting in the formation of 
 dextrin and glucose, and these subsequently undergo butyric 
 acid fermentation. This, however, cannot at present be con- 
 sidered proved. 
 
 Many other microbes than Bacillus amylobacter are capable 
 of giving rise to the butjnric fermentation. Among them may 
 be mentioned the Bacillus butylicus of Fitz, which can ferment 
 glycerine, liberating carbon dioxide and hydrogen, and leaving 
 in the solution butylic alcohol, butyric and lactic acids, propy- 
 lenic glycol, and traces of two other alcohols. This bacillus is 
 one of the few forms at present known to secrete invertase. 
 
XXI] PROPIONIC FERMENTATION. 371 
 
 Hueppe's bacillus is another interesting form, from the fact 
 that it not only forms butyric acid from lactates, but it 
 liquefies gelatin, and excretes the soluble enzymes trypsin and 
 rennet. 
 
 Bacillus ethylicus is capable of exciting butyric fermentation 
 in a thin starch-paste, provided the reaction is kept neutral by 
 the presence of powdered chalk. The fermentation is preceded 
 by hydrolysis of the starch, brought about by diastase which is 
 secreted by the microbe. The formation of the butyric acid in 
 this case is direct, no intermediate occurrence of lactic acid 
 being noticeable. 
 
 Among other bodies capable of undergoing butyric fer- 
 mentation by various microbes, may be mentioned mannite, 
 quercite, and glycerine. 
 
 The optimum temperature of butyric fermentation is a 
 little higher than that of lactic, being 39 — 40° C. 
 
 The products of the butyric fermentation are generally 
 complicated by secondary reactions taking place in the liquid. 
 The nascent hydrogen frequently effects processes of reduction 
 among the various bodies present. Succinic and valeric acids 
 can often be detected among these. 
 
 Propionic fermentation. 
 
 Under the action of certain bacilli, lactic acid, when in 
 combination with lime, undergoes a different decomposition, 
 being converted into several fatty acids, among which are acetic 
 and valeric, the most prominent of them however being propionic 
 acid. This decomposition was first observed by Strecker in 
 1854, in a fermenting liquid kept for several months at a low 
 temperature. Fitz has described a form of bacillus which can 
 bring about this transformation ; it is an elongated slender 
 organism, forming curved chains consisting of several cells 
 joined end to end. In the fermentation, the lactic acid splits 
 up into two parts of propionic and one of acetic acid, carbon 
 dioxide and water being simultaneously formed. It differs from 
 the butyric fermentation of the same acid in not disengaging 
 hydrogen. 
 
 24—2 
 
372 CITRIC FERMENTATION. [CH. 
 
 Another bacillus, also investigated by Fitz, decomposes 
 malic acid into almost the same products, but the relative 
 quantity of propionic acid is greater, and traces of butyric acid 
 and alcohol are simultaneously formed. 
 
 Citric fermentation. 
 
 During the year 1892, it was shown by Wehmer that under 
 certain circumstances glucose can be made to give rise to 
 citric acid. This change is brought about by two separate 
 species of moulds or filamentous fungi, to which their discoverer 
 gave the names of Gitromyces pfefferianiLS and C. glaber, respec- 
 tively. They form veils of dense mycelium of a green colour, 
 about five millimetres thick, on the surface of suitable solutions. 
 The most favourable sugar for their development is glucose, but 
 they are not dependent upon that form alone. The fermenta- 
 tion is accompanied by the evolution of carbon dioxide, and the 
 citric acid produced is identical in properties and composition 
 with that obtained from lemon juice. Under proper conditions 
 the quantity of acid formed corresponds to more than fifty per 
 cent, of the glucose employed. Eleven kilogrammes of sugar 
 yielded six kilogrammes of pure citric acid in one experiment, 
 without the appearance of any secondary products. To secure 
 this result, however, it is necessary to precipitate the citric acid 
 when it has reached a certain percentage, as a concentration of 
 20 per cent, is deleterious to the fungi and checks their growth. 
 
 The fungus cannot excite the fermentation in the absence 
 of air; it is deleteriously affected even by the accumulation 
 of the carbon dioxide which is evolved during the process. It is 
 independent of light, but the temperature needs to be carefully 
 regulated. 
 
 The presence of inorganic acids in the fermenting liquid ia 
 very deleterious, while neutral chlorides favour the production 
 of citric acid. 
 
 If the fermentation is allowed to proceed too far, the citric 
 acid itself is attacked and decomposed. 
 
 The process of acidification is not uniform, but its rate 
 describes a rather suddenly ascending and descending curve. 
 
XXl] OXALIC FERMENTATION. 373 
 
 which is in close relation to the amount of the decomposition 
 of the sugar effected by the fungus. 
 
 In connection with this fermentation it is interesting to 
 note that citric acid is occasionally present in the juice of the 
 sugar-cane, indicating an action of the protoplasm of the cells 
 of the latter similar to that of the fungi under discussion. 
 
 Oxalic fermentation. 
 
 Several fungi, particularly Penidlitmi and Sclerotinia, are 
 known to be capable of giving rise to oxalic acid when culti- 
 vated in solutions of sugar. The same power resides in certain 
 of the Saccharomycetes, one of which, S. Hansenii, has been 
 described by Zopf When cultivated in beer-wort, solutions of 
 sugar, or mannite, it forms a pellicle on the surface, which soon 
 sinks through the liquid. If chalk is added to the fluid in which 
 this organism is growing so as to neutralise the acid as it is pro- 
 duced, large quantities of oxalate of calcium are formed. The 
 yeast can form oxalic acid from galactose, glucose, cane-sugar, 
 lactose and maltose ; also from dulcite, mannite, and glycerine. 
 
 The oxalic acid fermentation contrasts somewhat sharply 
 with the citric in certain respects. While a rise of temperature 
 and the presence of neutral chlorides are advantageous to the 
 latter, they promote the destruction of the oxalic acid and 
 prevent its accumulation in the cultivations. 
 
 Besides these fermentations many others have been observed 
 in which various microbes are concerned, but no very complete 
 study has been made of the morphology of the latter, or the 
 exact nature of the changes they effect. Many of these fer- 
 mentations are probably very complex, several different microbes 
 taking part in them either simultaneously or successively, and 
 many interactions taking place between the various products. 
 
 A particular bacillus is said to decompose malic acid with 
 the formation of succinic and acetic acids, carbon dioxide and 
 water being simultaneously produced. Others which may be 
 prepared from hay have the power of fermenting erythrite, 
 giving rise to butyric acid with traces of formic and acetic 
 acids. Others again decompose erythrite into butyric, acetic. 
 
374 ACETIC FERMENTATION. [CH. 
 
 caproic and succinic acids, traces of alcohol also being formed. 
 Glycerine also can be split up by several of these organisms 
 besides the butyric microbe ah-eady alluded to. A particular 
 micrococcus is known, which slowly decomposes it with forma- 
 tion of ethyl and other alcohols, and butyric, formic and acetic 
 acids, the last two being in small quantity. The microbe of 
 pus, B. pyocyaneus, produces succinic acid in addition. Certain 
 other micro-organisms can decompose the calcium salt of quinic 
 acid ; some anaerobic forms yield formic, acetic, and propionic 
 acids at its expense ; others which are aerobic oxidise it to 
 pyrocatechic acid. Several other acid fermentations of a similar 
 kind are known. 
 
 Acetic fermentation. 
 
 When alcoholic liquids are allowed to remain exposed to the 
 air, they speedily become attacked by micro-organisms. Very 
 soon, if the concentration of the spirit is not too great, a kind 
 of pellicle or skin can be found upon the surface of the liquid, 
 which sometimes remains thin and delicate and easily broken, 
 and at other times attains a considerable thickness. In 
 some cases the formation of the pelKcle is accompanied by a 
 growth of the organism in the body of the liquid, when a large 
 amount of viscous matter may be produced. Coincidently with 
 the growth of the microbes, the alcohol of the liquid becomes 
 decomposed and is replaced partly or wholly by acetic acid. 
 
 The pellicle was named Mycoderma by Persoon in 1822 ; he 
 noted its botanical nature, and rightly concluding it to be a 
 fungoid growth, named it accordingly. 
 
 Kiitzing reinvestigated the subject in 1837, and described 
 the organism as consisting of a number of small circular bodies 
 arranged together in the form of chains. He was the first 
 observer to note that the development of the acetic acid was 
 connected with the presence of the microbe. He erroneously 
 classed the latter among the Algae, naming it Ulvina aceti. 
 
 About the year 1821 light was first thrown upon the 
 process of acetification. Before that date it was known in a 
 general way that acetic acid was formed &om alcohol, and that 
 
XXl] ACETIC FERMENTATION. 375 
 
 the access of air was necessary for the process, but no reasons 
 for the phenomena were known. Lavoisier stated that acetic 
 fermentation was effected by the absorption of oxygen from the 
 air. In 1821, however, Edmund Davy discovered that when 
 platinum black was moistened with alcohol, it became incan- 
 descent, and the consumption of the spirit thereby induced was 
 attended by the formation of acetic acid. His discovery was 
 confirmed two years afterwards by Doebereiner, who carried it 
 further by showing that the alcohol absorbed oxygen, and 
 became decomposed into acetic acid and water. 
 
 By most of the writers who succeeded Dcebereiner the 
 membrane of the mycoderma was compared with platinum 
 black as an excitant of the decomposition, and was held to act 
 in a similar way. Berzelius in 1829 taught that the action of 
 the pellicle was due to acetic acid enclosed within its pores. 
 Liebig considered the mycoderma to be devoid of life, a struc- 
 tureless precipitate of albuminous matter, and held that it was 
 a porous substance like platinum black and that its action was 
 connected with its porosity and the oxygen thus mechanically 
 entangled in it. He believed that the formation of the acetic 
 acid was due to a movement or vibration set up by matter 
 undergoing decomposition, as he taught in the case of alcoholic 
 fermentation. The vibration in the present case provoked the 
 oxidation of the alcohol. 
 
 Pasteur himself failed to see the actual part played by the 
 organism. He recognised the accuracy of Kiitzing's work and 
 showed experimentally that the opinion of the latter as to the 
 function of the mycoderma was accurate. He agreed however 
 with Liebig that the fungus acted after the manner of platinum 
 black. He showed moreover that not only was alcohol oxidised 
 during its activity, but that if the action was prolonged the 
 acetic acid also was decomposed, carbon dioxide and water being 
 the only products. 
 
 It was not till 1873 that it was clearly shown that the two 
 modes of producing acetic acid from alcohol are not comparable. 
 Von Knieriem and Ad. Mayer pointed out several differences. 
 Platinum black oxidises both concentrated and dilute alcohol, 
 while the mycoderma cannot attack alcohol of greater concen- 
 
376 ACETIC FERMENTATION. [OH. 
 
 tration than 14 per cent. Acetic fennentation proceeds most 
 actively at 35° C. and the organism cannot work at a point 
 higher than 40° C. ; platinum black becomes more and more 
 efficacious as the temperature rises, and the action may become 
 so violent that the metal becomes incandescent. 
 
 The phenomenon gradually came to be considered a bio- 
 logical process, corresponding in all essential features with 
 the alcoholic fermentation induced by yeast. 
 
 The action was at first held to be always due to one par- 
 ticular fungus, but later work carried out by many observers 
 has shown this to be erroneous. The form which is most 
 commonly met with was named Mycoderma aceti by Pasteur, 
 but was referred later to the genus Bacterium by Zopf Hansen 
 distinguished two species of this genus which are capable of 
 acetifying alcoholic solutions, and he named them B. aceti and 
 B. Pasteurianum. Two other species have been described by 
 A. J. Brown under the names of B. aceti and B. xylinum. The 
 former does not appear, in the light of later researches, to be 
 identical with Hansen's organism bearing the same name. 
 Lafar has isolated and cultivated an acetifjdng organism be- 
 longing to the Yeasts; he found it present in an acid beer. 
 
 Bertrand has described a fission fungus which has the power 
 of attacking sorbitol, an alcohol isomeric with mannitol; it 
 converts it into a ketose or ketone sugar known as sorbinose 
 or sorbose. This fungus has not however been accurately 
 identified. 
 
 A very careful investigation into the acetous fermentation 
 set up by Bacterium aceti was made in 1886 by Adrian J. Brown. 
 He obtained the organism in a pure condition by a combination 
 of the fractional method of Klebs and the dilution method of 
 V. Nageli. So cultivated, it formed a somewhat greasy pellicle 
 covering the surface of the liquid and inclined in its early 
 stages of growth to creep up the moist sides of the vessel con- 
 taining it. When grown on diluted claret the coating or veil 
 attained the thickness of stout paper. Slight agitation easily 
 broke the surface of the pellicle, and when the latter was com- 
 pletely wetted it sank to the bottom of the liquid. A fresh 
 growth however speedily appeared upon the surface. The 
 
XXl] ACETIC FEEMENTATION. 377 
 
 liquid below the veil became turbid from the presence of 
 isolated cells of the organism, and after some weeks a consider- 
 able quantity of the latter had sunk to the bottom of the vessel. 
 When the pellicle was examined under the microscope it 
 appeared to be a mass of cells averaging 2/t in length, but 
 showing a good deal of difference in this respect. The cells were 
 slightly contracted in the middle, and presented roughly the 
 appearance of a figure of 8. They were united together 
 in chains of variable length. Sometimes the cells were divided 
 in the middle, producing chains of micrococcus-like forms. In 
 the liquid below the surface-film longer cells were found, 
 sometimes reaching a dimension of 10 — lo/i; in some cases 
 their form was that of leptothrix threads of equal thickness 
 throughout; in others the long cells were swollen out in 
 two or three places along their length, giving them a very 
 irregular appearance. These cells were generally of a dark 
 grey colour. At their ends short chains of small rods or 
 micrococci were sometimes observed. Other forms frequently 
 seen were short rods of about 3/a in length and micrococci of 
 about 1/Lt in diameter, which floated freely in the culture liquid. 
 The shorter rods and cells were motile. 
 
 A litre flask half full of a 5 per cent, sterile solution of pure 
 ethyl alcohol in yeast water, possessing a neutral reaction, was 
 inoculated with a trace of a pure culture of the organism, and 
 kept at a temperature of 28° C. for 10 days. It was then found 
 to contain rather more than 1 per cent, of acetic acid and a 
 very slight trace of a non- volatile acid which appeared to be 
 succinic. 
 
 When the microbe was cultivated ia a solution containing 
 •76 per cent, of acetic acid but no alcohol, and was kept at the 
 same temperature for six weeks, two-thirds of the acid disap- 
 peared, being oxidised to carbon dioxide and water. 
 
 When an alcoholic solution was allowed to ferment until all 
 the alcohol was converted into acetic acid, and the experiment 
 then continued, the acid so formed was decomposed even more 
 quickly, the same products being formed. 
 
 The oxidation of the alcohol takes place in two stages; 
 there is first a formation of aldehyde, which is subsequently 
 
378 ACETIC FERMENTATION. [CH. 
 
 converted into acetic acid, the reactions being expressed by the 
 equations 
 
 2CH3CH,OH + 0, = 2CH,C0H + 2HJ0 ; 
 2CH3COH + O2 = 2CH3COOH. 
 
 B. aceti was found by Brown to be capable of oxidising 
 propylic alcohol to propionic acid, but it was not able to de- 
 compose either methylic, butylic, or amylic alcohol. 
 
 Boutroux stated in 1880 that when B. aceti was cultivated in 
 a solution of glucose it converted the latter into gluconic acid. 
 During the progress of the action the solution was kept neutral 
 by the presence of calcic carbonate, which neutralised the acid 
 as it was formed. 
 
 Blondeau also noted that sugar could be converted into an 
 acid by the organism, without passing through the stage of 
 alcohol. Brown confirmed these observations and suggested 
 the following equation as expressing the reaction — 
 
 2CHjOH(CHOH)4COH + O, = 2CH,OH(CHOH)4COOH. 
 
 The oxidation is thus seen to affect the aldehyde group in the 
 sugar just as in the case of the second stage of the oxidation of 
 ethylic alcohol. 
 
 The organism was found to be incapable of attacking cane- 
 sugar. 
 
 The alcohol mannitol, which corresponds to the aldehyde 
 glucose, was also examined. Brown cultivated the organism in 
 a 2 per cent, solution of mannitol, Pasteur's mineral medium 
 and a little gelatin being added as food for the bacillus. The 
 culture was continued for six weeks, at the expiration of 
 which time the solution had acquired a very sweet taste and 
 reduced Fehling's fluid freely. It was then found that the 
 mannitol had completely disappeared, having been oxidised to 
 levulose. The sugar was identified by its reducing power 
 and its optical activity, and further by its power of forming a 
 crystalline compound with lime, according to Dubrunfaut's 
 process. 
 
 The organism cannot decompose lactose, starch, dulcite, or 
 levulose ; it can convert glycol into glycollic acid. 
 
 Another organism which possesses the power of forming 
 
XXI] ACETIC FERMENTATION. 379 
 
 acetic acid at the expense of ethylic alcohol is the so-called 
 vinegar plant, which also has been investigated by Brown, 
 who has given it the name of Bacterium xylinum. This, like 
 B. aceti, is an aerobic organism, found growing on the surface of 
 the culture fluid, and forming there a jelly-like translucent mass, 
 sometimes as much as 25 mm. in thickness. This membrane is 
 slightly heavier than water and sinks when completely wetted, 
 a further growth commencing at once above the old layer. 
 Frequently 5 or 6 layers of growth are thus formed, so that 
 the veil or pellicle appears laminated. If the organism is 
 cultivated in a liquid unfavourable to its free growth, such as 
 yeast-water, it appears as a jelly-like transparent mass at the 
 bottom of the solution. This gradually increases in size till 
 the liquid is almost entirely filled with it. The jelly when 
 treated with sulphuric acid and iodine takes on a deep blue 
 coloration, indicating that it is composed of cellulose. It seems 
 to be made up of extremely difHuent membranes of the organism, 
 forming a kind of zooglcea. 
 
 When a film of the vinegar plant is examined microscopi- 
 cally it is found to consist of bacteria arranged more or less in 
 lines and lying embedded in a transparent structureless sheath. 
 These bacteria are most commonly found as rods about 2/a in 
 length, several often being united together. In old cultivations 
 the rods are frequently to a large extent replaced by micrococci 
 about 'S/i in diameter. When the organism has been cultivated 
 in an unsuitable medium, such as yeast-water, it appears as 
 long twisted threads, from 10 — 30/i in length and of a leptothrix 
 nature, differing however in appearance from the similar threads 
 of B. aceti. 
 
 The organism grows best at a temperature of about 28° 0. 
 Above 36° C. it ceases to develop. 
 
 The fermentations set up by B. xylinum are similar to those 
 caused by B. aceti. Ethylic alcohol is oxidised to acetic acid, 
 and the acid subsequently entirely destroyed. Glucose is oxi- 
 dised to gluconic acid and mannitol to levulose. The organism 
 has no oxidising action on cane-sugar, starch, or levulose. The 
 great difference between the two is the large amount of cellulose 
 formed by B. xylinum. 
 
380 NITRIC FEBMENTATION. [CH. 
 
 Nitric fermentation. 
 
 The decay of organic matter in the soil, whether it is 
 derived from the ddbris of vegetable or animal matter, is as- 
 sociated with the formation of ammonia or its compounds. 
 Ammonium salts, especially the sulphate, are constantly in use 
 among agriculturists for purposes of manure. The compounds 
 of ammonia are however not so easily made use of by green 
 plants as are the salts of nitric acid; It has long been known 
 that the soil is the seat of a variety of chemical changes, among 
 which the formation of such salts from compounds of ammonia 
 is especially prominent. 
 
 Nitrification, as this process is called, was formerly regarded 
 as a purely chemical process. Like the oxidation of alcohol, it 
 can be eifected through the agency of platinum black. If a 
 mixture of air and ammonia gas is heated gently in a tube 
 containing spongy platinum, the latter becomes white-hot, and 
 forms ammonium nitrate at the expense of the gaseous mixture. 
 The influence of porosity, and the free oxygen entangled in the 
 interstices of the porous body were held to be the explanation 
 of the oxidation in the soil as they were in the case of acetification. 
 
 That the phenomenon is to be regarded as biological and 
 not purely chemical may be attributed to the researches of 
 Schlosing and Miintz, which were published in 1877. They 
 found that the conditions of nitrification strongly suggested 
 bacterial agency. The process is hardly noticeable at a low 
 temperature, such as 5° C; as the temperature rises it becomes 
 more marked, and reaches a maximum at 37° C. Beyond this 
 point it gradually weakens and cannot be observed above 55° C. 
 They found that soil which had been heated to 100° C was not 
 subsequently capable of exhibiting the process. 
 
 By introducing small quantities of soil into a fluid medium 
 containing a salt of ammonium, preferably the chloride, nitrifi- 
 cation of the latter can be made to take place. 
 
 Experiments made subsequently by many observere have 
 shown that the compounds of nitrogen found in liquids and soils 
 which are the seat of nitrification are not always the same. In 
 some cases nitrites and in others nitrates are produced. A long 
 
XXt] NITRIC FERMENTATION. 381 
 
 series of investigations has shown that the action takes place in 
 two stages ; in the first the ammonium salt is oxidised to the 
 condition of a nitrite and subsequently further oxidation pro- 
 duces nitrates from the latter. 
 
 Schlosing and Mtintz originally attributed the action to a 
 single organism, which they considered oxidised the ammonium 
 salt to the condition of a nitrate, and they held that the 
 nitrites found were the result of purely chemical processes of 
 reduction. They described the microbe as consisting of cor- 
 puscles, round or slightly elongated, of varying dimensions, 
 largest when grown in media rich in organic matter, but at 
 all times very small ; occurring either singly or in pairs, and 
 multiplying by division. 
 
 The researches of Warrington in England and Winogradsky 
 on the continent have satisfactorily shown that two distinct 
 micro-organisms play a part in the process of nitrification, and 
 that probably more than one species of each exists. 
 
 One of these has the power of oxidising salts of ammonium' 
 to the condition of compounds of nitrous acid. When in a 
 pure culture this stage has been reached no further oxidation 
 takes place. Winogradsky has established two genera of these 
 organisms, Nitrosomonas and Nitrosococcus, the former being 
 somewhat strangely only found in the soils of Europe, Asia, 
 and Africa, the latter in those of America and Australia. 
 
 The form of nitrosomonas as cultivated by Winogradsky in 
 an ammoniacal solution is generally that of an elongated ellip- 
 soid, but the youngest cells are nearly spherical. The breadth 
 does not exceed 1/i nor the length 1-1 — 1-8/^. The longest are 
 those about to divide and they then exhibit a dumb-bell form. 
 More rarely the organism is spindle-shaped with blunt ends. 
 A chain of 3 — 4 individuals is very rare. 
 
 When the Nitrosomonas is cultivated in an aqueous solution 
 of sulphate of ammonium to which some carbonate of magnesium 
 has been added, three stages can be noted in its growth. After 
 about 4 days isolated colonies appear in the liquid, each of 
 which is included in a gelatinous mass of the nature of a 
 zoogloea. Three days or so later the liquid becomes turbid, and 
 is found to contain a motile form which is furnished with cilia. 
 
382 NITRIC FERMENTATION. [CH, 
 
 The colonies at this stage have broken up. After a further 
 2 days the liquid again becomes clear and the microbes are 
 found to have formed another zooglcea which lies as a gelatinous 
 layer at the bottom of the culture vessel. 
 
 The Nitrosococcus forms behave dififerently. They do not 
 form a zoogloea, nor do they possess cilia. They are slightly 
 larger than the others, ranging from 1'5 to 2/i in diameter. 
 
 Besides these organisms, which are responsible for the first 
 stage only of nitrification, there are others which have the 
 power of converting the nitrites therein formed into nitrates, 
 thus completing the work. These are among the smallest of 
 all living organisms so far discovered. The cells are elongated, 
 and oval or somewhat pear-shaped, being about 'o/i in length 
 and from "15 to '25/i in breadth. The name Nitrobacter has 
 been given to the genus. 
 
 Most of our knowledge of this microbe is due to the 
 researches of Winogradsky. It can be cultivated in Hquid 
 media, when it forms a thin mucinous skin which adheres to 
 the wall of the vessel. 
 
 The two kinds of organism are usually both present in the 
 same soil, those of the second type immediately oxidising the 
 nitrites which those of the first form from ammonium salts. 
 The Nitrobacter forms not only cannot oxidise the latter bodies 
 but they are very injuriously affected by the presence of free 
 ammonia. Except in this respect the two classes show great 
 similarity. Both flourish in inorganic solutions, and are aided 
 in their work by the presence of acid carbonates. Neutral or 
 alkaline carbonates hinder or prevent their development, and 
 the chlorides of potassium and calcium are injurious to their 
 action. Neither of them apparently will grow on gelatin. 
 
 The conditions of their action are remarkably similar : below 
 0° C. hardly any change can be initiated by either ; at about 
 12° C nitrification becomes sensible and increases rapidly as 
 the temperature rises to 37° C. At this point the rate of 
 change is ten times as great as at 14° C. Above 37° C. a 
 rapid decline of the rate may be observed, and according to 
 Winogradsky the fermentation stops at 55° 0. Warrington 
 failed to observe nitrification above 40° C. 
 
XXl] NITRIC FERMENTATION. 383 
 
 A very interesting peculiarity attaching to the bacteria of 
 nitrification is their marked distaste for organic nutriment. 
 Neither of them will grow upon gelatin or similar material, 
 apparently disliking all organic substances. They can be 
 cultivated readily on masses of gelatinous silica, impregnated 
 with the appropriate compounds of nitrogen. As a suitable 
 culture fluid for the Nitroso-forms Winogradsky recommends a 
 mixture of 2 — 2'5 grammes of ammonium sulphate, 2 grammes 
 of sodium chloride and a sufficient quantity of magnesium 
 carbonate per litre of well-water. The latter salt is needed to 
 neutralise the acids as they are formed. For Nitrobacteria 
 the ammonium salt must be replaced by sodium nitrite. 
 
 When either organism is cultivated in such a medium it 
 can grow and multiply, and the development is greatest in the 
 absence of light. The source of the carbon of the increased 
 bulk of the plant is carbon dioxide, derived partly from the car- 
 bonate in the solution, and, according to Godlewski, partly from 
 the air. We have in these plants therefore a power which 
 appears special to them, viz. that of decomposing carbon 
 dioxide and availing themselves of its carbon in the con- 
 struction of new cells, in the absence of light. They show 
 indeed that the power of decomposing carbon dioxide can 
 go on in these plants in the absence of any chlorophyll 
 apparatus. The steps by which this carbon dioxide is built up 
 into a compound capable of being assimilated by the living 
 substance are not known. The energy necessary for the process 
 appears to be supplied by the oxidation of the molecules con- 
 taining nitrogen, so that it is dependent upon such oxidation 
 taking place. Winogradsky has investigated this point with 
 much care, and he has come to the conclusion that about 
 35 milligrammes of nitrogen are oxidised for each milligramme 
 of carbon absorbed and fixed. 
 
 Besides the oxidation of ammonium salts, another process is 
 also constantly going on in the soil which is of exactly the 
 opposite character. We meet continually with a process of 
 denitrification, marked sometimes by the evolution of ammonia 
 and sometimes by the disengagement of free nitrogen. The 
 decomposition of proteid matter and its derivatives is effected 
 
384 VISCOUS FERMENTATION. [CH. 
 
 by many species of Schizomycetes, and by many of the higher 
 fungi. Of them all Bacillus mycoides especially may be men- 
 tioned, as it gives rise to a very considerable volatilisation of 
 ammonia. This process has very much in common with the 
 ordinary putrefactive processes, which will be discussed sub- 
 sequently. The production of free nitrogen is different, as the 
 sources from which it is derived are the nitrates and nitrites of 
 the soil. The Bacillus denitrificans a and /8 of Guyon and Dupetit 
 are especially noteworthy in this connection. Both species 
 reduce nitrates, a reducing them to nitric oxide and nitrogen, 
 yS liberating free nitrogen only. 
 
 Several other species possessing similar powers have been 
 described by various writers, among them being Bacterium coli 
 commune, which reduces nitrates to nitrites when cultivated in 
 a nutrient solution by itself and in the absence of air. When 
 in the soil it frequently grows symbiotically with a bacillus, 
 known as B. denitrificans I., and the two can then decompose 
 the nitrate completely, free nitrogen being evolved. Loew has 
 shown that the process of reduction goes so far as the formation 
 of ammonia. 
 
 Viscous fermentation. 
 
 A peculiar phenomenon occurs frequently in certain wines, 
 in beer-worts, and in some natural vegetable juices. The liquid 
 on exposure to the air becomes extremely viscous, in some 
 cases indeed it appears to be transformed into a jelly. 
 
 This phenomenon, which must not be confused with the 
 action of pectase, has been investigated by several observers, 
 and has been found to be due to the presence of microbes, 
 several of which have been isolated. Among the earlier workers 
 in this field may be mentioned Braconnot, Guy-Lussac, Fremy, 
 and Pasteur. 
 
 According to Pasteur this viscous fermentation is caused by 
 a particular organism which acts upou either glucose or invert- 
 sugar and transforms it into a kind of dextrin or gum, mannite 
 and carbon dioxide being formed at the same time. The gum 
 appears to resemble dextrin rather than gum-arabic, as nitric 
 acid oxidises it to oxalic and not to mucic acid. 
 
■^Xl] VISCOUS FERMENTATION. 385 
 
 H^ry found that the peculiar ropiness which is formed in 
 certain inks is due to similar agency. 
 
 The same phenomenon is often met with in the manufacture 
 of cane-sugar, masses of gelatinous consistency being formed 
 in the crude juice. These were iirst accurately investigated 
 in 1878 by Cienkowski, who found them to be composed of 
 microbes with extraordinarily swollen and gelatinised cell-walls, 
 which appeared as masses of jelly in which the organisms were 
 embedded. Van Tieghem, who also studied them, and who 
 followed out the life-history of the microbes, held that they 
 resembled in most important respects the alga Nostoc, except 
 that they did not contain any of the blue-green colouring 
 matter so characteristic of that plant. He gave to the microbe 
 consequently the name of Leuconostoc. Further investigations 
 were carried out by Liesenberg and Zopf in 1891. 
 
 The microbe, according to the last-named observers, does not 
 form the gelatinous material unless the nutrient material on 
 which it is cultivated contains either cane-sugar or glucose. 
 In the absence of these carbohydrates it is found in chains, 
 sometimes of considerable length, sometimes of only two or 
 three cells. When either sugar is present large sheets of 
 zooglcea are formed, in which the cells are arranged in pairs. 
 The cells have greatly swollen walls, forming mucinous cap- 
 sules, which gradually coalesce, giving rise to the gelatinous 
 masses. 
 
 The jelly does not consist of unchanged cellulose, but of a 
 material which was originally called dextran by Scheibler, and 
 which is probably a derivative of cellulose. 
 
 An organism which has recently been examined by the 
 writer in collaboration with Marshall Ward, and to which refer- 
 ence has been made in the preceding chapter, was also found to 
 have the power of forming a mucous or slimy material when 
 cultivated in solutions of cane-sugar. After about 3 days' 
 growth, the whole contents of a flask in which the culture was 
 conducted became extremely viscid. The solution was found 
 to contain two carbohydrates resembling in many respects 
 Scheibler's dextran. They were thrown out of solution by 
 the addition of alcohol, and were separated by subsequent 
 G. F. 25 
 
386 VISCOUS FERMENTATION. [CH. 
 
 treatment with water, in which one of them dissolved. The 
 other was soluble only in dilute alkali, 1 per cent, of caustic 
 soda taking it up slowly in the cold and more rapidly on 
 boiling. It was freely soluble in the cold in a 10 per cent, 
 solution of the alkali. 
 
 The first of these bodies had a specific rotatory power of 
 (a)jp = + 130 ia a 1 per cent, solution. It gave a pink colora- 
 tion when treated with iodine, and did not reduce Fehling's 
 solution. 
 
 The other, when in solution in 1 per cent, caustic soda, had 
 no action on polarised light. It was coloured violet by iodine, 
 and like the first had no cupric-reducing power. 
 
 Both bodies when boiled for some time with a dilute 
 mineral acid were converted into substances which reduced 
 Fehling's fluid. 
 
 From their reactions they appeared to belong to the group 
 of the hemicelluloses, but they did not yield oxalic acid when 
 oxidised by nitric acid. 
 
 Various organisms have been found to set up similar forma- 
 tions in milk, causing a peculiar "ropiuess " in the liquid. 
 Their influence appears to be chiefly confined to the milk- 
 sugar. 
 
 It is probable that in most of these cases the viscous matter 
 is nothing more than the extremely diffluent cell-walls of the 
 organisms, and it is therefore doubtful whether these phenomena 
 should be classed among such fermentations as we are con- 
 sidering. Whether or no enzymes having the properties of 
 cytase or pectase are secreted by the microbes we have no 
 evidence to show, nor whether the changes in the cell-walls 
 are due to cytasic or pectasic powers of the protoplasm. 
 
 There are however cases known in which a similar production 
 of viscous or mucous matter has been found to take place extra- 
 cellularly. A microbe was described in 1889 by Kramer as 
 possessing this property. Microscopic investigation shows it 
 to be a bacillus, occurring in short rods which are joined 
 together in chains. The cell-walls of the organism do not 
 swell up as in the formation of a normal zooglcea, but when it 
 is cultivated in the presence of cane-sugar a peculiar mucoid 
 
XX l] PUTREFACTION. 387 
 
 material is formed in the culture medium which does not seem 
 to include the bacilli. 
 
 Another organism was ascertained by Glaser to possess the 
 same property. He named it Bacteriv/m gelatinosv/m betce, from 
 its occurrence in the juice of the Beet. The mucous material 
 which it forms shows considerable resemblance to dextran. 
 
 Putrefaction. 
 
 Putrefaction is a process which is associated with the changes 
 which various micro-organisms set up in proteids or albuminoid 
 bodies. The various decompositions usually begin with the 
 formation of peptones in a manner which resembles the gastric 
 and pancreatic digestions already described. The changes do 
 not stop at this stage, nor do they follow the same course as 
 those brought about subsequently by trypsin; leucin and 
 tyrosin appear to be formed, but at the same time many 
 other bodies appear, probably simultaneously. Among these 
 are indol, skatol, phenol, and a variety of substances belonging 
 to the aromatic series. Besides such comparatively simple 
 bodies whose composition is known, we find others of more 
 complex nature, many of which are eminently toxic. These 
 include the so-called animal alkaloids or ptomaines, a great 
 number of which have been isolated from various putrefying 
 materials. The proteid molecule is broken down yet further as 
 the fermentations proceed, several of the higher fatty acids, 
 ammonia and its compounds, various amines, and different 
 nitrates, making their appearance. The sulphur of the albu- 
 minoid matter is generally evolved in the form of sulphuretted 
 hydrogen, though mercaptan has been detected in some 
 cases. 
 
 The putrefactive process is accompanied by the development 
 of a peculiar and characteristic odour, partly due to the pre- 
 sence of certain of the bodies mentioned, and partly to the 
 sulphuretted hydrogen, ammonia, and other gases which are 
 disengaged. This feature is especially noticeable when the 
 microbes carry out their work in the absence of air, the process 
 being comparatively inodorous when a free access of oxygen is 
 
 2.5—2 
 
388 PUTREFACTION. [CH. 
 
 permitted. Indeed putrefaction in the strict sense of the term 
 is largely an anaerobic process. 
 
 Many microscopic organisms take part in these putrefeetive 
 changes and their action is far from completely ascertained at 
 present. It is not known whether a particular series of changes 
 is due entii-ely to one kind of microbe, or whether the process 
 is initiated by one and carried further by another; whether 
 the decompositions take place simultaneously or successively. 
 
 The earliest researches on the question which may be 
 quoted are those of Ehrenberg, who was the first to notice, in 
 1830, the microbe long known as Bacterium termo. This was 
 described 11 years later by Dujardin as possessing a cylindrical 
 form about 2 — 3/i in length and 1 — 1-2/a in thickness. The 
 cells were said to be frequently joined together in pairs, and 
 the short chain so formed exhibited a tremulous movement. 
 Cohn stated in 1892 that putrefaction was a special process set 
 up by this bacterium. 
 
 Bacterium termo was the subject of study by many 
 observers during the next decade. Improved methods of 
 cultivation showed that several separate forms were associated 
 under the one name, and in 1884 Eosenbach described three 
 distinct species, under the names of Badllus saprogenes I, 
 II, and III. Hauser a year later described three species of 
 Schizomycetes taking part in putrefaction, which he referred to 
 a new genus, Proteus. These are eminently motile, the cells 
 being furnished with a variable number of cilia. P. vulgaris 
 is often met with in the form of elongated rods, ranging from 
 3 — 6/i in length, with a transverse diameter of 'Q/i. All the 
 forms however are very polymorphic. 
 
 Escherich has described another microbe under the name of 
 Bacterium coli commune, which is always present in the mam- 
 malian intestine and which can be detected in large numbers 
 in the evacuated faeces. It is a short rod ranging in length 
 from bfj. to 2 — 3/i and having an average breadth of '4/^. 
 
 Several other species, not so clearly identified, have been 
 described by many writers. All agree however in being ex- 
 tremely pol3niioi'phic, the form they take varying according 
 to conditions of cultivation and nourishment. The difficulty of 
 
XXl] PUTREFACTION. 389 
 
 ascertaining the limits of any species is consequently extremely 
 great. 
 
 The complicated nature of the digestive changes in the 
 mammalian alimentary canal must be associated with the 
 presence there of many of these bacterial forms. As we have 
 seen the final action of the pancreatic juice is to convert part 
 of the proteid molecule into leucin and tjrrosin. The occurrence 
 of indol, skatol, and other malodorous products in the large and 
 in part of the small intestines, can no doubt be explained by the 
 action of these microbes, which work simultaneously with the 
 trypsin of the pancreatic secretion and continue to act after the 
 enzyme has ceased to be effective. The absence of the microbes 
 from the region of the small intestine in which the bile remains 
 is explained by the antiseptic powers of this secretion. 
 
 Though no doubt the direct intervention of the protoplasm 
 of the microbe is in most cases the exciting cause of the 
 fermentation, or putrefaction, it must not be forgotten that 
 many of these organisms have been shown to excrete enzymes, 
 to the action of which definite decompositions have been traced. 
 The observations of Lauder Brunton and MacFadyen, and of 
 Wood, have been alluded to in a former chapter, and it has been 
 shown there that various microbes have been proved to secrete 
 trypsin. Various toxic albumoses have been shown by Hankin, 
 Martin, and others, to result from the activity of Bacillus 
 anthracis, and Hankin was able to extract from this microbe an 
 enzyme that had the property of forming albumoses from fibrin. 
 Another enzyme has been prepared by Oourmont and Doyon 
 from the bacillus of tetanus which effects a different decom- 
 position, producing a toxic substance which is comparable to 
 strychnine. This enzyme, which has received no name, is 
 destroyed on heating to 65° C. Its discoverers attribute the 
 peculiar symptoms of tetanus to its presence, and state that 
 immunity from this disease is the result of the action of causes 
 that inhibit the activity of the enzyme. It is hot itself toxic, 
 but produces the alkaloidal substance in the body, and the 
 deleterious material can be extracted from tetanised muscles and 
 from the blood and the urine of an animal which has been thus 
 poisoned. 
 
390 PTOMAINES. [CH. 
 
 The toxic products of putrefaction include not only albu- 
 moses, but a number of derivatives of various albuminoid 
 bodies to which the name of ptomaines was given by Selmi in 
 1878. There have been many workers in this field, chief among 
 whom may be named Panum, Nencki, and Brieger. 
 
 The majority of the ptomaines at present known were dis- 
 covered l)y the last-mentioned investigator, to whose labours we 
 are indebted for new methods of separating these substances from 
 putrefying liquids. Brieger isolated choline, jmtresdne, neuri- 
 dine, and cadaverine. From the decomposition of the first of 
 these rmiscarine may be obtained, the poison of the red Agaric. 
 Some of these toxic bodies occur in stored cheese which has 
 been attacked by certain microbes. 
 
 Bodies aUied to the ptomaines, though not identical with 
 them, which are produced in the body have been called leuco- 
 viaines. They differ from the ptomaines in being products of the 
 metabolic activity of the tissues and not of microbes. They are 
 produced, that is, by animal, and not by vegetable protoplasm. 
 
 Many of the ptomaines and similar bodies appear to belong 
 to the pyridine group of aromatic compounds. 
 
 The fermentative power of protoplasm is thus seen to be 
 constantly manifested and to conduct a great variety of de- 
 compositions, some relatively simple, and others extremely 
 complex. The living substance may be that of unicellular or 
 of multicellular organisms, of microbes of almost immeasurable 
 smallness, or of plants of very large dimensions, the differen- 
 tiation of the plant affecting the distribution but not the 
 character of the active protoplasm. Certain features connected 
 with the manifestation of this property by unicellular microbes 
 led to their being for a long time considered to be especially 
 "ferments" and to possess a property which was peculiar to 
 them as a class. Among these features was the phenomenon 
 that the changes they caused were on such a relatively large 
 scale that they seemed to be altogether out of proportion to 
 their biological needs, and hence to require an explanation of 
 a different kind. The prominent fact was the magnitude of 
 
XXl] THE FERMENTATIVE POWER OF PROTOPLASM. 391 
 
 the disturbance they caused, and this obscured the metabolic 
 ■work to which the induced changes ministered. 
 
 The same facts meet us even to-day and we are not yet 
 prepared with more than a tentative explanation of them. 
 But we can see from comparing these humble forms with 
 the higher plants that their activity is paralleled by the 
 activities of the cells of many of the tissues of the latter ; that 
 the decompositions set up by both are of the same character ; 
 and that in some cases the changes are brought about by both 
 through the agency of secreted enzymes. 
 
 The decompositions of these so-called organised ferments aire 
 then only peculiar in two respects : (1) the great and apparently 
 wasteful extent to which they are conducted, (2) the more 
 complicated character of the products produced by some of 
 them. The second of these statements must not however be 
 regarded as proved ; it may be that the complex products of 
 putrefaction, for instance, are the result of the energy of several 
 microbic forms working in succession. In this case the second 
 statement merely indicates that the protoplasm of dififerent or- 
 ganisms possesses very varied powers of exciting very different 
 decompositions. And even this view is incomplete, for the 
 number of enzymes known is continually increasing and may 
 ultimately be found to correspond to the variety of decom- 
 positions now known to be set up by living substance. 
 
 Other specific peculiarities have been associated with mi- 
 crobes, vindicating their claim to be regarded as organised 
 ferments. These have been brought forward with a view to 
 demonstrating that "organised" and "unorganised" ferments 
 are radically different. They have been especially emphasised 
 by Naegeli and Sachs, and may be mentioned here to show 
 how completely this old distinction has broken down, how 
 indeed two things have been put in comparison that are so 
 fundamentally different that comparison is impossible, whereby 
 the true relation between them has been entirely obscured. 
 
 Sachs states the first of these peculiarities in the following 
 terms: — "I find with Naegeli a further particularly striking 
 point of difference in the fact that (apart from split-ferments 
 such as emulsin and myrosin) the plastic matters are transferred 
 
392 THE FERMENTATIVE POWER OF PROTOPLASM. [CH. 
 
 by the action of unorganised ferments from the passive into the 
 active condition. Fermentation by means of Fungi has, as 
 Naegeli insists, just the opposite character; its products are, 
 without exception, less nutritious compounds, and it destroys 
 especially the most nutritious substances." 
 
 Naegeli states, " The contrast appears most striking in the 
 case of carbohydrates and proteid substances. While the 
 action of unorganised ferments produces from them glucoses 
 and peptones, fermentation by means of Fungi breaks up these 
 compounds into alcohol, mannite, lactic acid, and into leucin, 
 tyrosin, &c. — and in some cases several fermentations follow 
 one another; their products then became less nutritious step 
 by step. We may say, generally, that the yeast fungi render 
 the medium in which they occur chemically less suitable for 
 nutrition by every process of fermentation which they effect." 
 
 In the light of the knowledge which has been obtained 
 during recent years these statements can no longer be regarded 
 as marking differences between fermentation by microbes and 
 by enzymes respectively. Sachs's first criticism loses a good 
 deal of force on account of the exceptions he mentions. There 
 is no reason why emulsin and myrosin should be considered 
 exceptional. We have seen that the conditions under which 
 they are active are the same as those which are needed by 
 diastase and by pepsin. The action is similar in kind as far 
 as we can judge; diastase and emulsin both cause hydrolysis 
 and subsequent decomposition of the bodies which they attack. 
 The property of giving rise to less nutritious compounds does 
 not belong to. Fungi alone ; enzymic activity often results in 
 similar decompositions. Urease produces ammonium carbonate 
 from urea ; the lactic enzyme discovered by Hammarsten effects 
 the same change as the lactic microbe ; Buchner's zymase 
 produces alcohol from sugar. On the other hand, again, certain 
 microbes convert starch into sugar, while others peptonise 
 albumin. 
 
 The contrast drawn by Naegeli also fails. We have just 
 seen that the saccharification of starch and the formation of 
 peptone from albumin can be brought about by members of 
 both classes. 
 
XXl] THE FERMENTATIVE POWER OF PROTOPLASM. 393 
 
 The conversion of proteids into leucin, tyrosin, asparagin, 
 &c., is not a special property of Fungi; it is in fact the 
 characteristic feature of both animal and vegetable trypsins. 
 
 Both the botanists quoted lay stress on the formation of 
 nutritive substances from plastic ones by enzymes. The action 
 of the latter is by no means confined to effecting transformations 
 of this kind. We may mention here the recently discovered 
 group of the oxidases, which are not concerned with nutritive 
 processes at all. Nor so far as we know are the clotting enzymes 
 thrombase and pectase to be considered as forming nutritive 
 materials for the organism in which they occur. 
 
 Sachs says again, "Naegeli puts his view of fermentation 
 due to organisms as opposed to that produced by unorganised 
 ferments in the following statement : — ' Fermentation is there- 
 fore the transference of the movements of the molecules, 
 atomic groups, and atoms of various compounds composing 
 living protoplasm (which remain chemically unaltered in the 
 process) to the fermentable material, by which means the 
 equilibrium between its molecules is destroyed and it is broken 
 up.' " It has been shown by Fischer and other writers that 
 this statement, which presents Naegeli's theory of fermentation, 
 if true at all, applies equally well to enzymes. This point how- 
 ever will be more fully examined in a subsequent chapter. 
 
 If we turn again to the consideration of the fermentative 
 processes set up by the protoplasm in the higher plants we find 
 that the latter exhibit the same variety as the microbes in the 
 nature of the products that are formed. Boehm and de Luca 
 have shown that if any part of a living plant is insufficiently 
 supplied with oxygen, hydrogen and sometimes marsh-gas are 
 evolved from it. Boussingault and Schulz have observed 
 similar phenomena. Hydrogen is given off also from plants 
 containing mannite, while malic acid gives rise to acetic acid in 
 the fruits, flowers, and leaves of the Privet. In the decom- 
 position of proteids, Boehm found ammonia exhaled. The 
 condition under which these results are obtained, viz. the lack 
 of oxygen, is the normal condition of many microbes, they 
 being anaerobic in their mode of life. When oxygen is present 
 we find the same agreement. The inversion of cane-sugai- is 
 
394 THE FERMENTATIVE POWER OF PROTOPLASM. [CH. 
 
 the same process whether that action is brctught about by the 
 action of the living cell of a leaf or by invertase extracted from 
 either a higher plant or from yeast. 
 
 Sachs claims as a peculiarity of all fermentation set up by 
 fungi that carbon dioxide appears as a bye-product. • This, 
 however, we have seen to be rather an effect brought about 
 through an insufficient supply of oxygen, and easily made 
 evident under the same conditions in the fermentative actions 
 of the protoplasm of the higher plants also. We can see 
 therefore that in both the lower and the higher plants we have 
 to recognise essentially the same constitution, the differences 
 between them depending only on differentiation and consequent 
 division of labour. In the lowly forms the great prominence 
 of their metabolic decompositions has overshadowed all their 
 other functions, and they have therefore been regarded as 
 endowed with special properties. In the higher plants inves- 
 tigation has shown us that precisely similar decompositions can 
 be brought . about, not now by the whole plant-body, but by 
 special cells or parts of it. The agent in the decomposition is 
 the same, viz. protoplasm; the conditions are similar and the 
 resulting products are strictly comparable. The secretion of an 
 enzyme, which is a power exerted by both lowly and more 
 highly organised plants, is a mark of differentiation within the 
 living substance, just as in the slow movements of amoeboid 
 protoplasm we recognise something which in the higher and 
 more differentiated organism appears as the contraction of 
 muscular fibre. 
 
 A fact which at first appears to constitute the microbes into 
 a special class of fermenting organisms has several times been 
 incidentally alluded to. That is the power which many of 
 them exhibit of exciting more than one kind of decomposition. 
 Lauder Brunton, Wood, and other writers already quoted, have 
 shown that the same bacillus may saccharise stai'ch and pep- 
 tonise albumin ; many others can cause the clotting of milk in 
 addition. Bacillus mesentericus vulgatus has been shown to be 
 possessed of diastasic, inverting, cytasic, and peptonising power, 
 and to clot milk. 
 
 In these cases it is important to recognise the biological 
 
XXl] THE FERMENTATIVE POWER OF PROTOPLASM. 395 
 
 peculiarities of the organisms. They exercise these powers 
 according to the medium in which they are cultivated; many 
 of them carry out the changes by the excretion of enzymes, 
 and the formation of these depends in some way upon the 
 stimulus of the culture medium. Vignal, who investigated the 
 last-mentioned of these microbes, points out that the propor- 
 tions of the several enzymes it forms vary very greatly 
 according to the nature of the fluid in which it finds itself. 
 The fermentation in these cases is seen to be strictly sub- 
 ordinate to the metabolism. We have noticed in preceding 
 chapters that the secretion of particular enzymes by the higher 
 animals is very largely influenced by the character of their diet. 
 We may again mention in this connection the researches of 
 Vassilief on the influence of the latter on the relative propor- 
 tions of diastase and trypsin in the pancreatic juice. In fact 
 the production of enzymes shows a certain dependence on the 
 physiological and alimentary conditions of the organisms which 
 secrete them, as has been pointed out by Duclaux. 
 
 These phenomena recall also the observations of Schiff and 
 others as to the necessity of an absorption of some nitrogenous 
 compound or peptogen before the secretion of the gastric pepsin 
 takes place, which we have mentioned in an earlier chapter. It 
 also reminds us of the behaviour of an Amoeba, which only 
 forms a food vacuole with its digestive fluid round an ingested 
 nutritious particle. Any foreign substance which is not digest- 
 ible does not become enclosed in such a vacuole. 
 
396 
 
 CHAPTER XXII. 
 
 THE SECRETION OF ENZYMES. 
 
 We have seen incidentally in the case of many enzymes, 
 particularly those formed in well differentiated glands, that a 
 certain stimulus is necessary before secretion takes place. In 
 the case of saliva an increased flow can be excited by reflex 
 nervous agency, as when an afferent nerve is stimulated by 
 some sapid substance being placed upon the tongue, or when 
 some appetising odour reaches the olfiictory membrane of the 
 nose. The taking of food into the mouth is followed by an out- 
 pouring of both gastric and pancreatic juices. 
 
 Similar phenomena can be observed in the cases of some 
 vegetable secretions. The leaves of Drosera and of Dionsea do 
 not pour out their enzymes unless they are stimulated by con- 
 tact with the body to be digested. Clautriau has observed that 
 if a pitcher of Nepenthes is severed from the plant its contents 
 will not digest albumin, though another exactly similar one left 
 in its normal attachment will do so rapidly. He has suggested 
 that the secretion is controlled by the plant by something com- 
 parable to a nervous influence. 
 
 Apart however from actual nervous influences there is evi- 
 dence to show that the absorption of nutritive materials has 
 a very potent influence on the formation and discharge of the 
 secretion. This has been shown by many writers, among whom 
 may be mentioned Heidenhain, whose experiments on the 
 secretion of gastric juice in a portion of the stomach isolated 
 
OH. XXIl] THE SECRETION OF ENZYMES. 397 
 
 from the rest by Thiry's method, point to a material increase 
 in the quantity poured out, in consequence of the absorption of 
 food by the mucous membrane of the main portion of the organ. 
 Schiff also has called attention to the increase in the flow which 
 follows absorption of various nutritive bodies. 
 
 This form of stimulus has been observed to be also very 
 advantageous in the case of various vegetable organisms. The 
 formation of diastase in the pollen grains of Zamia was shown 
 by the writer to be directly dependent on the absorption of 
 sugar. 
 
 The observations already alluded to, which were made by 
 Lauder Brunton and MacFadyen on the enzymes secreted by the 
 bacilli they examined, also bear upon this point. They found 
 that the character of the medium in which the microbes were 
 cultivated determined whether the enzyme produced was dia- 
 stase or trypsin. The absorption of a carbohydrate in the one 
 case or a proteid in the other was apparently antecedent to the 
 production of the ferment. 
 
 A curious case of the influence of abundant nutrition on the 
 secretion of enzymes has been noticed by Dubourg. Certain 
 yeasts will not either invert or ferment cane sugar under 
 ordinary conditions. If however they are cultivated in a 
 liquid containing abundant supplies of nitrogenous material 
 together with glucose they become capable of doing both. 
 If the yeast is withdrawn from such a culture medium while 
 it is actively fermenting its sugar, and is washed free from all 
 saccharine matter, it will set up a fermentation on being mixed 
 with a solution of cane sugar. It appears in this case as if the 
 abundant supply of nutritive matter excited the formation of 
 the enzymes. 
 
 The apparently direct influence of the nature of the diet on 
 the secretion will also be remembered here. 
 
 In some cases however the stimulus is of a different kind. 
 Brown and Morris showed in their experiments on the secretion 
 of diastase by the scutellum of the barley-embryo, that no 
 enzyme is formed in its cells so long as the embryo is artifici- 
 ally supplied with solutions of nutritive carbohydrates, and is 
 not obliged in consequence to obtain its nourishment from the 
 
398 THE SECRETION OF ENZYMES. [CH. 
 
 staxch contained in its endosperm. Wortmann showed that 
 certain microbes produce diastase when starch grains are their 
 only available food, but do not if sugar is offered to them with 
 the starch. De Bary indicates a similar phenomenon in the 
 case of Bacillus amylobacter, which forms no cytase so long as 
 it can obtain glucose freely. If the supply of the sugar is 
 stopped, it attacks cellulose, and for that purpose secretes the 
 enzyme. Pfeffer has shown that the same thing can be noticed 
 in the cultivation of three organisms, Penidllium glauaiim, 
 Aspergillus niger, and Bacterium megatherium. The produc- 
 tion of diastase by aU three diminished as the percentage of 
 sugar was increased in the medium in which they were growing, 
 though Aspergillus proved itself less sensitive than the others. 
 H^rissey has shown that the secretion of emulsin by Aspergillus 
 is s imil arly influenced 
 
 In several of these cases the secretion of the enzyme appears 
 to be provoked by diminished nutrition, approaching indeed to 
 incipient starvation. 
 
 The process of secretion and the effect of these stimulations 
 upon it can only be advantageously studied in the case of well 
 differentiated glandular structures. Nor do all these offer equal 
 facilities, but those are most favourable in which the process 
 of secretion is both intermittent and fairly rapid when once 
 initiated. 
 
 For many reasons the cells of the pancreas are the most 
 suitable for a study of the secretory process. It is true that 
 this organ elaborates several enzymes, and so far as we know 
 they are aU formed together in the same cells. Their 
 formation is however a single process so far as can be 
 ascertained. 
 
 Heidenhain studied the appearances presented by pancreatic 
 cells under different conditions of nutrition. In the case of an 
 animal (dog) which had been festing for rather more than a day, 
 each cell was seen to consist of two zones ; one which abutted 
 on the lumen of the alveolus, and one at the back of the cell 
 towards the basement membrane. The iimer was considerably 
 the larger in area and was studded with fine granules. The 
 outer was narrow and its substance was homogeneous. The 
 
XXIl] THE SECRETION OF ENZYMES. 399 
 
 nucleus of the cell was shrunken and corrugated, and was found 
 at the border of the two zones. In the pancreas of another dog, 
 which was killed during full intestinal digestion, the same two 
 zones were evident, but the homogeneous outer one was much 
 wider and the inner granular one was contracted, the granules 
 being very much less numerous. The whole cell had suffered 
 a diminution in size, while the nucleus had regained a spherical 
 shape and was nearly central in position. A third pancreas, 
 excised at the time when digestion had ceased, showed the 
 outer zone again diminished and a marked increase in the 
 granularity of the inner zone. 
 
 The times when the granularity was most marked showed 
 that the latter was very probably connected with the presence 
 of the enzyme, which appeared to be formed duriag the period 
 of rest and extruded from the cell during that of activity. The 
 change in size of the cell which accompanied the pouring out 
 of the pancreatic fluid showed that the latter removed a con- 
 siderable quantity of some soluble substance with which the 
 cell had been distended. 
 
 The formation of the granules was synchronous with an 
 enlargement of the cell and indicated the absorption of some 
 material, nutritive in character, at the expense of which they 
 were constructed. 
 
 Kiihne and Lea watched the process of secretion in the 
 pancreas of the living rabbit, and noted similar appearances to 
 those described by Heidenhain ; they associated the preparation 
 of the enzyme with the formation of the granules, and the 
 pouring out of the secretion with their disappearance. 
 
 If we turn to the salivary glands we find similar phenomena. 
 In the mucous glands of the tongue of the frog we can see an 
 outer region in which the protoplasm is clear, and a much larger 
 inner region which is crowded with granules. In a serous gland 
 in a state of rest the whole cell appears granular, there being 
 no outer homogeneous zone. When the secretory nerve is 
 stimulated, an outflow of the secretion follows, and this is 
 attended by a diminution of the granularity. In the mucous 
 gland the granules nearly all disappear ; in the serous one they 
 are removed from the outer part of the cell, which becomes 
 
400 THE SECEETION OF ENZYMES. [CH. 
 
 homogeneous. As the granules axe carried away the whole 
 alveolus shrinks and the outlines of the constituent cells become 
 more evident. 
 
 The same round of changes can be observed in the chief 
 cells of the gastric glands, which secrete pepsin. In the various 
 regions of the stomach of a mammal we can observe an even 
 more definite relationship between the granules and the enzyme. 
 In the rabbit we have an animal which shows this relationship 
 remarkably well. In the cardiac or fundus region of its stomach 
 the chief cells are very coarsely granular, the tubular gland 
 appearing almost as a mass of granules. Towards the greater 
 curvature this becomes less and less obvious ; about the middle 
 of this region the cells possess an outer homogeneous border: 
 further on, as the pyloric region is approached the cells contain 
 fewer and fewer granules, and the pyloric glands themselves are 
 non-granular. This distribution corresponds exactly with that 
 of the pepsin in the mucous membrane. 
 
 The regularity of the disappearance of the granules, be- 
 ginning at the outer border of the cell and extending inwards 
 towards the lumen, is characteristic especially of mammalian 
 glands. In the lower vertebrates, including birds, snakes, 
 and frogs, the formation of a non-granular outer zone cannot 
 generally be observed, but there is instead a diminution of the 
 number and size of the granules throughout the cell. 
 
 The reconstruction of the granules takes place with great 
 readiness. The process appears to begin before the cell is 
 completely emptied, and to continue during the latter part of 
 the period of digestion, so that by the time this process is 
 finished the cells have almost regained their origrual granular 
 appearance. 
 
 In some cases, especially in the pyloric glands of the 
 stomach, the secretion does not appear as granules, but is 
 stored in a homogeneous form in the meshes of the proto- 
 plasm. 
 
 In comparing these different changes, which we see are m 
 the main the same for aU glands, we find that the process of 
 secretion consists of a certain sequence of events. Starting 
 with a cell almost entirely depleted, we have its growth 
 
XXIl] THE SECRETION OF ENZYMES. 401 
 
 at the expense of something absorbed from without; im- 
 mediately following this growth there is the manufacture 
 of the coarse granules which in the resting condition of the 
 gland fill the cells ; and lastly, at the onset of the outflow of 
 the secretion, we have their solution and extrusion in the liquid 
 which the gland pours out. 
 
 In some cells these processes go on so far successively that 
 the sequence can be observed. It may be that all are pro- 
 ceeding simultaneously but at relatively dififerent rates. In 
 others the round of changes is the same, but they progress 
 simultaneously and at rates so much alike that the sequence 
 of events becomes very indistinct. 
 
 This difficulty of recognising a sequence of changes is still 
 more marked in the case of vegetable secretions, which are usually 
 very prolonged and probably hardly at all intermittent. They 
 have been studied in a few cases by various observers, but our 
 information is not nearly so complete as in the instances of the 
 animal cells already described. The difference depends ap- 
 parently on the character of the metabolism of the plant and 
 the animal respectively. In the latter, digestion is an inter- 
 mittent process, continually repeated; in the former the cor- 
 responding process, the utilisation of reserve materials in such 
 bodies as seeds, tubers, &c., is a continuous and very gradual 
 one. 
 
 The observations of Brown and Morris on the changes in 
 the scutellar epithelium of the barley grain are among the 
 most complete which have been made. 
 
 The epithelium covering the scutellum of the barley embryo 
 is composed of columnar cells having a length of about '03 to 
 •04 mm. and a breadth of "01 mm. Their long axes are at 
 right angles to the surface. The cell-walls are very thin and 
 are not cuticularised. The cell-contents before the commence- 
 ment of germination are very finely granular, and the nucleus, 
 which is large and elliptical in shape, lies near the base of 
 the cell, with its longer dimension across it. Within a few 
 hours after germination begins the very fine granules in the 
 protoplasm become much larger and coarser, and increase in 
 number to such an extent that the nucleus, which was at first 
 G. F. 26 
 
402 THE SECRETION OF ENZYMES. [CH. 
 
 very conspicuous, is so obscured as to become almost invisible. 
 This process is complete in about a day, and the granularity 
 is maintained until the endosperm is almost exhausted of its 
 reserve materials. At this point the epithelium shows a great 
 fallin g off in its function as a secreting tissue ; the protoplasm 
 again becomes clear and transparent. A few granules only 
 remain, and they are very small and highly refractive, showing 
 very little resemblance to the coarse granulation existing during 
 secretion. Another remarkable fact now noticeable is that the 
 nucleus of the cell has entirely disappeared. 
 
 The continued secretion depends upon the cells being amply 
 supplied with food. This is derived from the reserve materials 
 of the endosperm, partly the carbohydrates on which the 
 enzymes work and partly reserve-proteids obtained from the 
 aleurone layer. 
 
 It is impossible to disregard the general similarity of 
 feature between this 'prolonged process and the intermittent 
 one characteristic of the pancreatic cells. 
 
 The secretory process has been studied by Gardiner in the 
 cells of the glands which are situated on the leaf of DioncBO. 
 As we have seen, when the leaf of this plant is stimulated by 
 contact of its surfece with some nutritive material, the leaf 
 folds over and encloses the exciting body, and the glands pour 
 out a digestive secretion which contains a proteolytic enzyme. 
 The process of secretion is much more rapid than that of the 
 barley embryo, and may be repeated more than once by the 
 same gland. 
 
 Gardiner distinguishes four periods in the act of secretion — 
 (i) one antecedent to secretion, when the cells are in the 
 resting condition; (ii) a period during which the secretion is 
 formed and extruded from the gland ; (ui) a time when absorp- 
 tion of the digested material is effected; (iv) a period of 
 recovery. In the first of these, a layer of protoplasm lines the 
 wall of the cell, and surrounds a large central vacuole, filled 
 with cell-sap. The protoplasm is extremely granular, especially 
 round the nucleus, which is situated at the base of the cell 
 In many cases it is quite obscured by the granules. At the 
 end of the second period the nucleus has moved to the centre 
 
XXIl] THE SECRETION OF ENZYMES. 403 
 
 of the cell and is surrounded by a layer of protoplasm, which 
 is connected by bridles with the parietal film. The protoplasm 
 has lost its granularity and has become clear and hyaline. The 
 nucleus itself is much smaller than before. 
 
 Gardiner noticed also in the glandular cells of the tentacles 
 of Drosera that in the resting condition of the glands the 
 protoplasm was much more granular than it became after a 
 period of activity or secretion. Similar results were obtaiued 
 by Miss Huie. 
 
 The secretion of rhamnase by the raphe of the seed of 
 Rhamnus infectorius is stated by Marshall Ward to be attended 
 with the formation of granules. The cytase of the hyphae 
 of Botrytis, as described by the same observer, appears to be 
 associated with a number of brilliant refiringent granules which 
 give proteid reactions. These are very apparent in the drops 
 of fluid which the hyphse exude when they come into contact 
 with a fresh leaf surface ; moreover they only appear when the 
 hyphse are secreting the enzyme. 
 
 If we pass from the more definitely glandular tissues to 
 those which are the seats of intracellular digestion, the process 
 of secretion is not nearly so easily traced. In a few cases it 
 has been found to be associated with granularity of the 
 protoplasm of the cell, but this is far from universal. Nor 
 need we expect to find the stages so evident iu these cases, 
 for the mechanism of secretion is not nearly so well differen- 
 tiated. The cell in which the process is to take place must not 
 merely furnish the enzyme; it has also to absorb and perhaps 
 to prepare the material on which the enzjrme must work, and 
 it has to dispose iu some way or other of the products of the 
 digestion. These various functions must necessarily push the 
 process of secretion somewhat iato the background, or at any 
 rate obscure the appearances. 
 
 We have in Guignard's researches upon myrosin some facts 
 brought to light which indicate an essential similarity of the 
 process to that already described. The cells iu the roots of the 
 horse-radish and other plants which contain the myrosin can 
 be recognised by their peculiar granular contents, which give a 
 very marked reaction with Millon's reagent, the colour produced 
 
 26—2 
 
404 THE SECRETION OF ENZYMES. [CH. 
 
 being much more intense than that which is developed when 
 this fluid comes into contact with proteids. Guignard has 
 demonstrated a close correspondence between the degree of 
 this granularity and the amount of enzyme in the cells. 
 
 The process of the secretion of diastase in the cells of 
 foliage leaves is hardly at all known. Some researches carried 
 out by A. Meyer seem to indicate that it does not arise in 
 the ordinary protoplasm of the cell, but is formed in the 
 chromatophore or chlorophyll grain. The same thing has been 
 noted in connection with the parts of the plant which are not 
 green, where the diastase appears to arise in the leucoplastids 
 which form the grains of starch. The hypothesis is largely 
 based upon the appearances presented by grains which have 
 been partially dissolved by the enzyme. Meyer describes many 
 cases in which the starch grains were attacked most energetic- 
 ally where the sheath formed by the stroma of the plastid was 
 thickest. In Iris he frequently found the side of the grain 
 eaten out to some extent where the leucoplast was in contact 
 with it. 
 
 Meyer's view has been confirmed by Salter, who made a 
 series of observations on the starch grains of Pellionia. He 
 says with reference to these, that when he examined grains in 
 process of solution, he found the action had proceeded farthest 
 at the point where the plastid was situated. The latter had 
 practically eaten its way, in many, cases, into the substance of 
 the starch grain. 
 
 Though in so many cases we are able to associate the 
 secretion of enzymes with the formation of the coarse granules 
 described, it by no means follows that the latter are the 
 ferments. Indeed evidence lies ready to hand that this is 
 not the case. If a pancreas is removed from a fasting dog 
 immediately after death and at once extracted with an 
 appropriate solvent, the resulting extract has Httle or no 
 fermentative activity. If however it is kept warm for a few 
 hours, and particularly if it is faintly acidified, the extract will 
 be very powerful. If the extract made immediately after 
 death is acidified and kept warm for some time, it will become 
 active. The material extracted from the gland cells, which 
 
XXIl] ZYMOGENS. 405 
 
 we have seen to be connected with, if not composed of, the 
 granules, is not the enzyme, but something which rapidly gives 
 rise to it when warmed with a trace of acid. This antecedent 
 substance has been named " mother of ferment " or zymogen. 
 
 There appears to be for each enzyme a distinct antecedent 
 or zymogen, in which form it is stored in the cells, ready to 
 be transformed into the active ferment directly it is required. 
 This change seems to take place immediately prior to, or 
 perhaps during, its extrusion from the cell. 
 
 We see thus that we can recognize in the secretion of en- 
 zymes a gradual process, at least one antecedent body being 
 formed. Are we able to investigate this process more closely ? 
 
 In the recital of the changes in the cells, we have noticed 
 almost always that the nucleus has appeared to play a part in 
 the secretion, though what its function is has not so far been 
 apparent. We may recall here the observation of Brown and 
 Morris on the scutellar secretion of diastase. This is a process 
 which is continuous after its first inception, and not inter- 
 mittent like that of animal glands. It will be remembered 
 that when the secretion has ceased and the granularity of the 
 cells has disappeared, the nucleus is found to be disintegrated. 
 
 Some extended researches upon this point have been carried 
 out by Macallum in connection with an enquiry into the distri- 
 bution of iron and phosphorus in cells generally. 
 
 His experiments were carried out on the pancreas of several 
 animals, by means of staining reagents which coloured differently 
 the various parts of the cytoplasm and nucleus, and also of cer- 
 tain special reagents capable of detecting iron and phosphorus. 
 Using eosin, he found that at a time coinciding closely with the 
 commencement of the deposition of the zymogen granules in 
 the cell, some material which stained conspicuously with that 
 dye disappeared from the nucleus, and the cjrtoplasm simul- 
 taneously developed an affinity for the eosin. In other words, 
 an eosinophilous substance diffused out of the nucleus into the 
 cytoplasmic zone. Later, this same substance appeared to be 
 removed from this region to be fixed in some way in the 
 zjrmogen granules. 
 
 Macallum noticed further that as the cell was filling up 
 
406 ZYMOGENS. [CH. 
 
 ■with zymogen granules the latter were largest at the border of 
 the lumen of the tubule, while the smallest were at the edge 
 of the gianukr area nearest to the nucleus. The granules 
 appeared to increase in size afber their first formation, at the 
 expense of a substance in the protoplasmic area of the 
 gland cell. 
 
 This relation between the nucleus and the zymogen granules 
 is supported by a striking resemblance in chemical composition. 
 The chromatin of the nucleus contains phosphorus in com- 
 bination. When nuclei are digested by artificial gastric juice 
 a residue containing phosphorus always remains undissolved. 
 This is associated with the nuclein which they contain, the 
 chromatin appearing to belong to the class of nucleo-proteids. 
 The zymogen granules in the cells of the pancreas of Die- 
 myctylus, afber being fi-eed fi-om adherent lecithin, were also 
 found to contain phosphorus, which however was hardly so 
 intimately combined in them as in the nuclear chromatin. 
 The protoplasm in which the granules lay in the inner zone 
 of the cell, also gave the phosphorus reaction, but much less 
 distinctly. The outer protoplasmic zone gave a reaction which 
 was intermediate in depth, and which appeared to be due not 
 to the cytoplasm itself, but to a substance contained in its 
 meshes. 
 
 All these various constituents of the cell were found to 
 contain iron in addition to phosphorus and in much the same 
 proportions. Macallum found that in a large variety of secreting 
 cells, glandular secretion was associated with the presence of an 
 iron-holding cytoplasm. Among them may be mentioned the 
 cells of the pancreas, the gastric and intestinal foUicles and the 
 parotid gland of Amhlystoma ; also the cells of the submaxillary 
 glands, and the chief cells of the cardiac gastric glands of the 
 dog and cat. In different stages of secretion the amount of 
 iron-holding substance varied. When the cell was exhausted 
 and the granules of zymogen were scanty the cytoplasm still 
 gave the reaction, though only faintly. In fact the iron-holding 
 area of the cytoplasm varied inversely as the granular zone. 
 
 As the result of his experiments Macallum puts forward 
 the hypothesis that the nucleus forms out of its chromatin a 
 
XXIl] ZTMOGENS. 407 
 
 material which he calls prozymogen, and extrudes it into the 
 cytoplasm. Before extrusion it may be diffused in the nuclear 
 substance, or collected into definite masses which he calls jpZossmo- 
 somata. When extruded into the cytoplasm some of it unites 
 with a constituent of the latter and becomes zymogen, which 
 is soon aggregated into granules. The increased size of the 
 granules as they lie in the imier zone is due to a farther 
 addition fi:om the prozymogen of that region. 
 
 Macallum holds further that the chromatin of the nucleus 
 is an iron-holding nucleo-albumin, in which the iron is attached 
 to the nuclein. 
 
 In such cells the process of secretion appears therefore to be 
 intimately associated with this constituent of the nucleus. At 
 the same time the zymogen is not purely a nuclear product, the 
 cytoplasm of the cell contributing to the later stages of its 
 construction. 
 
 Macallum has found also that the aleurone-layer of the 
 endosperm of the wheat grain gives the same reaction for iron 
 as do the cells of the pancreas. We have already seen reason 
 to attribute considerable secretory activity to this layer, parti- 
 cularly in connection with the formation of cytase. 
 
 Macallum noticed a similar distribution of iron ia some 
 Protozoa. We have seen that it is probable that these uni- 
 cellular organisms digest their food by the agency of enzymes 
 which are secreted into definite vacuoles which surround the 
 ingested particles. From his observations he holds it to be 
 probable that there is the same relation as m the gland-cells 
 between a zjrmogen and an iron-holding compound which is 
 contained iu the meshes of their cytoplasm. 
 
 The view that the secretion of the enzymes may thus be a 
 gradual process passing through several stages has been put 
 forward by other observers. It was suggested ia 1882 by 
 Langley ia the followiag words ^: "There are one or two 
 farther observations on the relation of pepsin to the gland cells 
 which are suggested by some experiments of Briicke. Briicke 
 pointed out that prolonged extraction with water does not take 
 out all the pepsin firom the gland cells; the remaining tissue 
 ' Journal of Physiology, 3. 290. 
 
408 ZYMOGENS. [CH. 
 
 when treated with dilute hydrochloric acid still gives a pepsin- 
 containiiig extract. He further pointed out that, when the 
 gastric mucous membrane is treated with dilute hydrochloric 
 acid until it is quite broken up, the residue nevertheless when 
 washed and again treated with dilute hydrochloric acid still 
 gives a pepsin-containing extract. We could scarcely imagiue 
 that this could be the case if the zymogen existed in the cells 
 in one state only. It necessitates a conception of the gland 
 cells which seems to me indeed on general grounds almost 
 necessary. I conceive the matter thus. The protoplasm of the 
 gland cells does not at one swoop form zymogen as it occurs 
 immediately previous to its conversion into pepsin, but forms 
 certain intermediate bodies in which the zymogen radicles 
 become more and more isolated. Since the zymogen contaros 
 the radicle of the ferment, the ferment will be obtained with 
 greater difficulty from the imperfectly elaborated zymogen, i.e. 
 as we ascend from the final mesostate to protoplasm, the 
 ferment will be split off less and less readily. The last traces 
 of ferment then which are obtained by repeated extractions, 
 I take to arise from the splitting up of substance which was on 
 the way to be converted into zymogen." 
 
 We may now turn our attention to the relation between 
 zymogens and enzymes, and to the differences which may be 
 shown to exist between them. 
 
 We have pointed out already that the e\idence in favour of 
 the granules consisting of zymogen and not of enzyme is based 
 upon a comparison of the fermentative power of a fresh tissue 
 before and after being warmed with a dilute acid. A more 
 convincing proof has been offered by Langley in connection 
 with his researches on the histology of the mammalian gastric 
 glands. He ascertained that pepsin is rapidly destroyed when 
 warmed gently with a 1 per cent, solution of sodium carbonate, 
 and applied this peculiarity in testing the contents of a mucous 
 membrane. In a very striking experiment he used an extract 
 of the mucous membrane of the fimdus and greater curvature 
 of the stomach of a rabbit, which was powdered and macerated 
 in water for 2J hours at 32° C. He divided the filtered ex- 
 tract into two parts, and warmed one of them at 32° C. for 
 
XXIl] ZYMOGENS. 409 
 
 50 minutes in the presence of "06 per cent, of hydrochloric 
 acid, neutralising it with dilute alkali at the end of that time. 
 This would effect the conversion into pepsin of any zymogen 
 present in the extract. At the same moment he added to 
 the other the same amount of acid and alkali simultaneously. 
 The two extracts are referred to by him as P and Z. They 
 only differed in that P had been warmed with the acid before 
 the alkali was added, while Z had not. He then added to 
 10 c.c. of each, an equal volume of 2 per cent, solution of sodium 
 carbonate, and kept them at 39° C. for 15 minutes. They 
 were then removed, neutralised, and made up to "2 per cent. 
 of hydrochloric acid, and some fibrin was added to each. P failed 
 to digest more than a trace, while Z was eminently peptic. 
 
 The substance extracted from the glands had been left un- 
 affected in ^ by preliminary treatment with acid; it was not 
 destroyed by the sodium carbonate, for it was rendered active 
 by the subsequent acidification and digested the fibrin; the 
 same substance in P had been acted on by the acid for some 
 time before being subjected to contact with the alkali, and was 
 evidently destroyed by the latter, as the neutralised and 
 subsequently acidified liquid then possessed no peptic power. 
 The extracted material was capable of giving rise to pepsin 
 on warming with an acid, as shown by Z. In P this pepsin, 
 formed during the first warming with dilute acid, was de- 
 stroyed by the alkali. The substance was not pepsin at the 
 time of its extraction, or it would have been destroyed in Z 
 also. In the condition in which it was present in the original 
 gastric extract, the sodium carbonate had no action on it. 
 
 By this experiment Langley showed that the granular 
 matter thus extracted from the glands is not pepsin but an 
 antecedent or zymogen, and farther demonstrated a difference, 
 between the latter and the enzyme with regard to their be- 
 haviour with dilute alkalis. 
 
 The zymogen, being the antecedent of pepsin, has been 
 called pepsinogen. 
 
 Langley showed by similar means that the gastric glands of 
 the dog, sheep, mole, snake, frog, and newt contain pepsinogen 
 and not pepsin. 
 
410 ZYMOGENS. [CH. 
 
 Pepsinogen can be converted into pepsin not only by dilute 
 acids but by dilute alkalis also. The latter must be very 
 dilute or the pepsin is destroyed almost as fast as it is 
 formed. 
 
 Heidenhaia showed in 1875 that the secretion of the 
 pancreas contained trypsinogen and not trypsin, and that the 
 former could be converted into the latter by the action of 
 acids. A watery extract of the gland slowly acquires tryptic 
 powers. 
 
 Hammarsten showed in 1872 that the rennet enzyme of the 
 stomach is also derived from a zymogen. Langley confirmed 
 this observation in 1881. He prepared an extract of the 
 mucous membrane, using "1 per cent, solution of sodium car- 
 bonate as the solvent. When a part of this extract was 
 made acid, warmed for 15 minutes, and subsequently made 
 alkahne agaia, it rapidly caused clotting iu milk, while the 
 original extract unwarmed with acid possessed no such power. 
 It was necessary to work with a faintly alkahne solution, as 
 a very little acidity causes precipitation of the casein, as 
 already shown. The alkabnity had however to be very slight, 
 as rennet zymogen is rapidly destroyed by as little as '5 per 
 cent, of sodium carbonate. There is not the difference in this 
 respect between the zymogen and the enzyme as is the case 
 with pepsin and its antecedent. 
 
 Lorcher has recently obtained similar results. He used a 
 glycerin extract of the dried gastric mucous membrane, which 
 he found able to coagulate in 17 minutes 20 times its volimie of 
 milk to which 1 part to 10000 parts of hydrochloric acid had 
 been added. When he allowed the acid and the gastric extrsict 
 to stand together for 2 hours before adding them to the milk, 
 (Coagulation took place in 2 minutes. 
 
 The evidence for the existence of the zymogen of salivary 
 diastase is not so complete, though analogy with other secretions 
 points to its existence, and it is strongly suggested by the 
 appearances in the cells of the salivary glands already de- 
 scribed. A certain amoimt of direct evidence however is not 
 wanting. Goldschmidt has demonstrated its existence in the 
 saliva of the horse. Certain facts which were ascertained by 
 
XXIl] 
 
 ZYMOGENS. 
 
 411 
 
 the writer point to its presence in human saliva also. It is not 
 easy to determine it in the latter, as the secretion contains so 
 much ready-formed diastase. Some saliva was secreted, freed 
 from mucin, and considerably diluted and 2 per cent, of potassic 
 cyanide was added to the liquid. This antiseptic was found to 
 be very valuable in preserving saliva, such a preparation having 
 been found to retain its activity unimpaired for several months. 
 The saliva so prepared was divided into two portions ; one of 
 them was kept for 21 days in an incubator, the temperature 
 of which was maintained at 38° C, while the other remained at 
 the laboratory temperature. At certain intervals 2 c.c. of each 
 were digested with 20 c.c. of 1 per cent, starch-paste and the 
 products of hydrolysis were titrated with Fehling's fluid. 
 The following table gives the result of the experiment. 
 
 Time of 
 exposure 
 to 38- C. 
 
 Cuprie oxide reduced 
 
 by products of action 
 
 of 2c.c. of the warmed 
 
 saliva on 20 c.c. of 
 
 starch-paste 
 
 Cupric oxide reduced 
 
 by products of action 
 
 of 2 C.C. of unwarmed 
 
 saliva on 20 c.c. of 
 
 starch-paste 
 
 days 
 
 grms. 
 
 grms. 
 
 1 
 
 •0565 
 
 •045 
 
 3 
 
 •0697 
 
 •045 
 
 8 
 
 •067 
 
 •062 
 
 9 
 
 •0706 
 
 •0708 
 
 15 
 
 •066 
 
 •095 
 
 21 
 
 •032 
 
 ■094 
 
 From these experiments, it is probable that the saliva 
 contained, in addition to a certain amount of diastase, a small 
 quantity of zymogen, which exposure to a temperature of 
 38° C. converted into the enzyme almost completely in three 
 days. Possibly a little remained unchanged till the ninth day. 
 After 15 days at this temperature the enzyme began to change, 
 losing its hydrolytic power, which at the end of the twenty-first 
 day was much less than at the beginning of the observation. At 
 the temperature of the laboratory, which would be about 18° C. 
 the zymogen did not begin to be converted into the enzyme till 
 after the third day, and it then progressed gradually for the 
 rest of the time it was under observation. There was no 
 destruction of the enzyme at that temperature during the 
 
412 ZYMOGENS. [CH. 
 
 whole of the period, so that the curve of its activity rose 
 somewhat higher than did the corresponding curve of the 
 warmed extract, in which, during part of the experiment, both 
 processes were at work. 
 
 Some experiments made by the writer upon the action of 
 light upon dilute saliva also point to the existence of zymogen 
 in the secretion. 
 
 A preparation of saliva made in the way already described, 
 and preserved by the same antiseptic as in the experiments 
 quoted, was exposed for several hours to the rays of the electric 
 arc at such a distance that the heating effects of the lamp were 
 imperceptible. 
 
 By the use of certain screens the effect of different parts of 
 the spectrum were examined separately. The screens were 
 prepared according to the directions of Landolt', given in 
 his work on rotation-dispersion. The coloured liquids em- 
 ployed for this purpose enabled the rays of the spectrum to 
 be divided into seven sets, which may be called roughly the 
 infra-red, red, orange, green, blue, violet, and ultra-violet. The 
 red rays included rays of wave-length 710 — GiB/ifi ; the orange 
 64,5— 585 fifjL, the green 685— 500/i/x, the blue 500— 430/*/*, and 
 the violet those of the visible spectrum beyond 430/*/*. 
 
 After the exposure, during which the temperature of the 
 saliva was registered by a maximum thermometer, the diastasic 
 power of the preparation was ascertained by allowing 5 c.c. to 
 digest with 20 c.c. of soluble starch in 1 per cent, solution. A 
 control preparation was in all cases examined, which was 
 composed of the same saliva suspended with the other in front 
 of the lamp but protected from the light by an opaque screen. 
 The table on page 389 shows the effect of the action of the light. 
 
 The results must be received with a certain amount of 
 caution, as with such different screens it was not at all easy to 
 get the intensity of the transmitted light the same in each 
 case, though as much care as possible was taken. Slight 
 differences in intensity in any one case were found however 
 
 1 Landolt. " Methods zar BestinunTing der Botationsdispersion mit Hiilfe 
 Ton Strahlenfiltern." Ber. d. deut. chem. Gesell. 1894, p. 2872. 
 
XXIl] 
 
 ZYMOGENS. 
 
 413 
 
 not to have a very marked efifect during the time of the 
 exposure. 
 
 Band 
 examined 
 
 Cupric Oiide 
 
 in grms. 
 reduced by 
 solution ex- 
 posed to band, 
 after correction 
 for infra-red 
 rays 
 
 Cupric 
 
 oxide in 
 
 grms. 
 
 reduced by 
 
 unexposed 
 
 solution 
 
 Increase or 
 diminution of 
 
 amount of 
 CuO reduced. 
 
 Diminution 
 
 indicated by 
 
 — sign 
 
 Increase or 
 
 diminution 
 
 of diastase 
 
 per cent. 
 
 Mean 
 
 increase or 
 
 diminution 
 
 per cent. 
 
 Red 
 720—640 /ip 
 
 Orange 
 640—585 ^11 
 
 Green 
 585—500 ft^ 
 
 Blue 
 500—430 ii/i 
 
 •0557 
 •0565 
 •0921 
 •0518 
 
 •0338 
 •0296 
 
 •0285 
 •0107 
 
 •0282 
 •0711 
 
 •0354 
 •0393 
 •0568 
 •0343 
 
 •0323 
 •0282 
 
 ■0333 
 •0129 
 
 •0242 
 •0568 
 
 + ■0203 
 + ■0172 
 + ■0353 
 + ■0175 
 
 + ■0015 
 + •0014 
 
 - -0048 
 -•0022 
 
 + •0040 
 + •0143 
 
 + 57^4 
 + 43-7 
 + 621 
 + 51^0 
 
 +4^6 
 +4^9 
 
 -14^4 
 -17-0 
 
 + 16^5 
 + 25^1 
 
 +535 
 
 +4^75 
 -15-7 
 + 20^8 
 
 
 Cupric oxide 
 reduced by 
 solution ex- 
 posed to 
 infra-red rays 
 
 Cupric 
 
 oxide 
 
 reduced by 
 
 unexposed 
 
 solution 
 
 Gain 
 
 Increase of 
 diastase 
 per cent. 
 
 Mean 
 
 Infra red 
 
 •0337 
 •0323 
 
 •0323 
 •0282 
 
 + •0016 
 + •0047 
 
 + 5^0 
 + 16^6 
 
 + 10^8 
 
 Less careful separation of the rays of light showed that the 
 red end of the spectrum also increased the amount of diastase 
 in a freshly-prepared extract of malt. 
 
 From these experiments it seems probable that saliva 
 contains a zymogen which can be converted into diastase with 
 considerable readiness by exposure to the red rays of the 
 spectrum. The infra-red, orange and blue rays have a similar 
 but less pronounced effect. 
 
 The other rays, particularly those of the violet and ultra- 
 violet regions, have the power of destroying diastase almost as 
 effectually as temperatures above 70° C. 
 
 The zymogens of the vegetable enzymes have not been very 
 closely studied up to the present time. Their existence was 
 
414 ZYMOGENS. [CH. 
 
 first established by Vines in his experiments on Nepenthes. 
 He treated some pitchers of this plant with dilute acetic acid 
 (1 per cent.) for 24 hours before extracting them with glycerine, 
 and at the same time extracted other similar pitchers with 
 glycerine without preliminary treatment with acid; the first 
 extract possessed greater proteolytic powers than the second, 
 leading him to infer that a zymogen was present in the glands, 
 which was converted into an enzyme by the acid. 
 
 The writer's experiments on the antecedent of the enzyme in 
 the resting seed of the Lupin also indicate a similar condition 
 in the cells, though its identification is not so easy, as the 
 acid treatment usually adopted for zymogen conversion is not 
 available, the digestion having to be a prolonged one and to be 
 conducted in an acid medium. The zymogen was ascertained 
 to exist by a modification of the treatment adopted by Langley 
 in the case of the pepsinogen of the stomach, based upon the 
 destruction of the enzyme by dilute alkalis, which do not 
 decompose the zymogen. 
 
 Inulase can be more easily shown to exist as a zymogen in 
 the resting tuber of the artichoke. In the writer's experiments 
 some pieces of full-grown tubers were kept at a temperature of 
 35° C. for 24 hours. An extract then prepared from them was 
 found to convert inulin into sugar, while an extract made from 
 other pieces of the same tubers without warming, was inert. 
 When some of this latter extract was subsequently warmed 
 for a time with a solution of acid-albumin in '2 per cent, 
 hydrochloric acid, some enzyme was developed in it, though 
 less than was obtained by warming the tubers alone before 
 extraction as described. Treatment with acid alone was useless 
 in the case of inulase, as the quantity needed to convert the 
 zymogen was sufficient to destroy any ferment liberated from it. 
 
 The lipase and rennet of the castor-oil seed have also been 
 shown by the action of acid to exist in the zymogen condition 
 until the onset of germination, the former of them being 
 convertible into the ferment also by the prolonged action of 
 water without acidification. 
 
 Brown and Morris mention that the quantity of diastase 
 secreted by the epithelium of the scutellum of the barley grain 
 
XXIl] ZYMOGENS. 415 
 
 is increased 20 per cent, by the presence of very dilute formic 
 acid. Baranetzky found that a freshly-prepared extract of the 
 leaves of MelianthviS major was incapable of hydrolysing starch, 
 but that after standing a few days it possessed diastasic powers. 
 He noted the same thing in the case of the tubers of the 
 potato. 
 
 Experiments made by Reychler, and by Lintner and 
 Eckhardt, point to the existence of a zymogen in the cells of 
 the grain of wheat. They found that the action of a dilute 
 acid upon the gluten of wheat gave rise to a diastasic enzyme. 
 The experiments do not seem however to be quite conclusive, 
 as the gluten might have contained a little of the diastase of 
 translocation, which we have seen is present in the ripe endo- 
 sperm, though in very small amount. 
 
 Frankhauser observed that during the germination of barley 
 small quantities of formic acid could be detected in the grains. 
 This may be regarded as important in discussing the increase 
 of diastasic power attending germination, as such an acid would 
 probably transform a zymogen into ferment. 
 
 The existence of a zymogen which is the antecedent of the 
 fibrin ferment or thrombase has been suggested by many 
 observers, but a satisfactory proof of it can hardly yet be said 
 to be forthcoming. 
 
 The most definite statements on the subject which have so 
 far been published are those of Pekelharing, who claims to have 
 been able to prepare a substance from oxalated plasma which in 
 many respects resembles a globulin, but is devoid of fibrino- 
 plastic properties ; after contact with calcium chloride it acquires 
 the power of inducing coagulation of fibrinogen solutions. 
 Pekelharing regards this as a zymogen ; he says that on com- 
 bustion it yields an ash which contains little or no calcium, while 
 thrombase itself is rich in that metal. Pekelharing holds this 
 substance, like Halliburton's thrombase, to be a nucleo-proteid. 
 He found it possible to precipitate it from plasma by the addition 
 of acetic acid. According to him, a number of bodies, first 
 examined by Wooldridge, and named by him tissue- fibrinogens, 
 which are capable of causing coagulation of the blood in the 
 living blood-vessels, will yield thrombase on treatment with 
 
416 ZYMOGENS. [CH. 
 
 calcium chloride. Pekelharing considers that these bodies 
 contain the zymogen. 
 
 He explains the action of peptone in inhibiting coagulation 
 of the blood by the hypothesis that this proteid has a strong 
 affinity for calcium compounds, and that when it is injected into 
 the blood it prevents the interaction between those which the 
 blood contains, and the zymogen which is also present. Con- 
 sequently thrombase is not formed and the blood does not 
 coagulate when shed. He quotes in support of this view a 
 statement of Munk that soaps which combine with calcium 
 compounds produce similar symptoms to those caused by 
 peptone when injected into the blood-vessels. All the dele- 
 terious effects which follow the injection of peptone are 
 obviated by injecting a solution of some salt of calcium at 
 the same time. 
 
 Pekelharing suggests that intravascular clotting is caused 
 by the rapid conversion of zymogen into ferment. He says 
 that peptone will restrain coagulation in intravascular plasma 
 if added so rapidly that the zymogen has not time to combine 
 with the calcium salt to form the ferment. 
 
 This view is not however in accordance with the fact which 
 has been noticed by more than one observer, that injection of 
 very strong solutions of thrombase into the blood-vessels of a 
 living rabbit is not followed by intravascular clotting. 
 
 According to Pekelharing the conversion of the zymogen 
 into thrombase is effected by an actual combination with the 
 calcium salt. He says further that fibrin itself is a calcium 
 compound, and that the main action of the ferment appears to 
 be the transference of the calcium to the fibrinogen. 
 
 Hammarsten endorses Pekelharing's view as to the relation 
 of the zymogen to the thrombase, but he opposes the theory 
 that fibrin is a calcium compound of fibrinogen, as he has 
 found that both fibrin and fibrinogen contain the same amount 
 of calcium. 
 
 The points of difference between zymojgens and the enzymes 
 to which they give rise have not been very completely examined. 
 Langley and Edkins have made a comparison between pepsin- 
 ogen and pepsin, and have found as might almost have been 
 
XXIl] ZYMOGENS. 417 
 
 expected that they have much in common, such differences as 
 exist being rather those of degree only. Their behaviour with 
 regard to dilute alkalis has already been mentioned. Both 
 are ultimately destroyed by alkalis and by alkaline salts, but 
 the destruction of pepsinogen is much slower than that of 
 pepsin. In the absence of acids, which rapidly convert the 
 zymogen into the ferment, pepsinogen is fairly stable. In 
 neutral and faintly alkaline solutions its conversion is very 
 slow, and in a glycerine extract it may remain unchanged for 
 years. 
 
 When carbon dioxide is passed through a solution containing 
 pepsinogen the latter is destroyed with some rapidity, but little 
 remaining when the stream of gas has been maintained for an 
 hour. The rate of destruction is increased by the presence of a 
 small quantity of magnesium sulphate, about '1 per cent, being 
 the most efficient proportion. Traces of acetic acid or of sodium 
 carbonate have the same effect. Peptone on the other hand 
 greatly delays the destruction, and when about "25 per cent, 
 is present, prevents it altogether. Other proteids have a 
 similar effect, but they are not so potent in this direction as 
 peptone. 
 
 Carbon dioxide has much less effect on pepsin. 
 
 Pepsinogen, like pepsin, is rapidly destroyed by heating its 
 solution to about 55° — 60° 0. 
 
 G. F. 
 
 21 
 
418 
 
 CHAPTER XXIII. 
 
 THE CONSTITUTION OF ENZYMES. 
 
 The knowledge which we have as to the constitution or 
 composition of enzymes is exceedingly scanty. This is owing 
 in great measure to two causes. In the first place they are 
 apparently very unstable bodies and undergo decomposition 
 with great readiness. The methods of preparation which have 
 been employed to isolate them show a considerable variety 
 of detail, but they seem to agree in this, that they are all 
 accompanied by a great loss of ferment power, the enzymes 
 being to a large extent destroyed during the preliminary 
 extraction or the attempts at subsequent purification. In the 
 second place a difficulty has to be encountered which is very 
 far reaching, and goes beyond manipulative details. We have 
 no criterion of their purity, and it is consequently impossible 
 to say whether any of the processes so far adopted has pre- 
 pared a really isolated product. 
 
 From the similarity of their action to that of the li^dng 
 protoplasm, which we have examined in a previous chapter, the 
 idea is at once suggested, that they are very possibly either 
 proteid in character, or at any rate not very different from 
 proteids. Their instability, the changes which they undergo 
 on heating, and their general behaviour with dilute acids and 
 alkalis, also favour this hypothesis. 
 
 So far as various observers have been able to prepare them 
 free from admixture with known substances, they have, with 
 only a few doubtful exceptions, been found to give the general 
 reactions for proteids. Their solutions at any rate assume the 
 
CH. XXIIl] THE CONSTITUTION OF ENZYMES. 419 
 
 yellow coloration on treatment with nitric acid and ammonia 
 which is known to physiologists as the xanthoproteic reaction. 
 
 Reactions which are peculiar to them, and so may be relied 
 on as distinctive, are altogether absent, and the only evidence 
 we can obtain of the presence of any of them in a solution is 
 the power of the latter to bring about the changes characteristic 
 of the particular enzyme it is suspected to contain. 
 
 Such characteristic reactions have been sought, and not 
 infrequently various observers have announced their discovery, 
 only however to find that further researches have demonstrated 
 the unsatisfactory character of the suggested method. 
 
 Among the writers who have claimed to have ascertained 
 definite reactions for the enzymes may be mentioned Weisner. 
 He says that a characteristic colour reaction may be obtained 
 by heating them with a solution of orcin in alcohol in the 
 presence of strong hydrochloric acid. Nor does he stop there ; 
 he says that different enzymes give distinctive colours, and that 
 they can be discriminated by his method, diastase in particular 
 giving a bluish-violet. 
 
 Guignard made investigations in the same direction, and 
 states that by the action of hydrochloric acid alone, the enzymes 
 can be distinguished as a class and many of them identified. 
 He boiled 1 centigramme of different enzymes with 1 c.c. of 
 pure hydrochloric acid, and says that he found diastase yield 
 a red which gradually turned brownish ; emulsin gave a viofetp 
 papain an orange-red, and trypsin a greenish yellow. 
 
 These colour reactions have been found however to be 
 characteristic of many other bodies besides the enzymes. 
 Reinitzer has shown that dextrin, maltose, and lactose, all give 
 similar colours when treated in the way suggested. Weisner's 
 colour reactions have been proved to be shared by a very large 
 number of substances, many of which occur especially in such 
 vegetable tissues as also yield enzymes. The orcin reaction 
 is shared by nearly all carbohydrates, and appears to be due 
 to the production of furfurol during the treatment, which is 
 necessarily somewhat drastic. Udransky obtained the same 
 reaction from various proteids. Probably in Weisner's prepar- 
 ations the colours were given by some of these bodies which 
 
 27—2 
 
420 THE CONSTITUTION OF KNZYMES. [CH. 
 
 had not been separated from the enzymes, and were not charac- 
 teristic of the latter at all. 
 
 Guignard again describes the action of hydrochloric acid 
 alone on various proteids, and shows that they give colour 
 reactions much like those which he has attributed to the 
 enzymes. 
 
 Guignard has called attention to certain reactions yielded 
 by the cells which contain myrosin. When a section in which 
 these cells existed was heated with a small quantity of Millon's- 
 reagent (which is a mixture of the two nitrates of mercury and 
 an excess of nitric acid), the cells in question rapidly assumed 
 a more or less vivid rose colour, while the protoplasm of the 
 surrounding cells was stained much more slowly, and so great 
 a depth of tint was never attained. The contents of the 
 myrosin-containing cells further became precipitated in granular 
 form under the action of the reagent, so that they stood out 
 quite distinctly among the rest. 
 
 Striking however as this reaction is, it cannot be regarded 
 as undoubtedly characteristic of the enzyme, for it might 
 equally well be due to some other constituent of the cells. 
 It may suffice to point out where the myrosin is, but it may be 
 attributed to something accompanying the ferment with as great 
 a plausibility as to the latter itself 
 
 It was ascertained by Schonbein in 1868, that when dias- 
 tase is mixed with a little of the resin of guaiacum and a little 
 peroxide of hydrogen added, a somewhat vivid blue coloration 
 is produced. Other observers have extended this observation 
 to other enzymes, and by many workers it has been assumed to 
 be an unfailing test for the various members of the group, 
 though it has not been thought to give distinctive reactions for 
 them individually. Lintner showed in 1886 that the reaction 
 is more delicate when an alcohoUc solution of the guaiacum is 
 employed instead of the resin. 
 
 The reaction, like the others described, has been ascertained 
 to be by no means characteristic of diastase. It has long been 
 one of the tests for blood-stains, and has been used in the 
 examination of urine for the presence of albuminous matter. 
 A careful examination of its validity as a reaction for enzymes 
 
XXIIl] THE CONSTITUTION OF ENZYMES. 421 
 
 was made in 1897 by Pawlewski, who found that a similar 
 coloration to that given by diastase was caused by peptone, 
 and by albumin and other native proteids, as well as by gelatin. 
 Even the addition of hydrogen peroxide alone to guaiacum 
 tincture gave a blue colour, which appeared at once if the 
 solution was warm, but took a little time to manifest itself 
 at a lower temperature. Other oxidising agents than peroxide 
 of hydrogen, such as ozone, chlorine, nitric acid, and perman- 
 ganate of potassium gave a blue colour with the tincture. 
 
 As a special test for diastase or any other enzyme the reagent 
 is useless, for no conclusion can be drawn as to the nature of the 
 body present, even when the blue tint appears. 
 
 If we survey all these reactions which have been put 
 forward as means of identifying enzymes, we see that they do 
 not give any evidence which can weigh very heavily against 
 the hypothesis of their being proteid in nature. Weisner's 
 preparations of some of the ferments were probably not very 
 pure, being mixed with carbohydrate as well as proteid 
 matters. 
 
 It has been suggested that the zymogens are proteids, and 
 that the enzymes arise from them by a process of decomposi- 
 tion consequent on oxidation. Heidenhain thought that the 
 zymogen consisted of the ferment in combination with an 
 albuminoid body. Many observers have advocated the view 
 that the enzymes are themselves proteids, Loew in particular 
 considering them as allied to the peptones. In support of the 
 hypothesis we may advance the fact that when an extract 
 contaiijing an enzyme has all its proteids removed by precipi- 
 tation, the filtrate possesses little ferment power, the latter 
 being diminished in many cases in proportion as the proteid 
 is thrown out of solution. 
 
 Too much stress must not be laid however on this observa- 
 tion, as it is constantly found that when a precipitate, even 
 of inert matter, is caused in a solution containing an enzyme, 
 the latter is generally carried down with the precipitate. We 
 have seen that this property is taken advantage of in severat 
 methods of purifying solutions of the enzymes, particularly in 
 Briicke's process ofJbreparing pepsin. 
 
422 THE CONSTITUTION OF ENZYMES. [CH. 
 
 A more striking fact is that the temperatures at which so 
 many enzymes are destroyed correspond very closely to the 
 points at which proteids occurring with them are coagulated. 
 
 Most of the methods of preparation which have been de- 
 scribed are such as would throw proteids out of solution. Two 
 very prominent means of precipitating enzymes we have seen are 
 the addition of excess of alcohol to the solution, and its satura- 
 tion by neutral salts. These methods precipitate the proteids, 
 and the enzymes are thrown down with them if any are present. 
 The experiments do not show whether the proteid and the enzyme 
 are identical, or whether the latter is merely mechanically 
 associated with the former. It is remarkable however that 
 prolonged exposure to strong alcohol coagulates the proteids, 
 and that the same treatment gradually destroys the power of 
 the enzymes. 
 
 In some cases observers have found the association of a 
 particular enzyme and a certain proteid to be so constant and so 
 close that they have been led to the view that the two are 
 identical. 
 
 Osborne has studied the association of diastase with proteids 
 in the cereals, wheat, rye, and barley. In them all he has 
 found an albumin which he has named leucosin, besides a 
 globulin and a proteose. All ferment extracts which he 
 prepared from any of these cereals contained a mixture of these 
 proteids, but when they were separated by various methods the 
 greatest diastasic power clung to the leucosin. The proteose 
 was found to be associated with a certain proportion of the 
 enzyme, but much less than the leucosin. The quantity of 
 leucosin and the fermentative activity also varied together, 
 though not with very great exactness. 
 
 As the result of his experiments Osborne came to the view 
 that the diastase is either the leucosin or a compound of the 
 latter with some other body, presumably the proteose ; and that 
 this compound breaks up on being heated and yields coagulated 
 albumin, some free albumin being coagulated at the same time. 
 
 The view cannot be considered proved, as certain of his 
 solutions which contained only the proteose possessed a certain, 
 though relatively feeble, diastasic power. 
 
XXIIl] THE CONSTITUTION OF ENZYMES. 423 
 
 Chittenden has shown that the bromelin and the rennet 
 ferment which are both present in the juice of the pineapple 
 are probably associated with two definite proteids, though 
 he does not assert that either enzyme is identical with such 
 proteid. Bromelin, the proteolytic enzyme, always occurs in close 
 connection with a proteid which exhibits most of the characters 
 of a proto-proteose. It is precipitated from a neutral solution 
 by saturation with either ammonium or magnesium sulphate, or 
 with sodium chloride. It is soluble in water and consequently 
 is not precipitated by dialysis. It is further not coagulated by 
 long contact with strong alcohol, and its aqueous solution is 
 very incompletely precipitated by heat. In its behaviour 
 towards nitric acid and heat and in some other ways it does 
 not show the typical proteose reactions, and it contains a 
 smaller amount of nitrogen than most proteids. 
 
 The rennet enzyme is more closely associated with a body 
 whose reactions agree in the main with those of hetero-proteose. 
 It is less soluble in water, requiring for complete solution a 
 little neutral salt. It is more completely precipitated by heat 
 than the proto-proteose described. 
 
 The closeness of this association in both cases may possibly 
 indicate that these proteids are actually the enzymes in question, 
 but the identity cannot be considered conclusively proved. 
 
 Lintner prepared diastase in what he considered to be a 
 pure condition, and from the results of analyses of his pre- 
 paration he concluded it to be a proteid, but not one of the 
 ordinary class. He found that it contained only two-thirds as 
 much nitrogen as, and less carbon than, the latter. It differed 
 also in not giving the biuret reaction. Lintner hence suggests 
 that the enzymes constitute a special class of proteids. 
 
 Wroblewski has come to the same conclusion. 
 
 Buchner associates his new zymase with a proteid in the 
 yeast which is coagulated at 45° 0. At this temperature the 
 zjnnase also is destroyed. When this proteid has disappeared 
 from the extract in consequence of the action of the proteolytic 
 enzyme of the yeast, the power of producing alcohol is lost. 
 Buchner does not however claim that the proteid is the zymase. 
 
 None of the researches quoted can be considered to go very 
 
424 THE CONSTITUTION OF ENZYMES. t*^- 
 
 far in the direction of proving the proteid nature of the enzymes. 
 All can apparently be explained equally well upon the hypo- 
 thesis that the enzyme in any case is closely associated with the 
 proteid. We have remarked several times that the soluble 
 ferments are very easily carried down by a precipitate formed 
 in the liquid in which they are dissolved. When we consider 
 further that there is every reason to believe that an extremely 
 small quantity of one of them is capable of causing the 
 transformation of an enormous amount of material, we may 
 well believe that such an association with a proteid would be 
 detected only with very great diflSculty by any method of de- 
 structive analysis. At the same time it is not impossible that 
 they may be proteids which have not yet been isolated, as the 
 very small quantity present mixed with other proteids might 
 very well escape separation. 
 
 It is very difficult to accept the view that they are the 
 proteids with which their particular activities have been asso- 
 ciated as already described. In many cases these can be 
 prepared without possessing any power of setting up the fer- 
 mentations. 
 
 The variety of proteids vri th which they have been found 
 militates still more forcibly against this view. According to 
 Osborne, diastase is associated with a particular albumin, leucosin, 
 and to a less extent with a proteose. Chittenden's enzymes 
 occur together with a proto-proteose and a hetero-proteose 
 respectively. Halliburton and Pekelharing associate thrombase 
 with a body which was at first considered a globulin, though 
 now it is held to be a nucleo-proteid. Many writers have 
 connected other enzymes with forms of coagulable proteids, 
 supporting their contention by the observation already quoted 
 that the enzymic power is lost at the temperature of heat- 
 coagulation, or precipitation, of the proteid. 
 
 It seems altogether improbable that some enzymes should 
 belong to one group of proteids, and some to another. It is 
 much more reasonable to predict uniformity of composition 
 for them, and to agree with Lintner that they must form a 
 separate class by themselves. 
 
 The connection between loss of fermentative power and 
 
XXIII] THE CONSTITUTION OF ENZYMES. 425 
 
 heat-coagulation cannot be supported in the case of proteoses, 
 which are not coagulated by even the temperature of boiling 
 water. We have no evidence so far that a proteose is changed 
 at all by heating it to such a point as would destroy an enzyme. 
 
 Osborne's definite identification of the diastase of cereals 
 with leucosin is open to still further objection. Leucosin is a 
 proteid which has only a limited range of occurrence, and many 
 tissues which yield active diastasic extracts do not contain it. 
 Leucosin can therefore represent only one form of diastase, and 
 as other forms exist there must be other diastasic proteids, if 
 the enzyme has a proteid constitution. When we consider the 
 wide distribution of the ferment and the very varied and 
 variable composition of the proteids in difierent plants, this view 
 almost forces us to the conclusion that diastase is not at all of 
 constant composition, a view which it is very difficult to accept. 
 
 From Osborne's experiments it appears that there is no very 
 satisfactory ratio between the amount of coagulable albumin and 
 the diastasic power; though the latter is higher the more albumin 
 is present, there is no numerical relation between them. This we 
 should hardly expect to be the case if the leucosin were itself 
 the diastase. 
 
 This lack of correspondence between the quantity of proteid 
 and the diastasic power is very striking when we compare malt- 
 extract and saliva. The latter is the more active in hydrolysing 
 starch, but the former • contains a far larger percentage of 
 proteids. The difference in behaviour between the two fluids 
 does not correspond to this difference in composition unless we 
 admit a great variation of hydrolytic power between unit 
 quantities of the two, which seems unlikely. 
 
 Other observers also have noticed that the ferment power, 
 particularly in weak extracts, does not vary directly with the 
 amount of proteid present. The writer investigated this point 
 with some care in the case of vegetable rennet prepared from 
 the castor-oil seed. The amount of proteid was determined by 
 regular dilution of a standard extract; and the result showed 
 that the coagulating action on milk diminished much more 
 rapidly than the quantity of proteid. In a typical experi- 
 ment it was found that "5 c.c. of a particular extract clotted 
 
426 THE CONSTITUTION OF ENZYMES. [CH. 
 
 5 CO. of milk in four minutes, while "25 c.c. took eighty-one 
 minutes and '125 c.c. took five-and-a-half hours. 
 
 Reychler's experiments have been considered to point to 
 proteid either as an antecedent of diastase or as the enzyme 
 itself. He found that by extracting the gluten of flour with a 
 dilute acid he obtained an amylolytic solution. His results are 
 open however to other interpretations than the one suggested. 
 His dilute acid may have extracted some of the residual 
 diastase of the endosperm from the flour, or some zymogen may 
 have been mixed with and incorporated in the gluten, and have 
 been converted into diastase by the solvent employed. 
 
 Recently Wroblewski has claimed to have shown that 
 diastase is proteid in nature. As he prepared it he found that 
 the greater part of his material consisted of carbohydrate 
 matter, which yielded arabinose when boiled with acids. If 
 freed from this, the active constituent was found to have all the 
 properties of a proteid. 
 
 From what has already been advanced, it is evident that 
 in discussing this point very little reliance can be placed upon 
 analyses of supposed pure products. So far however as 
 these analyses go they do not support the proteid theory very 
 satisfactorily. The difficulty of admitting that what was 
 analysed was the actual enzyme is very great. 
 
 The purest trypsin which Ktihne was able to prepare had 
 the following percentage composition: — 
 
 Carbon 
 
 47-22- 
 
 -48-09. 
 
 Hydrogen 
 
 7-15- 
 
 - 7-44. 
 
 Nitrogen 
 
 12-59- 
 
 -13-41. 
 
 Oxygen 
 
 31-31- 
 
 -29-20. 
 
 Sulphur 
 
 1-73- 
 
 - 1-86. 
 
 This differs from the analyses of the proteids in the per- 
 centages of the carbon and nitrogen especially, Henniger's 
 peptone containing 52-28 of the former and 16-38 of the 
 latter in 100 parts. 
 
 According to Wtirtz and Bouchut papain agrees much more 
 closely with proteids, containing 52-48 per cent, of carbon and 
 16-59 per cent, of nitrogen. 
 
XXm] THE CONSTITUTION OF ENZYMES. 427 
 
 Loew has concluded that malt diastase, and both the dia- 
 stase and the trypsin of the pancreas, have a percentage 
 composition very similar to that of proteids. On the other 
 hand Lintner's purest diastase only contained 9"9 per cent. 
 of nitrogen. 
 
 We may oppose to contentions based upon quantitative 
 analyses of this nature, certain considerations suggested by the 
 reactions of solutions of certain of the enzymes, when these 
 have been subjected to such processes of purification as have 
 been devised. 
 
 Pepsin as prepared by Briicke in the way described at 
 page 177, was stated by him to give none of the reactions 
 characteristic of proteids and to be precipitable only by the 
 acetates of lead. It yielded no trace of opalescence on the 
 addition of tannic acid, though this reagent is capable of 
 detecting one part of proteid in 100,000 parts of solvent. 
 
 Hammarsten states that after purification his preparation 
 of rennet did not show the xanthoproteic reaction; was not 
 precipitated by alcohol, nitric acid, tannin, iodine, or normal 
 lead acetate ; was precipitated by basic lead acetate. 
 
 Dastre has shown that the enzymes are not completely 
 insoluble in alcohol, which is inconsistent with their being 
 coagulable proteids. 
 
 Foster showed in 1867 that a diastasic solution could be 
 prepared from natural deposits of urates, with or without 
 previous washing in alcohol, and that this solution gave no 
 proteid reactions. 
 
 Other enzymes as we have seen can be prepared from the 
 urine. 
 
 Possibly in all these cases the quantity of enzyme was too 
 small to give the reactions, though sufficient to exert a certain 
 hydrolytic power. It must however be remembered that the 
 xanthoproteic reaction is exceedingly delicate. 
 
 Some serious objections to the view that enzymes are 
 proteids, can be based upon the action of light upon them. So 
 far investigations on this point have been carried out only in 
 the case of diastase, which was submitted to a close experimental 
 examination by the writer a few years ago. 
 
428 THE CONSTITUTION OF ENZYMES. [CH. 
 
 It was established by Brown and Morris in their paper on 
 the diastase of foliage leaves, that there is a diminution in the 
 amount of enzyme these organs contain after a period of 
 brilliant illumination. This is particularly noticeable when 
 comparative estimations are made from leaves gathered re- 
 spectively in the early morning and in the evening. The 
 latter always show a diminution of the amount of diastase, 
 and the diminution shows a certain relation to the amount of 
 bright sunshine which the leaves have received during the day. 
 
 The writer examined leaves ia the living condition, which 
 were gathered in the early morning and exposed to sunlight 
 for several hours, one half of each being shaded by a covering 
 of blackened paper. The sunlight was found to have a 
 powerfully destructive influence on the enzyme. The same 
 result was obtained when the light was allowed to pass through 
 a watery extract of the leaves, instead of through the living 
 tissue. The diastases of malt-extract and of saliva were 
 similarly affected. The light source also was varied, a very 
 powerful electric-arc lamp being sometimes employed instead 
 of sunlight. The effect was the same in this case also. 
 
 The deleterious effects of the illumination were chiefly due 
 to the violet and ultra-violet rays of the spectrum. 
 
 Light has not so far been ascertained to have any de- 
 leterious influence on proteids. It is very improbable that it 
 ever exercises such an influence, for in the experiments 
 described the writer ascertained that the addition of a co- 
 agulable proteid to the diastasic solution before exposure had a 
 very great protective effect, and up to a certain point, the 
 degree of protection increased pari passu with the amount of 
 proteid added. The proteid itself did not appear to be affected 
 by the illumination. 
 
 The difference of behaviour of enzymes and proteids towards 
 alcohol may also receive some attention. When coagulable 
 proteids are exposed to prolonged contact with this reagent 
 they pass into a condition which is almost if not quite 
 indistinguishable from heat-coagulation. Enzynfies are not 
 destroyed by alcohol with the facility with which they are 
 decomposed by heat. We have seen that one argument in 
 
XXIIl] THE CONSTITUTION OF ENZYMES. 429 
 
 favour of the hypothesis of their proteid constitution is that 
 they are destroyed at the temperature at which the heat- 
 coagulation of the proteid takes place. If the argument is 
 valid and the two bodies identical, the coagulation of the 
 proteid induced by alcohol ought also to destroy the enzyme. 
 But it does not do so, or at any rate a diminution of enzymic 
 power does not take place with the same speed as the co- 
 agulation of the proteid. In many of the methods employed 
 for the extraction of enzymes it is usual, as we have already seen, 
 to free them from large quantities of proteid matter, by pre- 
 cipitating both together by means of alcohol ; after allowing the 
 precipitate to stand under the spirit for a long time, a strongly 
 enzymic extract can be prepared from the coagulated proteid. 
 
 No extraction of an enzyme from proteid coagulated by 
 heat is possible. Apparently therefore we can draw a distinc- 
 tion between the two, from their behaviour with alcohol. 
 
 At the same time it is possible to push this argument too 
 far. Some proteids, such as peptones and certain albumoses, 
 are not coagulated by alcohol any more than they are by heat. 
 If the enzymes should prove to be proteids belonging to either 
 of these classes, alcohol would not destroy them. The action of 
 alcohol however seems to establish the fact that they cannot be 
 coagulable proteids. 
 
 Though enzymes are capable of prolonged resistance to the 
 action of alcohol this reagent does eventually destroy them. 
 They show great differences in their power of withstanding it, 
 some being destroyed in a few hours, others resisting decom- 
 position for months. 
 
 A very careful examination of the composition, properties, 
 and reactions of invertase was made in 1889 by O'Sullivan and 
 Tompson, and their results were published in the following 
 year. The source of the invertase which they used was the 
 liquor obtained from pressed yeast by allowing the latter to 
 stand for some weeks at a temperature of about 20° C, and 
 subsequently filtering. 
 
 Alcohol of 70 per cent, concentration was added to a 
 quantity of this yeast-liquor, very slowly and with frequent 
 pauses. When a precipitate appeared, the solution was allowed 
 
430 THE CONSTITUTION OF ENZYMES. [OH. 
 
 to stand for two hours, and then a further quantity of the same 
 alcohol was added, and so on till no further precipitate was formed. 
 The liquid was then computed to contain 47 per cent, of spirit. 
 The precipitate was so obtained in a finely-divided state and 
 with but little impurity. The solution was allowed to stand 
 over the precipitate for two days, when the latter was removed 
 by decantation and washed with alcohol of 47 per cent, con- 
 centration. It was next thrown on to a filter and again washed 
 with the same alcohol. The washed precipitate was then 
 transferred to a beaker, and water added in quantity equal to 
 that of the original yeast liquor. Part of the precipitate 
 remained insoluble and was found to consist of a peculiar 
 proteid, to which the authors gave the name yeast albuminoid. 
 The invertase dissolved, and the solution was found to have 
 an inverting power but little inferior to that of the original 
 yeast. The authors judged the invertase to be nearly pure, 
 and estimated that about 12 per cent, had been decomposed in 
 the process of preparation, so that what impurities were present 
 were only the products of its own decomposition. It contained 
 a certain amount of ash, which was principally a mixture of 
 potassium and magnesium phosphates. A separation of the 
 ash from the invertase was found to be possible by means of 
 dialysis, and the authors came to the conclusion that it did 
 not enter into the composition of the ferment. They do not 
 however appear to have freed the latter fi-om it completely, but 
 to have reduced the amount present to about ■45 per cent., 
 calculated on the weight of the solid matter. 
 
 So prepared and purified, invertase was found to be a white 
 substance, soluble in water and yielding a clear, slightly viscous, 
 solution which did not become turbid on boiling. It was in- 
 soluble in alcohol of "94 sp. gr. and could be precipitated from 
 its solution by adding alcohol to this degree of concentration. 
 The invertase was found to be destroyed to some extent by this 
 precipitation, and the more so the purer it was. If allowed to 
 stand under the alcohol the ferment power was soon lost. 
 When quite pure the solutions could not be precipitated by 
 alcohol without causing the destruction of the enzyme. 
 
 Invertase gave no colour with Millon's reagent in the cold 
 
XXIIl] THE CONSTITUTION OF ENZYMES, 431 
 
 but on heating the solution a pink tint was developed. The 
 solution of the enzyme had an optical activity of (a)j = 4- 80°. 
 Destructive analysis showed it to contain 46"41 per cent, of 
 Carbon, 6'63 per cent, of Hydrogen, and only 3'69 per cent, of 
 Nitrogen. In respect of the latter constituent it differed in a 
 remarkable manner from a proteid. 
 
 A study of the enzyme obtained from yeast-liquor in this 
 manner showed that when gradually and continuously decom- 
 posed, chiefly by fractionation with alcohol and acid, it gave rise 
 to a series of bodies, which the authors termed the invertan 
 series. They prepared and examined seven members of this 
 series, and described their properties under the designations of 
 o, /3, y, S, e, f, and t) invertan respectively, invertase itself being 
 the second member. 
 
 The two members at the extreme ends of the series, a and 17 
 invertan, were found to be very stable, but the intermediate 
 ones were very liable to spontaneous decomposition. The 
 bodies to which each of the latter gave rise under such circum- 
 stances were invariably a invertan, and the member next below 
 the one dissociated, a invertan itself yielded on decomposition 
 ^ invertan and yeast albuminoid. Omitting a invertan, each 
 succeeding member was found to possess more stability than 
 the one above it. 
 
 All the members of the series were non-crystallisable, 
 undialysable, and colourless ; a invertan was insoluble in water, 
 but the others dissolved readily, forming clear but viscous 
 solutions which did not become opalescent on boiling. Alkaline 
 or neutral solutions were transformed by the gradual addition 
 of alcohol into a milk-like fluid which would not become clear 
 on filtration, but from which a precipitate rapidly fell on the 
 addition of acid. The a invertan was precipitated in flocculent 
 masses, but the other members separated in the form of heavy 
 transparent syrups. 
 
 Their composition, so far as the Carbon, Hydrogen, and 
 Nitrogen are concerned, is given in the following table, in 
 which they are compared also with the yeast albuminoid found 
 in the original yeast-liquor. 
 
432 THE CONSTITUTION OF ENZYMES. [CH. 
 
 Constitaents per cent, on 
 
 Substance. 
 
 substance free &o 
 
 m asb. 
 
 
 C 
 
 H 
 
 N 
 
 Yeast albuminoid 
 
 54-06 
 
 7-35 
 
 14-53 
 
 a Invertan 
 
 4803 
 
 6-65 
 
 8-35 
 
 P „ (invertase) 
 
 46-41 
 
 6-63 
 
 3-69 
 
 y " 
 
 45-62 
 
 6-55 
 
 3-15 
 
 8 „ 
 
 46-50 
 
 6-82 
 
 2-43 
 
 £ » 
 
 44-45 
 
 6-36 
 
 2-07 
 
 ^ ., 
 
 44-73 
 
 6-40 
 
 1-61 
 
 V 
 
 — 
 
 — 
 
 1-05 
 
 From their analyses of these hodies the authors came to the 
 conclusion that the first six were compounds of yeast albuminoid 
 with the lowest member, r] invertan, and that the latter was 
 probably a combination of the same proteid with a carbohydrate. 
 They did not isolate the latter, but from their analyses they 
 calculated that it contained 43"22 per cent, of Carbon and 628 
 per cent, of Hydrogen. 
 
 Following up this hypothesis they computed that the lowest 
 member of the series, rj invertan, contained 18 parts by weight 
 of this carbohydrate to 1 part by weight of proteid, and that the 
 highest, a invertan, contained 3 parts of the carbohydrate 
 to 4 of the proteid. The other members of the series they held 
 to be formed by the union of these two substances according to 
 the general formula tj + On, where 17 represents three times 
 •jj-invertan and a represents a-invertan. Invertase on this 
 hypothesis is rja^ ; it split up into a- and 7-invertan according 
 to the formula r)tts = rja^ + a. r/a^ represents 7-invertan, and 
 this could be further transformed by the elimination of a 
 into ijas which is 8-invertan. 
 
 According to this view of the composition of invertase 
 the latter is' not a proteid, but as it contains proteid in its 
 composition it must give reactions characteristic of the proteid 
 group. It yields the pink colour on boiling with Millon's 
 reagent and it has the power of combining with copper salts. 
 The latter property, which is also possessed by proteids, is 
 shared by the other members of the invertan series with the 
 exception of the a-body. Several such copper compounds can 
 
XXIIl] THE CONSTITUTION OF ENZYMES. 433 
 
 therefore be formed, which have a similar appearance but 
 which contain different percentages of copper oxide. 
 
 Several observers have recently advanced the view that 
 the enzymes are nucleo-proteids. This has been urged by 
 Halliburton in the case of thrombase; as already noticed, he 
 associated this ferment with a body to which he gave the name 
 " cell globulin y8," and which he prepared from lymphoid cells 
 and tissues, such as lymphatic glands and thymus. He ex- 
 tracted it subsequently from the stromata of the red corpuscles 
 of blood. When a quantity of this material was submitted to 
 artificial gastric digestion it yielded an insoluble residue of 
 nuclein. Pekelharing has come to the same conclusion as 
 Halliburton as to the presence of nuclein in thrombase, but he 
 suggests that the nucleo-proteid is probably the zymogen of 
 the enzyme and that the latter is a calcium compound of 
 the nucleo-proteid. Pekelharing states that he has obtained 
 thrombase from nucleo-proteid by Schmidt's method of pre- 
 paration. Both he and Halliburton have shown that the ferment 
 prepared from serum by Schmidt's method gives a residue of 
 nuclein when submitted to artificial gastric digestion, and that 
 on analysis it is found to contain rather more than 1 per cent, 
 of phosphorus. 
 
 Pekelharing found that his purified pepsin also contained 
 phosphorus when prepared in the manner described in a previous 
 chapter, and freed from associated bodies by dialysis, and he 
 has advanced the view that this also is a nucleo-proteid. In 
 O'SuUivan and Tompson's researches on invertase also, the ash 
 of the preparation was found to contain phosphorus, as already 
 mentioned. Lintner ascertained too that the purest diastase 
 he could prepare contained a considerable quantity of phos- 
 phorus in its ash. 
 
 This hypothesis is considerably strengthened by the obser- 
 vations of Macallum, to which allusion was made in a previous 
 chapter. The nucleus has been shown by him to initiate the 
 process of secretion, and to excrete some material into the 
 cytoplasm, which there iindergoes further changes and ulti- 
 mately enters into the composition of the zymogen if it does 
 not actually form the principal part of it. The source of this 
 o. F. 28 
 
434 THE CONSTITUTION OF ENZYMES. [CH. 
 
 excreted material is the chromatin of the nucleus. Macallum 
 has shown how intimately phosphorus is associated with zymogen 
 from its inception in the nucleus till the time when the granules 
 are fully formed. 
 
 The chief constituent of the nuclear chromatin appears to 
 be a nucleo-proteid. It is consistent with this that the excreted 
 material may be formed of the same substance, and that the 
 changes it undergoes in the cytoplasm may not alter its 
 composition in any important respect. It may there become 
 associated with proteids of various kinds, which may exist side 
 by side with it in the granules, for there is no evidence that 
 these are zymogen alone. Indeed a consideration of their 
 dimensions and abundance seems to lead to the opinion that they 
 are mixtures of various substances. In the pancreatic cells, the 
 granules may indeed contain more than one zymogen, though at 
 present we have no means of testing whether this is the case. 
 
 According to the investigations of Jacobson several enzymes 
 possess, besides their specific powers, the property of liberating 
 oxygen from hydrogen-peroxide. Among them may be men- 
 tioned diastase, emulsin, and the ferments of the pancreas. 
 Many organs and tissues of the body have the same power. 
 
 These organs can also cause the conversion of salicyl alde- 
 hyde into salicylic acid. According to their degree of activity 
 some of the tissues have been arranged in the following 
 order: — blood, spleen, liver, pancreas, thymus, brain, muscle, 
 ovary, oviduct. We have seen that the blood and certain of 
 the tissues contain an oxidase which can destroy sugar. 
 
 Spitzer has recently investigated these various animal fluids 
 and tissues, and he finds that the property in question is 
 associated with nucleo-proteids. He prepared them from 
 several organs by the methods adopted by different observers, 
 and ascertained that they possessed the oxidising properties of 
 the tissues themselves. 
 
 Spitzer has come to the conclusion that all these nucleo- 
 proteids contain iron as well as phosphorus, his results agreeing 
 in this respect with the observations of Macallum. These 
 experiments support the view that the enzymes are members 
 of the nucleo-proteid group. 
 
XXIIl] THE CONSTITUTION OP ENZYMES. 435 
 
 The hypothesis of the nucleo-proteid nature of enzymes is 
 not contradicted by the considerations advanced in the earlier 
 portion of this chapter. Indeed it explains many things which 
 present considerable difficulty to an acceptance of the view that 
 they are proteid simply. If we adopt it, their resemblance in 
 composition to the latter group of substances will cause us to 
 cease to feel surprise at their close association with proteids, and 
 at the variety of proteids with which such association has been 
 noticed. The differences which have been noticed between the 
 mass of a supposed enzyme used in an experiment, and its 
 zymolytic activity, are explained if we have but a trace of the 
 nucjeo-proteid associated with a much larger quantity of the 
 proteid accompanying it. 
 
 An observation of Pekelharing's tells rather against this 
 view of the constitution of enzymes. He says that nucleo- 
 proteids are insoluble in dilute acids, and can be readily 
 precipitated from their solutions by faint acidification. This 
 is certainly not the case with many of the proteolytic enzymes, 
 nor with saliva and other diastasic preparations. 
 
 28—2 
 
436 
 
 CHAPTER XXIV. 
 
 THE MODE OF ACTION OF ENZYMES. 
 THEORIES OF FERMENTATION. 
 
 Before discussing the views at present held as to the 
 manner in which enzymes carry out their specific functions, 
 it will he well to trace the steps by which they have been 
 reached, and to review the theories which have been advanced 
 from time to time to explain the phenomena of fermentation. 
 
 We have already seen that it is only within comparatively 
 recent years that there has been brought forward a general rather 
 than a restricted view of this process. To the older writers 
 this term generally conveyed the idea of alcoholic fermentation 
 only, or at most such processes were included as involved the 
 evolution of a stream of gas. The processes of putrefaction 
 were held to be akin to this, though the idea was based only 
 upon general relationships, and did not include the view that 
 organic or living bodies played a leading pai-t in both operations. 
 The early theories of fermentation consequently sought to 
 explain only the phenomena which we now associate with 
 the formation of alcohol. 
 
 The subject is mentioned in writings dating from early 
 times, and ferments are spoken of frequently, but we have 
 very little to guide us in forming an opinion of what the 
 writers understood by the term. Basilius Valentinus, a German 
 alchemist, gives us the view that was current at the beginning 
 of the 15th century. In his opinion alcohol existed in the 
 wort, or extract of germinated barley, before fermentation, but 
 
CH. XXIV] THEORIES OF FERMENTATION. 437 
 
 it was held in combination by various impurities which masked 
 its special properties. Fermentation was a process of purifi- 
 cation in which the yeast or ferment, whose nature was 
 unknown, communicated to the liquid an internal inflammation 
 or disturbance, by which the initially turbid and discoloured 
 liquid was so improved and freed from its impurities that the 
 alcohol was enabled to show its true properties. The great 
 growth of yeast which accompanied the process, being de- 
 posited at the bottom of the vessels when the fermentation 
 was over, was thought to be composed of the impurities, and 
 by some writers was spoken of as fwces vini, or the excrement 
 of the fermenting liquor. 
 
 Libavius, about the end of the 16th century, held that the 
 ferment was a body which transformed the fermenting substance 
 into something of the same nature as itself, the chief agent ia 
 the change being found in the heat or zeal of the ferment. 
 
 A distinction between fermentation and effervescence, which 
 was for a long time thought -to be a similar phenomenon, was 
 drawn by de la Boe, who pointed out that in the former case 
 decomposition, and in the latter combination, were involved. 
 
 Lemery held less accurately that the chief difference between 
 the two processes was one of relative rapidity, fermentation 
 being the slower and more complicated operation of the two. 
 He considered that the spirit produced in alcoholic fermen- 
 tation was an oil refined by the essential salts which the must 
 of wine contained. The deposit of yeast accompanyiag the 
 process was composed of the grosser elements of the must, 
 which became separated during the action of the salts, and took 
 the form partly of a sediment, and partly of a scum floating on 
 the surface. 
 
 No intelligible view of the nature of the action appears 
 among the writers of this period until Willis in 1659, and Stahl 
 some forty years later, put forward the first physical hypothesis. 
 They suggested that a ferment was a body possessing an 
 internal movement which it transmitted to the fermentable 
 material. Stahl extended the idea to include putrefaction, of 
 which he held alcoholic fermentation to be a particular case. 
 He considered all fermentable material to be composed of 
 
438 THE MODE OF ACTION OF ENZYMES. [CH. 
 
 particles formed by a loose combination of salt, earth and 
 oil; under the influence of the internal movement set up by 
 the ferment the various heterogeneous particles were separated 
 from each other, and were subsequently recombined in various 
 ways, gradually forming compounds of increased stability, 
 containing indeed the same constituents but in different pro- 
 portions. 
 
 This idea of a physical movement or vibration shows a 
 great advance upon any earlier hypothesis, and has occupied a 
 leading position in later speculations based upon accurate 
 chemical knowledge. It held its place much as Stahl stated 
 it, until the beginning of the present century. 
 
 In 1810 Gay-Lussac put forward the view that fermentation 
 was set up in the must of wine and other saccharine fluids by 
 the presence of oxygen. He did not consider it necessary that 
 the gas should be present during the whole process, but held 
 that it could not be started without contact with oxygen or 
 air. 
 
 About 1837 the physical conception of fermentation was 
 superseded by the vitalistic theory of Cagniard de Latour, 
 which was based upon his discovery of the true nature of yeast, 
 a discovery independently made about the same time by 
 Schwann at Jena and by Kiitzing at Berlin. He suggested 
 as the result of his observations that the yeast-ceUs disengaged 
 carbon dioxide and formed alcohol by some effect of their own 
 vital processes. This idea had previously occurred twenty 
 years before to Erxleben, but he laid no stress upon it in his 
 published writings. 
 
 Astier also even earlier (1813) foreshadowed the vitalistic 
 theory. Though he held that the globules of the ferment 
 were animal in nature, he said that they were generated and 
 nourished at the expense of the sugar, and that they gave rise 
 to a disturbance of equilibrium among the elementary particles 
 of that body. 
 
 Schwann's views, put forward simultaneously with de Latour's, 
 are quoted by Lafar' in the following words: — ^"Vinous fer- 
 mentation must therefore be regarded as the decomposition 
 - Lafar. Technical Mycology, Eng. Ed. p. 16. 
 
XXIV] THEORIES OF FERMENTATION. 439 
 
 occasioned by the sugar-fungus extracting from the sugar and 
 a nitrogenous body the materials necessary to its nutrition and 
 growth, whereby such elements of these bodies (probably among 
 other substances) as are not taken up by the plant unite by 
 preference to form alcohol." 
 
 Kiitzing, whose name is also associated with the investigation 
 into the nature of yeast and with the consequent theory of its 
 action, was the discoverer of the nature of the organism causing 
 acetous fermentation of alcohol, and in the expression of his 
 views he groups the two fermentations together. Lafar quotes 
 from his writings the following passages : " It is well-known 
 that chemistry explains vinous fermentation by the reaction 
 of the so-called gluten on the amylura (starch) and sugar. 
 I must firmly maintain that the explanation does not give 
 me a clear idea of the process, and I am inclined to doubt 
 whether others are more fortunate in this respect. It is 
 however certain that the entire process of alcoholic fermen- 
 tation is dependent on the formation of yeast, and the acid 
 
 fermentation on the growth of the vinegar plant Along 
 
 with the increased growth of these organisms, the reproductive 
 impulse also increases, and concurrently, their reaction on the 
 
 liquid present In so far as fermentation is synonymous 
 
 with a reciprocal reaction of organic and inorganic bodies on 
 the constituents of a given liquid which may be regarded as 
 forming the nutrient medium of the organic product, so is it 
 necessarily synonymous with every organic vital function ; 
 wherefore organic life = fermentation. On the other hand, such 
 processes as lead to the production of vinegar from alcohol by 
 the use of platinum black, or other similar methods, cannot be 
 compared with fermentation, being purely chemical, whilst 
 fermentation is an organo-chemical process, as is also the life 
 process of any organic body^" 
 
 The upholders of the older view combatted the new theory 
 with considerable vigour. Liebig, who was the most conspicuous 
 champion of the physical or physico-chemical hypothesis, some- 
 what modified Stahl's statement, bringing it into accordance 
 with the discoveries that had been made in the field of 
 ' Lafar. loc. cit. p. 17. 
 
440 THE MODE OF ACTION OF ENZYMES. [CH. 
 
 chemistry in the years between them. He drew a more 
 definite distinction between putrefaction and fermentation 
 than Stahl had done, while admitting that they presented 
 many features in common. He states his opinion in the 
 following sense — " The j'east of beer, and in general all animal 
 and vegetable matter undergoing putrefaction, transmit to 
 other bodies the state of decomposition in which they are 
 themselves; the vibration which by disturbance of their 
 equilibrium has been imparted to their own particles is 
 communicated equally to the particles of the bodies which 
 are in contact with them." The difference between putrefaction 
 and fermentation consisted in that in the former case the 
 decomposition was transmitted by the decomposing matter itself, 
 so that it continued to proceed even if the original cause became 
 inactive ; while in fermentation the sugar was incapable by 
 itself, even while in a state of incipient decomposition, of 
 transmitting the movement or vibration to still undecomposed 
 substance. Hence the necessity for the ferment not only to 
 originate, but to continue, the decomposition. While however 
 admitting the necessity of the presence of the yeast, Liebig did 
 not at all agree that its action was related to its vegetative 
 powers, as was claimed by the vitalists. He regarded it simply 
 as a source of nitrogenous matter, by the decomposition of 
 which fermentation was maintained, just as putrefaction is 
 connected with the decomposition of similar albuminous ma- 
 terial. 
 
 Liebig's views found many sympathisers, for his hypothesis 
 seemed to account for not only alcoholic fermentation and 
 putrefaction but certain other fermentations which had hitherto 
 been unexplained, particularly the formation of lactic and 
 butyric acids from sugar. Though we know now that these 
 processes are the work of organic beings, at that time the 
 latter had not been observed, and the only explanation that 
 seemed possible was that they resulted from the action of 
 decomposing nitrogenous matter in the sugar solutions. Some 
 of the writers of this school, among them Fremy, held that the 
 character of a particular fermentation varied according to the 
 degree to which the decomposition of the albuminous exciting 
 
XXIV] THEORIES OF FERMENTATION. 441 
 
 body had proceeded. At first it would set up alcoholic fermenta- 
 tion, but as its decomposition became more advanced this would 
 be replaced by lactic or butyric fermentation, and so on. 
 
 The vitalistic theory of fermentation put forward by de 
 Latour, Schwann, and Kiitzing was supported by Pasteur, to 
 whose brilliant researches reference has already been made. 
 He carried it much further than its original propounders, and 
 sought to show the reason why the vegetative life of the yeast 
 is associated with such a complete decomposition as takes place. 
 In the course of his work he very greatly strengthened his 
 position by the discovery that the formation of alcohol and 
 carbon dioxide is not the only transformation taking place in 
 the sugar, but that about four per cent, of the latter is disposed 
 of in another manner, giving rise to succinic acid and glycerine. 
 He showed too that other alcohols than the ordinary one are 
 formed in varying quantities during the metabolic activity of 
 the yeast. 
 
 As the outcome of his researches Pasteur was led to the 
 view that the larger decomposition of the sugar was the 
 expression of the effort of the yeast to obtain a supply of 
 oxygen, and to him therefore the phenomenon became one of 
 the intramolecular respiration of the yeast plant. As already 
 mentioned this view was strengthened by the researches of 
 Lechartier and Bellamy on alcohol-production in fruits kept 
 in an atmosphere of carbonic dioxide. 
 
 Other later observers, particularly Schiitzenberger, contro- 
 verted Pasteur's intramolecular respiration theory, and argued 
 that the sugar was an alimentary substance for the yeast, a 
 view which is at present held. 
 
 The controversy between the physical and the physiological 
 schools was maintained with considerable vigour. Liebig largely 
 modified his views to bring them into harmony with the facts 
 established by Pasteur, while still maintaining that fermentation 
 is essentially vibration or movement among the molecules of 
 the fermenting substance. He went so far as to admit that 
 the cause of the vibrations was the living organism rather than 
 any decomposing nitrogenous matter. 
 
 Efforts were made to harmonise the two views somewhat 
 
442 THE MODE OF ACTION OF ENZYMES. [CH. 
 
 on the lines of Liebig's latest opinion. According to Pasteur 
 the action was essentially intracellular ; the decompositions took 
 place in the cells of the yeast plant, in direct relation to their 
 metabolic processes, and the products of the fermentation left 
 the cells as excreta. The advocates of Liebig's opinion, while 
 admitting that the living cells set up the vibrations they 
 contended for, suggested that the decomposing forces them- 
 selves proceeded beyond the cells and attacked the fermenting 
 matter in the medium around them. The most noteworthy 
 advocate of this view was Naegeli, who held that in fermen- 
 tation there was a transference of vibrations or movements in 
 the material composing the living substance of the yeast to 
 the fermenting material outside, the living substance itself 
 remaining chemically unchanged. By such transferred vibra- 
 tions the equilibrium of the molecules of the fermenting 
 substance became destroyed and so its decomposition took 
 place. Naegeli computed what he thought to be the distance 
 through which such vibrations might be transmitted, and 
 concluded that the sphere of action of each cell extended 
 to a distance of from 20 to 50/i from its centre. 
 
 While this controversy was going on a new discovery, or 
 rather series of discoveries, was being made, the influence of 
 which soon began to be felt, and which was destined to very 
 largely modify and extend the phj'siological hypothesis of the 
 vitalists. The work of Payen and Persoz in 1833, and of Schwann 
 in 1836, led to the recognition of a class of substances capable 
 of setting up fermentations of very much the same order as the 
 alcoholic fermentation of yeast, marked by very similar features 
 of their action, but yet not depending on the presence of any 
 living organism. These bodies, which we know now as enzymes, 
 were in all cases derived from living cells, and their action was 
 readily held to be very similar to that of the yeast cell and 
 other organisms which were beginning to be known. They 
 were ascertained to act as ferments, and the term unorganised 
 ferment was given them, to indicate at once their mode of 
 action and the distinction existing between them and the living 
 cells of yeast. 
 
 The theory of fermentation in favour clearly had no room 
 
XXIV] THEOEIES OF FERMENTATION. 443 
 
 for these new bodies, and for a time no effort was made to 
 bring them into harmony with the popular views. Indeed 
 efforts were made to show that though at first sight similar, 
 they were really fundamentally distinct from the organised 
 ferments. In his Theory of Fermentation, published in 1879, 
 Naegeli dwells particularly on this point. He says, "Fer- 
 mentation by means of fungi only takes place in immediate 
 contact with the protoplasm and so far as its molecular action 
 extends." He finds a point of difference between the two 
 classes of ferments in the fact that the enzymes convert plastic 
 material from the passive into the active condition, so that 
 it is rendered suitable for nutrition, while the products of 
 fermentation by means of fungi are without exception less nu- 
 tritious compounds, and such fermentation destroys especially 
 the most nutritious substances. "The contrast appears most 
 striking in the case of carbohydrates and proteid substances; 
 while the action of unorganised ferments (enzymes) produces 
 firom them glucoses and peptones, fermentation by means of 
 fungi breaks up these compounds into alcohol, mannite, lactic 
 acid, and into leucin, tyrosin, &c. In some cases several 
 fermentations follow one another ; their products then become 
 less nutritious 'step by step. We may say generally that the 
 yeast-fungi render the medium in which they occur chemically 
 less suitable for nutrition by every process of fermentation 
 which they effect." 
 
 Sachs, writing in 1882, supports the views of Naegeli. 
 In his "Lectures on the Physiology of Plants" he says, "In 
 fermentation by means of unorganised ferments the chemical 
 transformation proceeds smoothly and completely; dextrin is 
 entirely transformed into grape-sugar, cane-sugar entirely into 
 inverted sugar, and albuminates entirely into peptones. In the 
 alcoholic fermentation, on the other hand, the products of 
 which alone have hitherto been quantitatively determined, only 
 the greater part of the sugar is broken up into alcohol and 
 carbon dioxide, while according to Pasteur about 5 per cent, of 
 the sugar is decomposed by the way into glycerine, succinic 
 acid, and carbon dioxide. It is likewise certain that in the 
 lactic acid fermentation, not all the sugar is decomposed into 
 
444 THE MODE OF ACTION OF ENZYMES. [CH. 
 
 lactic acid. Carbon dioxide especially appears to be a by- 
 product of all fermentations due to the action of fungi and of 
 all putrefactive processes. The alleged difference between 
 fermentation by means of unorganised ferments and that due 
 to the action of fungi would be unintelligible if both processes 
 were due to the same cause. Every difficulty vanishes, on the 
 other hand, if fermentation by means of fungi is caused not by 
 a contact substance (ferment) but by living protoplasm. We 
 then apprehend that while the ferment as a simple chemical 
 compound alters another chemical compound in a simple and 
 equable manner, so that all molecules suffer the same kind of 
 decomposition, an organised substance with its various molecular 
 movements and molecular forces produces more complicated 
 decompositions." 
 
 Both Naegeli and Sachs lay emphasis on the fact that at 
 the time they wrote enzymes which could effect fermentation 
 in the absence of the cells had not been extracted from any of 
 these fungi. Naegeli also denied that any of the intracellular 
 fermentative changes in any organism, whether animal or 
 vegetable, were due to unorganised ferments, and attributed 
 them always to the fermentative activity of the protoplasm. 
 
 This view was however controverted almost from the first. 
 In 1858 Moritz Traube made the origin and influence of these 
 enzymes the basis of a new theory of fermentation, which 
 all subsequent investigations have gone far to support and 
 which is continually growing into favour. This view is that 
 all fermentation is not instigated so much by the organisms 
 themselves as by enzymes which are formed as products of 
 their vitality, and are either excreted by them to work on 
 substances outside them, or retained in the cells in which they 
 are secreted to effect intracellular digestion. This hypothesis 
 is of great value as. again bringing all fermentative phenomena 
 into line with one another, and in showing clearly that alcoholic 
 fermentation is not something sui generis, but only one mani- 
 festation of a very wide-spread power. The fact that it can be 
 excited by an enzyme extracted from the yeast cell strongly 
 supports the theory, already greatly helped by the extraction 
 of enzymes from so many other micro-organisms. 
 
XXIV] THEORIES OF FERMENTATION. 445 
 
 Berthelot was one of the first chemists to accept this 
 view, being led to endorse it by the consideration, based on 
 experiment, that in certain cases production of alcohol took 
 place without the intervention of yeast at all. The experiments 
 of Lechartier and Bellamy and of Pasteur on this point have 
 already been referred to. The weight of the opinion of 
 Hoppe-Seyler has also been given in favour of this theory, 
 reasons for the acceptance of which have already been advanced 
 in one of the earlier chapters of this volume. 
 
 Before turning to consider how far we can explain the 
 action of the enzymes it seems desirable to examine a little 
 more closely the objections to this view which Naegeli and Sachs 
 based upon the nature of the products of enzymic action as 
 contrasted with those arising from the activity of the organised 
 ferments or Fungi. The peculiar features of the latter which 
 appeared to them radical find their representatives among the 
 metabolic phenomena of other plants than Fungi, and are really 
 the expression of the vital activity of plants in conditions which 
 are common among the lowly forms, but which occur abnormally 
 among the higher ones. As we have seen, the marked feature 
 of the life of these plants is the fact that the products of their 
 activity become less and less nutritious as the life goes on, or as 
 the authors quoted put it, as the fermentation proceeds. When 
 we consider that the fermentation in question is the expression 
 of the striving of the cell to procure nourishment from the 
 environment in which it finds itself, this ceases to be a matter 
 for surprise and becomes* only what might be expected. But 
 under certain conditions the higher plants behave similarly, and 
 they ought therefore to be considered as ferments also, if this 
 feature is a specific mark of fermentation. Boehm and de Luca 
 have shown that if any part of a living plant is insufficiently 
 supplied with oxygen, hydrogen and sometimes marsh-gas are 
 evolved from it. Boussingault and Schulz have observed similar 
 phenomena. These exhalations are ordinary features of the 
 putrefaction set up by micro-organisms. From plants con- 
 taining mannite, hydrogen is given off, while according to 
 de Luca acetic acid is formed from malic acid in the fruits 
 flowers, and leaves of the Privet. In the decomposition of 
 
446 THE MODE OF ACTION OF ENZYMES. [CH. 
 
 proteids again Boehm found ammonia exhaled. The condition 
 under which these results are obtained, viz. the absence of 
 oxygen, is the normal condition of many of the microbes, they 
 being anserobic in their mode of life. When oxygen is present 
 we find the same agreement. The result of the action of 
 invertase is the same whether that action is brought about by 
 living yeast or by the enzyme extracted from a higher plant. 
 The decomposition set up by trypsin in the formation of 
 proteoses, peptones, and amides is similar to that induced by 
 some of the proteolytic bacteria which have not at present been 
 found to secrete an enzyme. Sachs claims as a peculiarity of 
 all fermentation set up by Fungi that carbon dioxide appears as 
 a by-product. This is not to be wondered at, for Fungi like all 
 other living beings exhale this gas in the course of their 
 respiration. The quantity exhaled can in many cases be very 
 greatly increased by the simple process of allowing the plant 
 an insufficient supply of oxygen. 
 
 The difference drawn between the two so-called classes of 
 ferments is therefore unsupported by evidence and breaks down 
 as soon as it is carefully scrutinised. 
 
 The exact mode of action of the enzymes is still to a large 
 extent a matter of speculation and must remain so until we 
 arrive at some definite conclusion as to their exact nature and 
 constitution. This, as we have seen, is beyond our grasp at 
 present, for even if we accept the view that they belong to the 
 group of nucleo-proteids, we have no conception either of their 
 chemical composition or of the manner in which their groups of 
 molecules are arranged. There are however features of their 
 action which are more or less clearly established. 
 
 We have seen that in many cases this action is one of 
 hydration, especially in the cases of the simplest transformationa 
 To recall several of these which have been discussed in preceding 
 chapters, we have among others the reactions expressed by the 
 equations : 
 
 Invertase d^J^n + H2O = CeHiA + CsHuOa. 
 
 Emulsin C^H^NOn -t- 2H,0 = CeH.COH + HON -i- 2C.H,,05. 
 
 Lipase C^H,„0, + 3H,0 = SCsH^O, + C3H.(HO)3. 
 
 Urease CON2H4 + 2H2O = (NH^XCO,. 
 
XXIV] THEOEIES OF FERMENTATION. 447 
 
 The oxidases exhibit another process of a similar character, 
 but instead of inducing hydration . they set up oxidation. In 
 the case of the action of laccase upon hydroquinone we have 
 the reaction expressed by the equation 
 
 2C8H4(OH), + O2 = 2H,0 + 2CeHA. 
 
 The enzyme differs from those of the other group by trans- 
 ferring oxygen to the fermenting body instead of water. 
 
 These two classes of reaction suggest that the enzyme is 
 concerned with the transference of some molecule to the 
 fermenting body which it could not otherwise combine with, 
 and a fairly simple chemical hypothesis might be framed upon 
 observations such as those quoted. But it would be unsafe to 
 generalise so hastily, for other decompositions due to enzjrmes 
 are not apparently of the same order. One noteworthy 
 instance at least is known in which decomposition of the 
 fermenting substance takes place apparently without the 
 intervention of either water or oxygen. Buchner's zymase 
 splits up sugar according to the equation 
 
 CoH^^Oe = 2CO2 + 2GH3CH,OH. 
 
 We must not however conclude that even the first reactions 
 quoted do more than express the final results of the actions of 
 the particular enzymes in question. We have no ground for 
 supposing the latter to be of so simple a nature as the 
 equations would indicate, but on the contrary there are many 
 reasons for entertaining the opposite view. We know that the 
 action of diastase, which is capable of being represented by 
 almost as simple an expression, is a progressive one and that 
 many stages of the process have been demonstrated. 
 
 Certain observations of O'SuUivan and Tompson on the 
 behaviour of invertase lead to the conclusion that the hydrolysis 
 of cane-sugar by the enzyme is complex rather than simple. 
 In studying the effect of a rise of temperature on the enzyme 
 they ascertained that there was a great difference in its power 
 of resisting decomposition by heat according to the conditions 
 in which it was placed with regard to cane-sugar. When it was 
 
448 THE MODE OF ACTION OF ENZYMES. [OH. 
 
 heated in the absence of the latter, the enzyme was almost all 
 destroyed at 50° C. ; while if the sugar was present this effect 
 was not produced till the temperature was 75° C, a difference 
 of 25° 0. The authors advance as an explanation the view 
 that the invertase enters into combination with the sugar as a 
 stage in the hydrolysis and that the compound so formed can 
 resist the heat more successfully than can the invertase alone. 
 They hold further that the new compound is unstable and 
 becomes decomposed when its molecule meets with another 
 molecule of cane-sugar. 
 
 Whether we endorse their explanation or not, their observa- 
 tion on the influence of cane-sugar on the temperature of 
 destruction must lead us to believe that the reaction is com- 
 plicated in some manner by the invertase entering into it. 
 
 This observation does not stand alone. The writer has 
 observed a similar difference of behaviour in the case of 
 vegetable rennet, though in the opposite direction. This 
 enzyme is weakened and destroyed at a lower temperature 
 if heated in the presence of caseinogen than when it is 
 similarly treated in the absence of the proteid. 
 
 Buchner's zymase, again, is more stable in the presence 
 than in the absence of sugar. 
 
 Chittenden has observed that if neutralised pine-apple 
 juice, containing bromelin, is heated to 60° C. in the absence 
 of any proteoses or peptones, the enzyme is rapidly destroyed, 
 whereas this temperature is the one at which it is most active 
 if proteids are present during the warming. 
 
 Biernacki found similarly that albumoses or peptones raised' 
 the temperature at which trypsin was destroyed by 5° C. or 
 more, and that while pepsin . in the absence of peptone was 
 destroyed in acid solutions by a temperature of 55° C. it was 
 active after being heated to 70° C. if peptone was present. 
 
 We may also note in this connection the possible significance 
 of the inhibitory effects of traces of acids or alkalis in the 
 solution in which an enzyme is working. The minute trace 
 of the reagent required to cause inhibition seems to correspond 
 to the extremely small amount of the enzyme usually present, 
 and it does not seem a wild speculation to suppose that it may 
 
XXIV] THEORIES OF FERMENTATION. 449 
 
 • 
 
 affect the action of the latter by entering into some combination 
 with it, the resulting body not being capable then of uniting 
 with the substance which the enzyme would ordinarily trans- 
 form. That acids and enzymes do in some cases combine we 
 know from a consideration of the relations between pepsin 
 and hydrochloric acid, the ferment being absolutely inoperative 
 without hydrochloric or some other acid. Biemacki has shown 
 that pepsin in the absence of an acid is destroyed at a tem- 
 perature 5° 0. lower than that which is required for similar 
 destruction in its presence. Chittenden found that bromelin 
 on the other hand would withstand a higher temperature in 
 neutral than in acid solutions. In this case the compound, if 
 such exists, is less stable than the enzyme alone. 
 
 As we have seen the decompositions set up by enzymes are 
 very varied. In some cases the action appears to be hydrolytic, 
 in others oxidative, and in others disruptive without previous 
 hydrolysis or oxidation. 
 
 In endeavouring to form a hypothesis which shall be 
 capable of accounting for all these transformations there seems 
 to be no necessity to draw any distinction in this respect 
 between (i) the fermentative activity of protoplasm, (ii) the 
 action of enzymes which act only intracellularly, and which 
 have only a transient existence, being decomposed as soon as 
 their immediate duty is discharged, and (iii) the action of those 
 which are excreted from the cells, and are much more stable, 
 being capable of preservation by the various methods already 
 described. The difference between these three agents is 
 probably one of degree only and not of kind. 
 
 It seems clear however that any hypothesis which shall be 
 satisfactory must go back in some way to a physical or physico- 
 chemical theory, although such a view need not have much or 
 indeed anything in common with that which gave place to the 
 physiological one. Admitting that the problem is physiological 
 as regards the functions of the enzymes and their position with 
 regard to the metabolism of the organism, we must recognize 
 that the changes which we call metabolic, and in which these 
 enzymes take part, are fundamentally connected with chemical 
 or physical processes going on in the cells. 
 
 G. F. 29 
 
450 THE MODE OF ACTION OF ENZYMES. [CH. 
 
 We cannot now be satisfied with the statement that fer- 
 mentation is connected with the life of the organism and is a 
 symptom or manifestation of that life. We must pass beyond 
 this to enquire into the nature of the processes by which it 
 assists to maintain that life and takes part in that metabolism. 
 Such a physical or chemical hypothesis will have very little in 
 common with that of Liebig beyond perhaps embracing the 
 view that molecular vibration plays a part in the transfor- 
 mations, if indeed any hypothesis based upon vibration will 
 survive examination. 
 
 Before discussing the question of a physical as distinct from 
 a chemical process the recent researches of Emil Fischer 
 demand some attention. They are based upon the great 
 development of stereo-chemistry during the last decade, and 
 develop the idea that in the reactions of various chemical 
 bodies the molecular configurations of those taking part must 
 show a certain correspondence. 
 
 This view appears to have been suggested by the anomalous 
 behaviour of dififerent species of Yeast or Saccharomyces when 
 brought into contact with different sugars. Hansen's very com- 
 plete researches into the biology of the Yeast plant enabled him 
 to determine at least ten definite species of this genus, whose life- 
 history and capabilities of inducing fermentation he ascertained 
 with extreme accuracy. Of these ten species, six hydrolyse 
 both cane-sugar and maltose, forming hexoses as described 
 in a preceding chapter. They then further carry on alcoholic 
 fermentation at the expense of these hexoses. Saccharomyces 
 Marsdanus cannot attack maltose, but can hydrolyse cane-sugar. 
 S. memhranoBfaciens does not invert either sugar and is not 
 capable of setting up alcoholic fermentation. With a particular 
 species of Yeast again there are conspicuous differences among 
 the sugars as to their power of undergoing fermentation. Of 
 the biose polysaccharides, cane-sugar, maltose and trehalose are 
 capable of producing alcohol, but they must first be hydrolysed 
 to hexoses. Of the latter glucose and fi:uctose are much the most 
 readily fermented ; galactose is very refi-actory, but is said by 
 Bourquelot to be fermentable when a small quantity of either 
 glucose, fructose, or maltose is present also, but not under other 
 
XXIV] THEORIES OF FERMENTATION. 451 
 
 conditions. Lactose again is said by Duclaux and by Kayser 
 to be capable of fermentation by three species of yeast. 
 
 Fischer was led from a consideration of such facts as these 
 to suggest that these ferments can only attack those sugars 
 which possess a molecular configuration corresponding to their 
 own. Researches which he carried out, using various enzymes, 
 enabled him to put forward this view as applying to fermenta- 
 tion in general, whether set up by living protoplasm or the 
 enzymes it secretes. His first investigations were directed 
 towards the behaviour of glucase when brought into contact 
 with various sugars and glucosides. 
 
 When a solution of a sugar in methyl alcohol is saturated 
 with hydrochloric acid it loses the power of reducing Feh- 
 ling's fluid, and a crystallisable compound having the formula 
 CeHuOeCHs is formed as the result of a reaction represented by 
 the equation GeHiaOs + CH3OH = CsHnOeCHa + H^O. The same 
 reaction takes place with all the alcohols which are capable 
 of dissolving sugar, and the compounds formed correspond to 
 natural glucosides. The sugar entering into the reaction need 
 not necessarily be glucose, so that series of such artificial 
 metbyl-glucosides may be' prepared. These bodies are not 
 altered by Fehling's solution, phenylhydrazine or caustic 
 potash, but when boiled with dilute acid they take up water 
 and are decomposed into sugar and alcohol. 
 
 Theory indicates the existence of two glucosides, both of 
 which are formed by the action of hydrochloric acid on a 
 solution of d glucose in methyl alcohol. According to Fischer 
 they have respectively the formulae 
 
 H— C— O— Ri and iR—O— C— H 
 
 /cHOH /cHOH 
 
 ^\ CHOH *^\ CHOH 
 
 ^CH ^CH 
 
 I I 
 
 CHOH CHOH 
 
 CHjOH CHjOH 
 
 1 E denotes CHj 
 
 29—2 
 
452 THE MODE OF ACTION OF ENZYMES. [CH. 
 
 Fischer calls them respectively a and /8 methyl-glucoside. 
 He acted upon these new compounds (which only differ in the 
 configuration of the one asymetric carbon atom) with a prepara- 
 tion of glucase made by digesting yeast with 15 times its 
 weight of water for 15 hours at 30— 35°C. and filtering. 
 The a body was decomposed by the enzyme after 20 hours 
 action. The quantity of glucase solution was one-twentieth 
 part of the solution of the glucoside, and the temperature was 
 30 — 35° C. About half of the glucoside was transformed into 
 glucose and alcohol. 
 
 The P body was tested in the same way, but remained 
 altogether unaffected. 
 
 Fischer prepared the a- ethyl-glucoside and found that 
 glucase decomposed it as it did the corresponding methyl 
 one. 
 
 When the artificial glucoside was prepared with I glucose, 
 glucase was found to be without action on it. When d glucose 
 was replaced by galactose again, no decomposition was effected 
 by glucase. 
 
 When ordinary d fructose (levulose) was used instead of d 
 glucose, the resulting glucosides of the a type were decomposed 
 by glucase. Invertase could not effect these decompositions. 
 
 Fischer attributes these differences of behaviour to the 
 difference of configuration of the sugar molecules in the 
 several cases. This view gains some support from the fact that 
 each of the biose polysaccharides is hydrolysed by a special 
 enzyme, cane-sugar by invertase, maltose by glucase, milk-sugar 
 by lactase, and others by other enzymes, as described in 
 Chapter ix. of this volume. If the reason is a correspondence 
 in configuration between the sugar and the enzyme it would 
 apply equally to the failure of glucase to decompose the 
 methyl-galactoside, as lactose, which yields galactose and glucose 
 on hydrolysis, is unaffected by glucase. 
 
 Fischer carried his observations further and experimented 
 on the action of glucase on the aromatic glucosides. He found 
 it unable to attack many of these, but to be capable of setting 
 up a decomposition of amygdalin, which affected the sugar 
 radicle of the complex molecule only. Instead of acting like 
 
XXIV] THEORIES OF FERMENTATION. 453 
 
 emulsin and forming bitter almond oil and hydrocyanic acid 
 with two molecules of glucose, it was only capable of splitting 
 off one molecule of glucose, leaving a new glucoside containing 
 the other molecule of the hexose. 
 
 Fischer studied the action of emulsin as well as glucase on 
 the artificial glucosides. He found that like glucase, emulsin 
 attacked the glucosides of d glucose, but it was incapable of 
 decomposing those of galactose and similar sugars. It was 
 also without effect upon the methyl I glucosides. Its action 
 was however easily distinguishable from that of glucase, for 
 it only decomposed the glucosides possessing the /3 consti- 
 tution, while glucase attacked only the a group. 
 
 Fischer found that emulsin was without action on methyl- 
 d-mannoside, methyl-sorboside or methyl-galactoside. It would 
 not decompose either the a or /3 form of methyl-glucoside 
 prepared from I glucose. 
 
 Myrosin showed similar differences ; it split up the a methyl- 
 d-glucoside but not the /S one. 
 
 Glucase prepared from yeast did not attack methyl-sorboside, 
 nor methyl-mannoside prepared from reserve cellulose. 
 
 On the basis of these researches of Fischer we maj' arrive 
 at the following view, which was in the main suggested by him. 
 The enzymes being probably nucleo-proteid in nature, are like 
 true proteids optically-active substances, and on account of this 
 their molecules have an asymmetric structure. As the work 
 described shows that the action of enzymes is a selective one, 
 it follows that those substances with which they come into such 
 contact as will enable them to provoke decompositions must 
 have a confiiguration corresponding to their own. The two 
 molecules may be taken to have such a mutual relationship as 
 that existing between a key and the lock for which it is con- 
 structed, the shape of the key enabling it only to unfasten the 
 particular lock to the arrangement of whose wards it corre- 
 sponds. 
 
 The configurations of the two bodies, the sugar and the 
 enzyme, fitting as it were into each other, the disruption of the 
 former by the latter becomes conceivable. Fischer lays stress 
 especially on the behaviour of the a and /S methyl-glucosides 
 
454 THE MODE OF ACTION OF ENZYMES. [CH. 
 
 with glucase. The two glucosides have the same composition, 
 are formed from the same alcohol and the same sugar, and 
 differ only in the configuration of a single carbon-atom which 
 is rendered asymmetric by the introduction of the methyl-group 
 into the sugar. 
 
 The same relationship or correspondence of form may well 
 exist between other enzymes than those decomposing sugars 
 and the bodies whose decompositions they effect. The latter 
 are for the most part asymmetric substances. 
 
 We can now return to the consideration of the question 
 whether the actual processes of fermentation can best be, 
 explained on a physical or a chemical hypothesis. 
 
 The original position of Liebig demanded a vibratory 
 movement of the molecules of the body exciting fermentation, 
 and he associated such a movement with the decomposing 
 nitrogenous matter in the solution. This was modified partly 
 by himself and partly by his successors to embrace the idea 
 that the vibrations were set up by living cells or by something 
 excreted from them. Fischer's hypothesis does not appear 
 inconsistent with this view ; indeed it supplies a reason for the 
 selective fermentations set up by different excreted bodies or 
 enzymes, showing that only such bodies can be thrown into 
 increased molecular vibration as have a similar configuration 
 to the enzyme. 
 
 But a view based upon molecular vibration does not seem 
 satisfactory, in the face of what is now known about the nature 
 of enzymes, little as it is. They are not continually undergoing 
 decomposition as was the case with Liebig's organic nitrogenous 
 material. On the other hand they are fairly stable substances, 
 even when in aqueous solution. Moreover in the light of the 
 observations noted already, there is reason to believe that they 
 enter into some form of chemical combination with the bodies 
 which they hydrolyse. 
 
 Berzelius long ago rejected Liebig's view of molecular vibra- 
 tion in favour of a theory that the action was one of contact, 
 comparable to the effect which spongy platinum produces on 
 water holding oxygen in solution. Not only spongy platinum, 
 but many other bodies, such as finely powdered charcoal, or 
 
XXIV] THEORIES OF FERMENTATION. 455 
 
 manganese dioxide, or finely precipitated silver, when added to 
 water saturated with oxygen cause the latter to be given off in 
 bubbles. Many of the enzymes will produce the same effect, 
 having this property in common, as well as possessing the 
 specific powers already described. Schoenbein, who first noticed 
 this peculiarity, thought it was especially connected with their 
 fermentative power. Jacobson has shown that this is not the 
 case, for if gently and gradually heated their solutions lose this 
 property before they part with the power of exciting their 
 specific actions. 
 
 The suggestion does not however throw any light upon the 
 mode of action which can be associated with them. Nor is 
 there such a similarity between enzymes and a body like 
 spongy platinum as would allow us to regard their behaviour 
 as identical. 
 
 A hypothesis has been advanced that fermentation is due 
 to electric hydrolysis; that as the result of electrical action 
 in the liquid the elements of water are withdrawn firom 
 the sugar and added on again in a different order, so that 
 there is going on in the liquid an electric hydration and 
 dehydration. The suggestion has been brought forward by 
 Armstrong, who has pointed out that in adopting this view 
 it is not necessary to assume that there is ever any actual 
 separation of the elements of water fi-om the molecule and 
 their subsequent readmission, but that the action may be 
 brought about by a kind of electrolytic change taking place 
 locally within the molecule. On this hypothesis the part 
 played by the enzyme or the organism might well be to 
 complete the galvanic circuit and so to allow the electrolytic 
 current to pass. This is not out of harmony with Fischer's 
 views, as a close approximation of the two sufficient to form a 
 pathway for the current might well depend upon their possessing 
 a corresponding configuration. This hypothesis demands how- 
 ever that enzymes shall be electrolytes, and of this so far 
 there is no evidence. 
 
 A physical hypothesis of a different nature has been 
 advanced by de Jager. Starting with Naegeli's view that 
 fermenting yeast-cells emit vibrations which pass out of the 
 
456 THE MODE OF ACTION OF ENZYMES. [CH. 
 
 cells and decompose the sugar in the solution surrounding 
 them, de Jager suggests that the enzymes may be regarded not 
 as substances at all, but as the vibrations themselves, that is as 
 properties of substances rather than material bodies. In this 
 way he compares them to other physical forces, such as light, 
 electricity, and magnetism. Fermentation so would come to 
 consist of chemical transformations set up by physical forces 
 and not in any sense dependent on the chemical action of a 
 molecular substance. 
 
 This theory has been recently taken up and elaborated by 
 Maurice Arthus, who has very ingeniously compared the 
 properties of enzymes with those of various physical forces. 
 He lays stress particularly on two facts: 1st, that the com- 
 position of the enzyme so far has eluded the investigations 
 of the most eminent chemists, and that all their results 
 have only shown that these bodies if they exist cannot be 
 separated from impurities. 2nd, that the amount of any of 
 them which efifects a decomposition is infinitesimal in com- 
 parison with the amount of material which it can decompose. 
 He regards the " carrying " theory of Bunsen as totally 
 inadequate to account for this enormous disproportion. Further 
 he endeavours to show that their various so-called " properties " 
 are paralleled by properties of the different physical agencies 
 with which he is disposed to rank them. 
 
 Though the theory is ingenious it seems impossible to recon- 
 cile it with the very definite processes of formation or secretion 
 which we have seen can be observed in both animal and 
 vegetable tissues. The different reactions which they present, 
 their respective powers of solubility and precipitation, their 
 varying capability of dialysis, &c.,,also point much more strongly 
 to their being definite material entities. 
 
 The view that the action of enzymes is chemical rather 
 than physical in its character has met with considerable 
 support. The changes which so many of them set up we have 
 seen are hydrolytic, the fermenting body taking up water through 
 their instrumentality and subsequently undergoing decom- 
 position. Most of the changes so effected can be brought about 
 in the laboratory by ordinai-y chemical reagents, — starch can be 
 
XXIV] THEORIES OF FERMENTATION. 457 
 
 hydrolysed to sugar by dilute mineral or organic acids; the 
 same bodies will produce glucose and fructose from cane-sugar; 
 fats can be decomposed by alkalis or by superheated steam; 
 peptones can be formed by heating proteids to a high tempera- 
 ture in a Papin's digester. Moreover when the changes set 
 up by the enzymes are investigated carefully, they show 
 considerable correspondence in detail with changes which are 
 distinctly chemical. In the course of the observations upon 
 invertase to which allusion was made in the last chapter, 
 C. O'Sullivan and Tompson showed that the rate of inversion 
 of cane-sugar which it brings about may always be represented 
 by a definite time curve which " is practically that given by 
 Harcourt as being the one expressing a chemical change of 
 which no condition varies, excepting the diminution of the 
 changing substance''." It thus obeys the law formulated by 
 Harcourt that in the case of a substance undergoing such 
 change under such conditions, the amount of change occurring 
 at any moment is directly proportional to the quantity of the 
 substance. J. O'Sullivan also has found that the hydrolytic 
 action of yeast at the ordinary temperature follows the same 
 course as that of a simple chemical interchange. 
 
 But if we adopt the view that we are dealing with an 
 ordinary chemical reaction, leading in these cases to hydration 
 and subsequent decomposition we have still to enquire what is 
 the exact nature of that reaction. Any explanation of the latter 
 must be such as to include also those more complex changes 
 which involve the actual disruption of the molecule, and which 
 we find in the cases of myrosin and Buchner's zymase. 
 
 In considering the question of the chemical changes set up 
 by enzymes, the first thing that strikes an observer is the ex- 
 tremely small amount of one of them that is needed to bring 
 about the transformation of an enormous amount of the body 
 which it attacks. Thus O'Sullivan and Tompson showed in 
 one of their experiments that a sample of invertase induced 
 inversion of 100,000 times its own weight of cane-sugar. More- 
 over it was not destroyed or even greatly injured by its activity. 
 The decomposition of an enzjrme during the course of its activity 
 > O'Sullivan and Tompson, ' On Invertase.' Jour. Gliem. Soc, 1890, p. 926. 
 
458 THE MODE OF ACTION OF ENZYMES. [CH. 
 
 has been shown by Tammann to be very slow and gradual, ajid 
 to be influenced by differences of temperature in the same way 
 as the activity itself These considerations lend a good deal of 
 plausibility to a suggestion which has been made by Bunsen and 
 by Hlifiier that enzymic action may be similar to the behaviour 
 of nitric oxide in the manufacture of sulphuric acid or of sul- 
 phuric acid in the preparation of ether. In the former process 
 a stream of sulphur dioxide mixed with oxygen in the presence 
 of water is presented to nitric oxide, and the further oxidation 
 of the sulphur dioxide is effected with great rapidity. A small 
 proportion of the oxide of nitrogen will effect the combination of 
 an almost indefinite amount of sulphurous anhydride and oxygen. 
 Nitric oxide in the presence of oxygen immediately becomes 
 nitrogen peroxide, and the latter when mixed with sulphurous 
 anhydride and a large quantity of water gives up oxygen to the 
 sulphurous anhydride, which becomes sulphuric acid, while the 
 original amount of nitric oxide is again formed. This process 
 continues so long as any sulphurous anhydride remains in the 
 presence of oxygen uncombined with it. 
 
 The action of sulphuric acid in the manufacture of ether firom 
 alcohol is of a similar kind. An interaction of the acid with the 
 alcohol takes place, which can be expressed by the equation 
 
 C AHO + H2SO4 = C AHSO4 + H,0, 
 Alcohol Snlphnric acid Ethyl sulphate Water 
 
 and the ethyl sulphate reacts with another molecule of alcohol 
 in a manner indicated by the further equation 
 
 C AHSO4 + QH^O = (C AXO + H^04. 
 
 Ethyl sulphate Alcohol Ether Snlphnric acid 
 
 On this view we may suppose the enzyme to attach itself to 
 the body on which it acts, with or without the elimination or 
 addition of water. The new compound meeting with a further 
 molecule of the same body will then be decomposed with the 
 formation of the body found to result from the action of the 
 enzj-me, the latter being again liberated unchanged or perhaps 
 reformed. 
 
 This view must remain at present hypothetical, as no one 
 so far has isolated a compound of the enzyme and the body it 
 
XXIV] THEORIES OF FERMENTATION. 459 
 
 attacks. But as we saw in the last chapter, O'Sullivan and 
 Tompson have found reason to believe that such a combination 
 exists, their view being based on the effect of heat on invertase 
 in the presence and in the absence of cane-sugar in the solution 
 under examination. Matthieu and Hallopeau were led by some 
 of their observations to the view that in the action of pepsin in 
 the stomach the chlorine of the hydrochloric acid enters into 
 combination with the albuminoid substances, and even with the 
 peptone. If this conclusion is well-founded the acid may be an 
 intermediary between the pepsin and the proteid, for we know 
 it is in very close association, if not union, with the former. 
 
 If the hypothesis is a valid one, the behaviour of enzymes 
 must fall into line with other chemical actions. One of the 
 fundamental features of the latter is that if two compounds 
 capable of chemical interaction are present in the same solution 
 the resulting interchange will not be complete unless one of 
 the products formed is removed as fast as it appears. A certain 
 amount of all four possible bodies will result, the relative 
 quantities depending on various conditions, and the chemical 
 interchange will only cease when a definite point of dynamical 
 equilibrium between them is reached. This point must depend 
 among other things upon the relative amounts of the reacting 
 bodies in the solution and will therefore vary accordingly. 
 
 The behaviour of sulphuric acid and alcohol will illustrate 
 this point. So long as alcohol is in excess the ethyl sulphate 
 first formed will combine with it as described, giving rise to 
 ether. But if water is in excess a reaction will occur, which 
 will lead to the decomposition of the ethyl sulphate into the 
 compounds firom which it was formed, 
 
 C,H3S04 -l-H,0 = CAHO -t-H3S04. 
 
 Ethyl sulphate Water Alcohol Sulphuric acid 
 
 In the process therefore all four bodies, alcohol, sulphuric 
 acid, ether, and water will be found together, and the ultimate 
 equilibrium point will depend on the relative proportions of the 
 alcohol, the water, and the sulphuric acid. 
 
 We must therefore expect that in the action of enzjTnes 
 the continuous and complete decomposition of the original 
 
460 THE MODE OF ACTION OF ENZYMES. [CH. 
 
 substance on which the enzjnne works cannot take place unless 
 the ultimate product formed is removed as fast as it appears. 
 If this is not done the action should go on rapidly at first, but 
 gradually more slowly until the equilibrium point is reached, 
 when the action should cease. This is exactly what has been 
 found in many cases. Tammann has shown that a considerable 
 inhibition of the action of emulsin on amygdalin is caused by 
 the accumulation of the products of the decomposition, and that 
 this is not due at all to a destruction of the enzyme. Brown and 
 Morris have shown that it is almost impossible to completely 
 hydrolyse starch by means of diastase, generally nearly 20 per 
 cent, of dextrin remaining. Lea has found that the hydrolysis 
 can be completed if the sugar is removed as fast as it is formed. 
 
 Again, if the relative amounts of the interacting bodies 
 regulate the course of the action, an enzyme should be capable 
 of effecting a certain amount of a reverse action, when the final 
 instead of the initial body is presented to it. Certain investi- 
 gations made recently have shown that this also is one of the 
 features of the behaviour of enzjnnes. 
 
 An extended research upon the action of maltase or glucase 
 upon malt-sugar was made by Croft Hill in 1897 and 1898. 
 The enzyme was prepared from dried yeast with appropriate 
 antiseptic precautions and the experiments were safeguarded 
 by the use of toluene in all the digestions. Hill found at the 
 outset that the presence of glucose had a marked inhibitory 
 eflfect upon the hydrolysis of maltose, and this effect was greater 
 as the sugar solutions were more concentrated. When the 
 enzyme was digested with a 40 per cent, solution of glucose at 
 30° C. a slow conversion of the latter into maltose was detected. 
 As the action proceeded its amount was measured by observing 
 the rotation of a ray of polarised light in a polarimeter and by 
 ascertaining the cupric-oxide-reducing power of the solution. 
 After five days 3"25 per cent, of the glucose had undergone con- 
 version, which was increased to 10 per cent, after 28 days, and 
 to 145 per cent, after 70 days. Beyond this point no change 
 occurred, so that an equilibrium point for these two sugars was 
 reached when there was 85'5 per cent, of glucose and 14'5 per 
 cent, of maltose present. The quantitative determinations were 
 
XXI V] THEORIES OF FERMENTATION. 461 
 
 checked by treatment of the contents of the flask with phenyl- 
 hydrazine acetate, when two osazones were formed, having the 
 properties of glucosazone and maltosazone respectively. 
 
 In a subsequent experiment, in which a mixture of the 
 two sugars was used, consisting of 75 per cent, of glucose and 
 25 per cent, of maltose, the concentration of the total sugar 
 being as before 40 per cent., the same equilibrium point was 
 reached, the digestion being continued 56 days. 
 
 With a smaller percentage of sugars in the digesting liquid 
 the equilibrium point was not the same. Using a 20 per cent, 
 concentration Hill found that on the conclusion of the experi- 
 ment 90'5 per cent, of the total sugar was glucose and 9'5 per 
 cent, maltose ; with a 10 per cent, concentration the equilibrium 
 point was reached when the proportion of glucose to maltose 
 was 94"5 : 5'5. In more dilute solutions the transformation of 
 glucose was still less — with 4 per cent, concentration the 
 ratio became 98 : 2, and with 2 per cent. 99 : 1. Hill found 
 therefore that with very dilute solutions of maltose hydrolysis 
 to glucose by the enzyme was almost complete: with a con- 
 centration of 20 per cent, a reverse action could be detected 
 which was well marked when the concentration was 40 per cent. 
 
 These experiments of Croft Hill afford considerable support 
 to the view that the action of glucase is a chemical one, and 
 that the transformations which it effects do not differ in any 
 essential feature from ordinary chemical changes. 
 
 Croft Hill's results have been challenged by Emmerling, 
 who claims that the sugar produced from the glucose is iso- 
 maltose and not the original sugar. 
 
 Hanriot states that he finds lipase capable of setting up a 
 reversing action and forming fat from fatty acid and glycerine 
 when appropriate proportions are present. The same observa- 
 tion has been made by Kastle and Loevenhart. 
 
 The action of other enzymes needs investigation from this 
 point of view. The decompositions which many of them set up 
 are more complex than those just described. The hydrolysis 
 effected by diastase for instance we have seen is progressive and 
 not complete in one operation, dextrins of different degrees 
 of complexity making their appearance simultaneously with 
 
462 THE MODE OF ACTION OF ENZYMES. [CH. XXIV 
 
 maltose, as the starch molecule is gradually broken down. 
 Trypsin also does not efifect the conversion of proteid into 
 peptone and amides by a single operation but by a succession 
 of stages, each characterised by the appearance of definite inter- 
 mediate bodies. The complexity of the decompositions in these 
 cases, however, does not negative the idea that the course of 
 action is the same as in the cases already examined. 
 
 Fischer's hypothesis of the dependence of the action upon the 
 configuration of the enzymes may throw some light upon their 
 destruction by heat. It has generally been considered that they 
 are entirely broken up, possibly by hydrolysis, at the temperature 
 which we have called their maximum point. This view is sup- 
 ported by the observation that when perfectly free fi:om water 
 they may be heated to a much higher point without destruction. 
 Pavy has shown that this is the case when the water is removed 
 by alcohol instead of by desiccation. He found liver diastase 
 active after being boiled in absolute alcohol. The process 
 appears thus as if it might be due to hydrolysis. On the 
 other hand it may be that the heating causes only such a change 
 in their configuration as to cause their molecules no longer to 
 correspond to those of the bodies they decompose. Tammann 
 has shown that an enzyme loses its activity at a low temperature, 
 though much more slowly than at a high one. As the tempe- 
 rature at which it is kept is raised it shows a greater proneness 
 to become inactive. This is consistent with the view that then- 
 so-called decomposition is only a change in configuration. 
 
 We are acquainted with numerous cases in which simply 
 heating an optically active substance changes its configuration. 
 We may mention especially the classical experiment of Jung- 
 fleisch in which ordinary tartaric acid is transformed by this 
 means into racemic acid, which is optically inactive. Many 
 other instances of this phenomenon are to be met with in 
 Fischer's researches on the sugar series. 
 
463 
 
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 Sharp, Gordon (217). Papain digestion. Pharmaceutical Journal 3 ser. 24 
 
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 785. 
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 32—2 
 
600 BIBLIOGRAPHY. 
 
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505 
 
 INDEX. 
 
 Acetic fermentation 374 
 Achroodextrin 53, 54 
 
 „ from glycogen 62 
 
 Acids, effect of on diastasic action 
 
 69, 70 
 Actinosphserium 11 
 
 ,, intracellular diges- 
 
 tion in 202 
 Aethalium, diastase in 24 
 Alcoholic fermentation 333 
 „ in ripe fruits 348, 351 
 „ in vegetative parts of plants 350 
 „ relation to respiration 358 
 „ „ to nutrition 354 
 
 Aleurone layer of grasses 28 
 „ work of Haberlandt 28 
 „ „ of Brown and Morris 29 
 ,, „ of Brown and Escombe 29 
 Alizarin 163 
 
 Alkalis, influence on diastase 71 
 Allyl sulpho-cyanate 155 
 Amide-acids 173 
 
 ,, source of in digestion 177 
 
 Ammonia, liberated by tryptic en- 
 zymes 233 
 Amoeba 11 
 
 „ intracellular digestion in 202 
 Amphopeptone 179 
 Amygdalin, decomposition of 6, 147 
 Amylase 15 
 
 (of Duclaux) 61 
 Amylodextrin 53, 54 
 Amyloln 52 
 Anthozymase 114 
 Antialbumate 173 
 
 „ identity with parapep- 
 
 tone 175 
 
 Antialbumid 173 
 
 , , identity with hemiprotein 
 
 175 
 ,, resemblance to dyspep- 
 
 tone 179. 
 Antialbumose 178 
 Antipeptone, jreactions of 179 
 
 „ possible nature of 184 
 
 Anti-residue of proteid digestion 178 
 Araban 49 
 Arbutin 148 
 Argenin 185, 199, 200 
 Arrack 346 
 
 Aspergillus, enzymes in — diastase 23, 32 
 ,, inulase 81 
 
 ,, invertase 121 
 
 ,, trehalase 138 
 
 ,, rafSnase 141 
 
 ,, melizitasel43 
 
 ., emulsin 151 
 
 „ tannaee 168 
 
 „ trypsin 230 
 
 „ lipase 248 
 
 Asphyxiation, its relation to alcoholic 
 
 fermentation 353, 355, 357 
 Atmid-albiunin 183 
 Atmid-albumose 183 
 Atraotylate of potassium 153 
 Atropine, effect on diastase 71 
 
 Bacteria, diastase in 24, 31 
 
 „ proteolytic enzymes of 231 
 ,, relations of enzymes of to 
 
 temperature <!fcc. 233 
 „ species producing alcohol 347 
 Beet, metabolism of root of 116 
 Betulase 166, see Gaultherase 
 
506 
 
 INDEX. 
 
 Botrytis, oytase in 91 
 
 Bromeliii 209 
 
 mode of preparation of 210 
 action on fibrin 210 
 „ on albnmin 212 
 „ on myosin 212 
 effect of reagents on 212 
 
 Butyric fermentation 369 
 
 Bynedestin 223 
 
 Bynin 223 
 
 Cadaverine 390 
 Calcium pectate 291 
 Calcium salts, influence on rennet 256 
 „ „ on thrombase 272 
 
 „ . II on pectase 290 
 Cane-sugar, relation to protoplasm 115 
 ,, formation ip plants 117 
 
 ,, relation to starch 117 
 
 Carchesium 11 
 
 „ digestive changes in 203 
 
 Caroubin 105 
 Caroubinase 105 
 Casease 233 
 Casein 251 
 Caseinogen 251 
 
 ,, digestion by pepsin 194 
 
 Gasse of wines 320 
 Cellulose, products of hydrolysis of 89 
 „ digestion of by animals 102 
 Cell-wall, composition of 86, 297, 299 
 Choline 390 
 
 Chromogeu produced by pepsin 193 
 „ contained in various fungi 
 
 316 
 Chymosin 251 
 Citric fermentation 372 
 Coagulated proteid 270 
 Coagulation of blood 266, 271, 273 
 „ „ inhibition of by 
 
 certain salts 
 275 
 ,1 „ inhibition of by 
 
 peptone 275 
 „ „ intravascular 
 
 277, 278, 416 
 Coniferin, action of emulsiu on 152 
 Cradina 224 
 
 Cytase 29, 89 
 
 „ preparation of 94, 96 
 
 „ properties of 97 
 
 „ localisation of in grasses 97, 
 
 98 
 „ „ „ in palms 99 
 
 „ products of action of 103 
 
 Deutero-proteose, reactions of 180 
 Dextran 345, 385 
 Dextrin 50 
 
 „ primary and secondary 54 
 Dextrinase (of Wijsman) 60 
 „ (of Duclaux) 61 
 
 Dextrose, see Glucose 
 Diastase, discovery of 6, 15 
 ,, sources of 16 
 „ distribution of 16, 22, 23, 43 
 ,, varieties of 17, 65 
 ,, analysis of 47 
 „ course of action of 49, 51, 59 
 ,, relations to temperature &c. 
 
 65 
 ,, influence of acids and alkalis 
 
 on 70, 71 
 , , conditions of secretion of"73 
 „ inhibition of 74 
 ,, methods of preparation of — 
 Payen and Persoz 6, 15 
 Mialhe 16, 46 
 BonrquelotandH6rissey24 
 Cohnheim 45 
 Krawkow 46 
 von Wittich 46 
 Lintner 46 
 Loew 47, 48 
 Muscnlus 47 
 Wroblewski 48, 49 
 „ from salivary glands of 
 
 insects 43 
 Diastase, animal 15, 16 
 
 » ,, distribution of 83, 
 
 34, 35 
 ,, „ secretion of 36 
 
 .! » varying amounts of 
 
 38 
 .. „ its existence in the 
 
 liver 39 
 
INDEX. 
 
 507 
 
 Diastase, animal, identity of with vege- 
 table diastase 44 
 Diastase of secretion 24 
 
 " i> work of Brown 
 
 and Morris and 
 of Haberlandt 
 25 
 >> )> distribution of 25 
 
 >> ,, formation of 27 
 
 i> ,, increase during 
 
 germination 28 
 )> ,> action on starch 
 
 paste 64 
 Diastase of translocation 17 
 
 ij ,, distribution 
 
 of 18, 19 
 I, ,, development 
 
 of 18 
 !, ,, compared 
 
 with animal 
 diastase 38 
 Digitalin 153 
 Dionsea, enzyme in 229 
 Drosera, enzyme in 229 
 
 „ secretion in 402 
 Dyspeptone 175, 176 
 
 „ probable identity with 
 
 antialbumid 179 
 Dys-proteose, reactions of 180 
 Dystropodextrin 63 
 
 Edestin 223 
 Emulsin 145, 147 
 
 actions of 147, 152, 153 
 distribution of 148 
 preparation of 152 
 occurrence with lactase 145, 
 
 154 
 Guignard's reaction for 149 
 secretion by animals 154 
 relation to temperature 154 
 conditions of action 154 
 suggested action on oil 171, 
 246 
 Emulsive enzymes 237 
 Enzymes, meaning of the term 7 
 „ secretion of 8 
 „ intracellular action of 11 
 
 Enzymes, features of their action 12 
 ,, classification of 14 
 „ action of light upon 428 
 Erythrite, fermentation of 373 
 Erythrodextrin 50, 54 
 
 ,, from glycogen 62 
 
 Erythrozym 147, 162 
 
 „ relation to emulsin 164 
 
 Ferment, old' ideas of 2 
 „ unorganised 6 
 „ organised 7, 9, 391 
 , , organised nature ofaotionof 
 10, 12 
 
 Ferment glueosique of Berthelot 112 
 
 Fermentation, definition of 9 
 „ intracellular 10 
 
 Fibrin 267 
 
 Fibrin ferment, see Thrombase 
 „ (Crustacean) 280 
 
 Fibrine soluble of Denis 277 
 
 Fibrinogen 267 
 
 Fibriuo-plastiu 2G7 
 
 Fishes, diastase in 43 > 
 
 Galactase 207 
 
 Galactose, derived from lactose 144, 154 
 „ from Xanthorhamniu 165 
 „ alcoholic fermentation of 
 337 
 Gaultherase 147, 166 
 Gaultherin 166 
 
 Gelatin, digestion of by pepsin 193 
 „ „ trypsin 201 
 
 „ „ galactase 208 
 
 „ „ enzymes of 
 
 microbes 233 
 Gentianose 127 
 Ginger-beer plant 342 
 Glucase 56, 111, 128 
 
 ,, distribution of 130 
 
 „ capacity of hydrolysis 132 
 
 „ amounts of in different tissues 
 
 134 
 „ optimum point of 135 
 „ action on amygdalin 136 
 Glucose, reactions of 55 
 
 „ produced in liver 55 
 
508 
 
 INDEX. 
 
 Glucose, method of separating it fiom 
 
 fructose 111 
 Glucosides 146 
 
 „ nutritive value of 171 
 „ relation to bouquet of wines 
 
 326 
 Glycerine, formation of in alcoholic 
 fermentation 4 
 ,, produced in the hydrolysis 
 
 of fat 237 
 ,, an antecedent of alcohol 
 
 847 
 „ fermentation of 374 
 
 Glycogen, its function 38 
 „ its digestion 89 
 „ action of diastase on 61 
 Glycolysis in blood 327 
 Gum-ferment 109 
 
 Hadromase 107 
 
 Hsemolymph of Crustacea 281 
 
 „ ,, coagulation 
 
 of 280, 283 
 Helioin 148 
 Hemialbnmose 173 
 
 „ decomposition of 177 
 
 „ reactions of 178 
 
 „ correspondence with 
 
 Meissner's and pep- 
 tone 179 
 „ ' a mixture 180 
 
 Hemipeptone 174 
 
 „ reactions of 179 
 
 Hemi-residue of proteid digestion 178 
 Hesperidin 1S3 
 
 Hetero-proteose, reactions of 180 
 Hippuric acid, decomposition by 
 
 microbes 302 
 Histozyme 307 
 Hordein 223 
 
 Hydrocyanic (Pmssie) acid, from 
 amygdalin 147 
 ,, from linamarin 170 
 ,, nutritive value of 171 
 Hydrogen, evolved from plants 367 
 Hydroquinone, formed from arbutin 
 148 
 „ actionoflaccaseon312 
 
 Hypotheses of fermentation, of Fischer 
 451 
 
 „ of Liebig 454 
 
 „ of Berzelius 454 
 
 „ theory of electric hydrolysis 455 
 
 „ of de Jager 455 
 
 „ of Bunsen 458 
 Hystidine 185 
 
 Indican, action of emulsin on 153, 170, 
 
 326 
 Indiglucin 171, 326 
 Indigo-white 326 
 Indol 387 
 
 Insecta, enzyme in 198 
 Insectivorous plants 225 
 Intercellular protoplasm of Eussow 298 
 Intestine, digestive changes in, work 
 
 of Brown and Heron 129 
 Intramolecular respiration 355 
 Inulase 79, 80 
 
 ,, zymogen of 85 
 „ conditions of working 85 
 Inulin, distribution of 77 
 „ properties of 78, 82 
 „ decomposition-products of 83, 
 84 
 Invertan 481 
 Invertase 111 
 
 „ discovered by Berthelot 112 
 
 „ distribution of 113, 117, 118 
 
 „ extraction of 123 
 
 ,, action of 124 
 
 „ conditions of action 124 
 
 ,, relations to temperature 125 
 
 „ effect of heating in presence 
 
 of cane-sugar 126 
 „ action on other polysaccha^ 
 rides than cane-sugar 127 
 „ nature of 429 
 Invertin 111, 115 
 
 ,, distributionof 115, 122. Also 
 see Invertase 
 Invert-sugar 111 
 Isomaltose (of Fischer) 56 
 „ (of Lintner) 56 
 „ (of Ling and Baker) 57 
 
 „ (of Brown and Morris) 57 
 
INDEX. 
 
 509 
 
 Jalapin 153 
 
 Kephir 195, 340, 341 
 Khuther 169 
 Koji 345 
 Koumiss 840 
 
 Lab 251 
 Laccase 309 
 
 ,, distribution of 313, 315 
 
 ,, association with manganese 
 314 
 Laoool 311 
 Lactase 111, 144 
 Lactic fermentation 367 
 Lactose 144 
 
 „ oxidationofbyenzymeSSl, 369 
 Leaven, action of 2 
 Leech extract, action of 271 
 Leguminosse, richness in diastase 21 
 Lencin 174 
 Leucodextrin 60 
 Lencomaines 390 
 Leueosin 223, 422, 424 
 Linamarin 170 
 Linum, enzyme in 170 
 Lipase 236 
 
 „ vegetable 242 
 Liver enzymes 39, 41 
 Liver of Crustacea and Mollusca a 
 
 hepato-panoreas 43, 198 
 Lotase 169 
 Lotoflavin 170 
 Lotusin 170 
 Lysatinin 199 
 Lysin 199 
 
 Malic acid fermentation 373 
 Maltase (of Wijsman) 60. Also see 
 
 Glucase 
 Malto-dextrin (Herzfeld) 51 
 
 „ (Brown and Morris) 52, 
 
 53 
 Maltose, the ultimate product of dias- 
 tasio action 50, 51, 54, 35, 
 58, 59, 63 
 „ bi-rotation of 53 
 „ reactions of 55, 129 
 
 Maltose, inhibitory effect of on hydro- 
 lysis of starch 72 
 „ fate of in animal body 128 
 „ products of its hydrolysis 129 
 
 Manganese, relation of to laccase 314 
 
 Manna 143 
 
 Mannitol, fermentation of 378 
 
 Melibiase, see BafQnase 
 
 Melibiose 141 
 
 Melizitase 111, 142 
 
 Melizitose 143 
 
 Metacasein 201, 259 
 
 Metamaltose 54 
 
 Metapectic acid 87, 88 
 
 ,, relation to arabinose 89 
 
 Metapectine 88, 289 
 
 Metapeptone 175, 176 
 
 Methyl-glucosides 451 
 
 Methyl-salicylic acid 166 
 
 Middle lamella 89 
 
 ,, nature of 298 
 
 Morozymase 114 
 
 Mulberry, enzymes in 114 
 
 Myo-albumin 285 
 
 Myoglobuhn 285 
 
 Myosin-ferment 283 
 
 Myosinogen 278, 285 
 
 Myrosiu 147, 155 
 
 ,, special secreting cells 157 
 „ localisation of 156 
 ,, actions of 160 
 , , chemical and physical proper- 
 ties of 160 
 „ action of reagents on 161 
 „ suggested action on oU 171 
 „ Guignard's reaction for 420 
 
 Myxomycetes, enzyme in 230 
 
 Nepenthes, enzyme in 227 
 Neuridine 390 
 Nitria' fermentation 380 
 Nucleo-histon 279 
 Nucleo-proteids 194 
 
 „ digestion of by pepsin 
 
 194 
 
 , , relation to coagulation 
 
 of blood 279 
 
 ,, ,, to glycolysis 330 
 
510 
 
 INDEX. 
 
 Nucleo-proteids, relation of enzymee to 
 
 433 
 Nucleus, its relation to secretion 405 
 
 (Enoxydase 320 
 
 „ temperature relations 822 
 
 Oil of winter-green 166 
 Optimum point 13 
 Organised ferments 391 
 Oxalic fermentation 373 
 Oxidases 308 
 
 „ relation to bouquet of wines 
 
 325 
 „ action on mUk-sngar 331 
 Oxycelluloses 87 
 
 „ hydrolysis of 103 
 
 Oxy-urnshic acid 310 
 
 Palms, cytase in 99 
 Pancreas, relation to glycolysis 329 
 Pancreatic juice, enzymes in 195 
 „ process of secretion 203 
 „ proportion of enzymes influenced 
 
 by diet 232 
 „ action in digestion of fat 236 
 „ action on milk 201 
 Papain, occurrence 214 
 
 „ conditions of action 215 
 „ course of action 216 
 Paraglobulin 268 
 Paramyosinogen 285 
 Parapectine 88, 289 
 Parapeptone 175, 176 
 
 „ resemblance to antial- 
 
 bumose 179 
 Pectase 287 
 
 ,, relations to calcium 290 
 „ to barium and strontium 292 
 ,, influence of reaction on work- 
 ing 292 
 „ distribution of 293 
 Pectic acid 87, 88, 287, 289, 297 
 Pectic compounds, reactions of 288 
 Pectinase 104 
 Pectinate 292 
 Pectine 87, 88, 287, 289 
 Peotoses 87, 88, 287 
 
 „ products of hydrolysis of 103 
 
 Pectosic acid 287 
 
 Fenicillium, enzymes in — diastase 32 
 „ emulsin 151, 153 
 
 „ trypsin 230 
 
 „ lipase 248 
 
 Pepsin, discovered by Schwann 6 
 prepared by Briicke 7 
 points of difference from 
 
 tiypsin 99, 186, 205 
 modes of preparation of 187 
 distribution of 189 
 conditions of working of 191 
 relations to temperature 191 
 effect of various reagents on 
 
 192 
 course of action 193 
 reactions of 427 
 Pepsinogen 409, 417 
 Peptogens 205 
 
 Peptone, influence on diastasic action 
 68 
 „ (of Meissner) 176 
 „ action of dehydrating agents 
 on 184 
 Peziza, cytase in 90 
 Phagocytes 367 
 Phenol 387 
 Phillyrin 170 
 Phloretin 148 
 Phlorizin 148, 153 
 
 Phosphates, influence on diastase 70 
 Pialyn 237 
 
 Pine-apple juice, proteids of 210 
 Pinguicula, enzyme in 229 
 Plasmase 266 
 Plasmine 267, 277 
 Plasmosomata 407 
 Polyporus, diastase in 23 
 
 „ emulsin in 158 
 
 Polysaccharides 110, 336 
 
 ,, condition of ferment- 
 
 ation of 336 
 Populin 153, 170 
 Presuxe 251 
 
 Propionic fermentation 371 
 Protamines 200 
 
 Proteid, decomposition by acids 173 
 )i „ by enzymes 175 
 
INDEX. 
 
 511 
 
 Proteid, decomposition by water heated 
 under pressure 183 
 „ relation to peptone 184 
 Proteinoohromogen 200 
 Proteolysis, Kuhne's schemes of 174, 178 
 „ „ modification of these 
 
 by Neumeister 181 
 „ compared with hydrolysis 
 
 of starch 182 
 „ objections to Eiihne's 
 
 views of 184 
 „ course of in cereals 222 
 
 Proteolytic enzymes, course of secre- 
 tion of 203 
 „ existence in bacteria 231, 232 
 Proteoses 180 
 
 „ mode of separation from 
 
 peptone 180 
 „ stages in hydrolysis of pro- 
 
 teids 182 
 „ comparative powers of dia- 
 lysis of 182 
 Prothrombin 274 
 
 Protoplasm, the active agent in fer- 
 mentation 5 
 „ theory of arrangement 7 
 
 „ properties of 7 
 
 Proto-proteose, reactions of 180 
 Prozymogen 407 
 Ptomaines 387, 390 
 Ptyalin 15, 16 
 Ptyalose 55 
 Putrefaction 887 
 Putrescine 390 
 Pyrogallol, action of laecase on 312 
 
 Baffinase 111 
 Eafanose 127, 141 
 Baggi 346 
 
 Eeactions of enzymes, with orcin 419 
 with tincture of 
 guaiacum 420 
 Beducing enzymes 331 
 Bennet 195, 250 
 
 ,, source of 252 
 
 , preparation of 252 
 
 ,, reactions of 254 
 
 , efiect of various reagents on 255 
 
 Eennet, influence of calcium salts 256 
 „ theories of action 258, 264 
 „ peculiarities of pancreatic 
 rennet 259 ; differences from 
 gastric rennet 260 
 „ inhibition of action 261 
 „ (vegetable) 261 
 
 Respiratory enzymes 308 
 
 Eeverse action of enzymes 460, 461 
 
 Bhamnase 147, 165 
 
 Bhamnetin 165 
 
 Bhamniu 165 
 
 Bhamnose 165 
 
 Bhus, tannase in 169 
 
 Bubiadipin 168 
 
 Eubiafin 163 
 
 Bnbiagin 168 
 
 Bubian 162 
 
 Bubiretin 163 
 
 Saccharomyoes 4 
 Salicin 148 
 
 „ action of emulsin on 152 
 Salicylic aldehyde 148 
 Saligenin 148 
 
 Salivary glands, of mammals 35 
 „ of insects 43 
 
 jj of molluscs 44 
 
 ,, process of secretion 
 
 in 76 
 Salts, effect on diastasic action 66, 69 
 Saponifying enzymes 237 
 Scutellum, the seat of formation of 
 diastase 25 
 „ secretion in 401 
 
 Secretion, process of 37, 398 
 Seminase 107 
 Seminose 103 
 Sinalbin 160 
 Sinigrin 155 
 Skatol 387 
 Solanin 158 
 
 Sponges, enzyme in 197 
 Steapsin 237 
 Stomata, action of diastase in opening 
 
 76 
 Succinic acid, formation of in alcoholic 
 fermentation 4 
 
512 
 
 INDEX. 
 
 Sugars, conetituiion of 110 
 
 „ formation from fat 245 
 Synaptase, see Emulsin 
 
 Taka 24 
 
 Tamus, oytase in 98 
 
 Tannase 168 
 
 Tannin, relations to diastase 71 
 
 „ decomposition by tannase 168 
 ,, oxidation of 324 
 Thrombase 266, 268 
 
 „ time of formation 269 
 „ nature of 269, 271 
 , , action of calcium salts on 272 
 „ influenced by other neutral 
 
 salts 273 
 ,, sources of 278 
 Thrombin 266 
 Thrombosin 279 
 Tissue fibrinogens 279, 415 
 Torula urese 302 
 Touranose 143 
 Trehala 138 
 Trehalase 111, 136 
 
 „ mode of preparation of 137 
 „ conditions of action 138 
 „ difierencesfrom glucase 139 
 „ distribution of 140 
 Trehalose 136 
 
 Trypsin, points of difference from 
 pepsin 186, 195, 199, 205 
 preparation of 193 
 distribution of 197 
 conditions of action 198 
 (vegetable) 209, 219, 222, 224 
 action of 220 
 
 identity of animal and vege- 
 table trypsins 234 
 Trypsinogen 410 
 Tryptophane 200 
 Tyrosin 174 
 Tyrosinase 317 
 
 „ mode of separation from 
 
 laccase 319 
 „ temperature relations 319 
 
 Uraster, enzyme in 197 
 
 Urease 301 
 
 ,, discovery of 303 
 
 „ action on urea 303 
 
 „ action on asparagin 307 
 
 Urine, fermentation of 301 
 
 Umshi 309 
 
 Umshio acid 309 
 
 Verantin 163 
 Vesicnlase 286 
 Yiscons fermentation 384 
 ViteUin, digestion of 181 
 
 Worms, enzyme in 197 
 
 Xanthine 162 
 Xanthorhamnin 165 
 
 Teast, nature of 3, 4 
 
 diastase In 24 
 
 excretion of invertase by 127 
 
 glucase in 135 
 
 rafiSnase in 142 
 
 lactase in 145 
 
 trypsin in 230 
 Yeast-extract, inverting action of 6 
 
 Zymase (of Bechamp) 114 
 „ (of Buchner) 359 
 ,, in fruits 361 
 Zymogen 38, 405 
 
 „ of inulase 85, 414 
 
 „ contained in resting glands 
 
 204 
 „ of vegetable trypsin 221, 
 
 414 
 „ of thrombase 274, 415 
 „ of pepsin 408 
 „ of rennet 410, 414 
 „ relation to nucleus 405 
 „ of saliva 410 
 „ effect of light on 412 
 ,, of lipase 414 
 Zythozymase 114 
 
 CAMBRIDGE : PBINTED BY J. & 0. F, CIiAY, AT THE UNrVERSITY PBESS. 
 
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