iJii^. %^iW:^i^l ■<■ ■■ ., z f^A s:ft-7i^f:=:^** ■■'^x 3i' ^')i*%^' ./ •wi' ■^ ^ -ifi.'^'y'^' •;.<& -v Digitized by tine Internet Arcliive in 2010 witli funding from Open Knowledge Commons (for the Medical Heritage Library project) http://www.archive.org/details/ondigestiveproteOOchit CARTWRIGHT LECTURES 1894 ON DIGESTIVE PROTEOLYSIS BEING THE CARTWRIGHT LECTURES FOR 1894 DELIVERED BEFORE THE ALUMNI ASSOCIATION OE THE COLLEGE OF PHYSICIANS AND SURGEONS OF NEW YORK, K H. CHITTENDEN, Ph.D. Professor of Physiological Chemistry in Yale University NEW HAVEN, CONN. : TUTTLE, MOEEHOUSE & TAYLOR, PUBLISHERS. 1895 THE TUTTLE, MOREHOUSE & TAYLOR PRESS, NEW HAVEN, CONN. PREFACE The present volume, as explained by the title, consists mainly of a rejDi'int of the Cartwright Lectures for 1894. These lectures were originally printed in the current num" bers of the Medical Kecord, but so many requests have been made for their publication in a more convenient and accessible form that they are now re-issued, through the courtesy of the publishers of the Record, in book-form. It is hoped that these lectures may prove of value not only in calling attention to some of the fundamental chemico-physiological facts of digestion, but in stimulating closer investigation of the many questions which are so intimately associated with a j^roper understanding of the processes concerned in the digestion and utilization of the proteid food-stuffs. E. H. CHITTEISTDEE". COISTTENTS. LECTUEE I. The general nature of Proteolytio Enzymes and of Proteicls. Introductory observations, ....... Early history of gastric digestion, .... The proteolytic power of the pancreatic juice, . The general nature of proteolytic enzymes, .... Origin of proteolytic enzymes, Preparation of pepsin, ...... Eeactions and composition of proteolytic enzymes, . The proteid nature of enzymes, ..... Conditions modifying the action of enzymes, The influence of temperature on proteolytic action, . The influence of acids, alkalies, and other substances on the activity of enzymes. Action of chloroform on pepsin, Theories of enzyme action with special reference to catalysis, ..... The general nature of Proteids, Classification of proteids, . Chemical composition of some of the more teids occurring in nature, Chemical constitution of proteids, prominent pro 10 13 15 17 18 20 31 33 37 39 31 33 X CONTENTS. The general nature of Proteids : — The presence of hemi- and anti-groups in all typical , , proteids, 34 Cleavage of the albumin-molecule with dilute sulphuric acid, ". 34 Hydration and cleavage of albumin by the action of super- heated water, with formation of atmid-albtimoses, etc. , 37 Action of powerful hydrolytic agents on proteid matter, 39 Initial action of pepsin-acid on proteids, .... 40 Scheme of the general line of proteolysis as it occurs in pepsin -digestion, with a view to the structure of the albumin-molecule, . 41 CONTENTS. XI LECTUKE II. Proteolysis hy pepsm-hydrochloriG acid, with a considera- tion of the general nature of proteoses and peptones. Proteolysis by pepsin-acid, ........ 44 Formation of hydrochloric acid in the gastric glands, . 45 Liebermann's theory regarding the formation of the acid of the gastric juice, ....... 46 Differences in the action of free and combined acid, . . 47 Proteolysis in the presence of combined acid, ... 49 The combining power of various forms of proteid matter with hydrochloric acid, ...... 51 Quantitative estimation of the affinity of the products of digestion for acid, 53 Richet's theory regarding the conjugate character of the acid of the gastric juice, ...... 54 Proteolysis in the presence of amido-acids, ... 55 Necessity for knowing the amount of combined acid in the stomach-contents, ....... 57 Antiseptic action of the hydrochloric acid of the gastric juice, .......... 58 The maximum action of pepsin exerted only in the presence of free hydrochloric acid, . . . . . 59 Division of the products of pepsin-proteolysis into three main groups, ........ 60 Detection of the products of digestion, .... 61 Separation of proteoses and xaeptones from a digestive mix- ture or from the stomach-contents, .... 62 Some of the chemical properties of peptones, . . . 64 The so-called propeptone a mixture of proteoses, . . 65 XU CONTENTS. Proteolysis by pepsin-acid : — Pepsin-proteolysis synonomoiis with a series of progressive hydrolytic changes, 66 Chemical composition of proteoses and peptones, . . 67 Pepsin-proteolysis a true hydrolytic and cleavage process, 71 Schutzenberger's results on the formation of fibrin-peptone, 72 Amphopeptones the final products of gastric digestion, but proteolysis never results in complete peptonization, 73 Solution of a proteid by pepsin-acid not synonymous with peptonization, 75 Influence of the removal of the products of digestion on the activity of the ferment, 75 Lack of complete peptonization by pepsin-acid not due to accumulation of the products of digestion, . . 76 The diffusibility of proteoses and peptones, ... 77 Absorption of peptones from the living stomach, . . 79 Differences between natural digestion in the stomach and artificial proteolysis, ....... 80 Eelative formation of proteoses and peptones in the living stomach, ......... 81 Gastric digestion merely a preliminary step in proteolysis, 81 Intestinal digestion alone capable of accomplishing all that is necessary for the complete nourishment of an animal, 83 Some physiological properties of proteoses and peptones, . . 83 The experiments of Schmidt-Miilheim and Fano on the action of peptones when injected into the blood, . 84 Physiological action of albumoses, ..... 85 Introduction of albumoses into the blood, ... 87 Proteose-like nature of the poisons produced by bacteria, . 89 The acrooalbumoses formed by the tubercle-bacillus, . 90 Toxic nature of proteoses and peptones, .... 91 CONTENTS. XIU LECTURE III. Proteolysis hy trypsin — Absorption of the main products of proteolysis. Proteolysis by trypsin, . 93 Comparison of pepsin and trypsin, ..... 94 Trypsin especially a peptone-forming ferment, . . 95 The primary products of trypsin-proteolysis, ... 95 Scheme of trypsin-digestion, showing the relationship of the products formed, ....... 96 The fate of hemi-gronps in trypsin-proteolysis, . . 97 The primary products of trypsin-digestion mainly anti- bodies, 98 Character and composition of antipeptones, ... 99 Antialbumid as a product of pancreatic digestion, . . 100 The peculiar action of trypsin in the formation of amido- acids, etc., . , . . . . . . . 101 Formation of lysin and ly satin in pancreatic digestion, . 103 The relationship of lysatin to urea, 105 Formation of tryptophan or proteinochromogen by trypsin, 105 Appearance of ammonia in trypsin-proteolysis, . . 107 Eelationship between artificial pancreatic digestion and proteolysis in the living intestine, . . . . 109 Leucin and tyrosin products of the natural pancreatic digestion in the intestine, ...... 112 The physiological significance of leucin and tyrosin, . 113 Absorption of the main products of proteolysis .... 116 Absorption of acid-albumin, alkali-albuminate, etc., . 117 Absorption limited mainly to the intestine, very little absorption from the stomach, . . . . . 119 XIV CONTENTS. Absorption of the main products of proteolysis : — The change which the primary products of proteolysis undergo in the process of absorption . . . . Peptones not present in the circulating blood . The change which peptones and proteoses undergo by con- tact with the living mucous membrane of the small intestine, Eetrogression of peptones by contact with other living cells, etc., . . . . . . . . Functional activity of leucocytes in absorption. Digestive leucocytosis incited by nuclein, . Shore's experiments on the ability of lymph-cells to assimi late either proteoses or peptones, Lymph a true secretion from the blood-vessels. Direct excitatory effect of peptones when present in the blood on the endothelial cells, .... Selective activity of endothelial cells, 120 121 122 125 128 131 133 134 136 137 DIGESTIVE PROTEOLYSIS LECTURE I. THE GENEEAL NATURE OF PROTEOLYTIC ENZYMES AND OF PROTEIDS. INTEODtrCTORY. In digestive proteolysis we have a branch of physiological study which of late years has made much progress. Chem- istry has come to the aid of physiology and by the combined efforts of the two our knowledge of the digestive processes of the alimentary tract has been gradually broadened and deepened. That which at one time appeared simple has become complex, but increasing knowledge has brought not only recognition of existing complexity, but has enabled us, in part at least, to unravel it. By digestive proteolysis is to be understood the trans- formation of the proteid food-stuffs into more or less soluble and diffusible products through the agency of the digestive juices, or more especially through the activity of the so- called proteolytic ferments or enzymes contained therein ; changes which plainly have for their object a readier and more complete utilization of the proteid foods by the system. In selecting this topic as the subject for this series of Cartwright Lectures I have been influenced especially by the opinion that both for the physiologist and the physician there are few processes going on in the animal body of greater importance than those classed under the head of digestion. Further, few processes are less understood than those concerned in this broad question of digestive prote- olysis, especially those which relate specifically to the diges- tion of the various classes of proteid food-stuffs, and to the 2 "Ji DIGESTIVE PE0TE0LY8IS. absorption and utilization of tlie several products formed. Moreover, the subject lias ever bad forme a strong attraction- as presenting a field of investigation where chemical work can advantageously aid in the advance of sound physiologi- cal knowledge ; and certainly every line of advance in our understanding of the normal processes of the body paves the way for a better and clearer comprehension of the pathological or abnormal processes to which the human body is subject. You will pardon me if I specially emphasize in this connection the fact that advance along the present lines was not rapid until physiologists began to appreciate the impor- tance of investigating the chemico-physiological problems of digestion by accurate chemical methods. Something more than simple test-tube study, or even experimental work on animals, is required in dealing with the changes which complex proteids undergo in gastric and pancreatic diges- tion. The nature and chemical composition of the proteids undergoing digestion, as well as of the resultant products, are necessary preliminaries to any rightful interpretation of the changes accompanying digestive proteolysis ; but physiology has been slow to appreciate the significance of this fact, and, until recently, has done very little to remedy the noticeable lack of accurate knowledge regarding the composition and nature of the proteid and albuminoid sub- stances which play such an important part in the life-history of the human organism, either as food or as vital constituents of the physiologically active and inactive tissues. This is to be greatly deprecated, since our understanding of the nature of proteolysis, of the mode of action of the enzymes or ferments involved, and of the relationships of the prod- ucts formed, is dependent mainly upon an accurate deter- mination of the exact changes in chemical composition which accompany each step in the proteolytic process. EAELT HISTOEY OF GASTRIC DIGESTION. 6 How otherwise can we hope to attain a proper appreciation of the real points of difference between bodies so closely related as those composing the large group of proteids and albuminoids ? Surely, in no other way can we measure the nature or extent of the changes involved in the various phases of proteolysis than by a thorough study of chemical composition and constitution, as well as of chemical reactions and general properties. In the early history of physiology there was, quite nat- urally, little or no thought given to the nature of proteolytic changes. The gastric juice, as one of the first digestive fluids to be studied, was recognized as a kind of universal solvent for all varieties of food-stuffs, and this even long before anything was known regarding its composition, but beyond this point knowledge did not extend. Active study of the gastric juice, as you well know, dates from 1Y83, when the brilliant Italian investigator Spallanzani com- menced his work on digestion. The names of Carminati, Werner and Montegre' are also associated with various phases of work and speculation in this early history of the subject, especially those which pertained to the possible presence of acid in the stomach juices. In 1824, however, Prout showed conclusively that gastric juice was truly acid, and, moreover, that the acidity was due to the presence of free hydrochloric acid, and not to an organic acid. Still, many observations failed to show the presence of an acid fluid in the stomach, and it was not until Tiedemann and Gmelin's^ masterly researches were published that the cause of this discrepancy was made clear. It was then seen that the secretion of an acid gastric juice was dependent upon stimulation or irritation of the mucous membrane of the ^ See Berzelius's Lehrbuch der Chemie, Band 9, p. 205, 4te Auflage, for an account of these early discoveries. ^ Tiedemann und Gmelin : Die Verdauung nach Versuchen. Heidel- berg und Leipzig. 1826. 4 DIGESTIVE PEOTEOLYSIS. stomach, and that so long as the stomach was free from food . or other matter capable of stimulating the mucosa, if con- tained very little fluid, and that neutral or very slightly acid in reaction. These early observers also recorded the fact that the amount or strength of acid increased "with the outpouring of the secretion, incidental to natural or artificial stimulation, thus giving a hint of the now well-known fact that any and every secretion may show variations in com- position incidental to the character and extent of the stimu- lation which calls it forth. The period between 1825 and 1833 was characterized especially by the presentation of the many results bearing on gastric digestion obtained by Dr. Beaumont on Alexis St. Martin, followed a little later, in 1842, by a long period of experimentation by many physiologists, as Blondlot,* Bassow,^ Bardeleben,^ Bernard,* Bidder and Schmidt,^ and many others on methods of establishing gastric fistulas on animals, by which many interesting results were accumu- lated regarding the physiology of gastric digestion. Up to 1834, however, there was no adequate explanation offered of the solvent power of the stomach juice ; aside from the presence of hydrochloric acid, nothing could be discovered by the earlier chemists to account for the remarkable diges- tive action. Eberle,^ however, attributed to the mucous membrane of the stomach a catalytic action, and claimed that it only needed the presence of a small piece of the stomach mucosa with weak hydrochloric acid for the mani- festation of solvent or digestive power. It remained for Schwann,' to show the true explanation of this phenomenon, ^ Traite analytique de la Digestion. Paris, 1842. '^ Bulletin de la Societe des Naturalistes de Moscou, vol. 16. 1842. ^ Archiv fiir physiol. Heilkunde, vol. 8. 1849. * Lecons de Physiologie de la Digestion. Paris, 1867. ^ Die Verdauungssafte. • Physiologie der Verdauung. Wiirzburg, 1834. '' Ueber das Wesen der Verdauungsprocesse. Miiller's Archiv, 1836, p. 90. EABLT HISTOET OF GASTRIC DIGESTIOK. 5 and althougli lie was unable to make a complete separation of the active principle which he plainly believed existed, he gave to it the name of pepsin. Wassmann, PajDpenheim/ Valentin, and later Elsasser,* all endeavored to obtain the substance in a pure form, and Wassmann,' in 1839, surely succeeded in obtaining a very active preparation of the fer- ment — one capable of exerting marked digestive action when mixed with a little dilute acid. Thus, a true under- standing of the general nature of gastric juice was finally arrived at, and the cause of its digestive power was right- fully attributed to the presence of the ferment pepsin and the dilute acid. Further, the analysis of human gastric juice made by Berzelius,* in 1834, showed that the secretion contains very little solid matter (1.26 per cent.), thus call- ing attention to the fact that the digestive power of this fluid is out of all proportion to the amount of pepsin, or even to the amount of total solid matter present, and consequently paving the way for a general appreciation of the peculiar nature of the dominant body, i.e., the pepsin. The original conception regarding the manner in which gastric juice exerts its solvent power on proteid foods was apparently limited to simple solution ; chemical solution if you choose, brought about by catalytic action, but without any hint as to the possible nature of the soluble products formed. Mialhe," however, recognized the fact that this transformation, by which insoluble and non-diffusible pro- teid matter was converted into a soluble and diffusible prod- uct, was a form of hydration, comparable to the change of insoluble starch into soluble sugar, and he named the hypothetical product albuminose. Mialhe's study of the ^ Zur Kenntniss d. Verdauung. Breslau, 1839. '^ Magenerweichung der Sauglinge. Stuttgart und Tiibingen, 1846. 2 Lehmann's Lehrbuch. d. physiol. Chem., Band 3, p. 41, 2te Auflage. ^Lehrbuch der Chemie, Band 9, p. 209. ^Canstatt's Jahresbericht d. Pharm., 1846, p. 163. 6 DIGESTIVE PROTEOLYSIS. matter in 1846 Avas followed by Lelimann's ^ investigation of the subject, and the coining of the word peptones g-s an • appropriate name for the soluble products of gastric diges- tion. The peptones isolated by Lehmann were described as amorphous, tasteless substances, soluble in water in all pro- portions and insoluble in alcohol. They were likewise pre- cipitated by tannic acid, mercuric chloride, and lead acetate, and were considered as weak acid bodies, having the power of combining with bases to form salts of a more or less indefinite character. Twelve years later, in 1858, Mulder^ gave a more complete description of peptones, but his study of the subject failed to advance materially our knowledge of the broader questions regarding the nature of the proc- ess, or processes, by which the so-called peptones were formed. A year later, in 1859, Meissner' brought forward the first of his contributions, and during the following three or four years several communications were made representing the work of himself and pupils upon the ques- tion of gastric digestion, or more especially upon the character of the products resulting from the digestive action of pepsin-hydrochloric acid. The general tenor of Meissner's results is shown in the description of a row of products as characteristic of the proteolytic action of pepsin-acid on proteid matter. In other words, there was a clear recognition of the fact that proteid digestion in the stomach, through the agency of the ferment pepsin, is something more than a simple conver- sion of the proteid into one or two soluble products. The several bodies then isolated were named parapeptone, metapeptone, dyspeptone, «2, /9, and ;' peptone ; names now seldom used, but significant as showing that at this early ' Lehmann's Physiologisclie Cheinie, Band 2, p. 318. ^Archiv f. d. Holland. Beitr., 2, 1858. sZeltschr. f. rat. Med., Band 7, 8, 10 und 14. PROTEOLYTIC POWER OF PANCREATIC JUICE. 7 date there was a full appreciation of the fact that digestive proteolysis as accomplished by the ferment pepsin is an intricate process, accompanied by the formation of a series of products which vary more or less with the conditions under which the digestion is conducted. This was the commencement of our more modern ideas regarding digestive proteolysis, but only the commence- ment, for it ushered in an era of unparalleled activity, in which Briicke, Schiitzenberger, and Kiihne each contrib- uted a large share toward the successful interpretation of the results obtained. Further, knowledge regarding the proteid-digesting power of the pancreatic juice was rapidly accumulating, thus broadening our ideas regarding diges- tive proteolysis in general. Corvisart' had called attention to the proteolytic power of the pancreatic juice in 1857, and although his observations were more or less generally discredited for a time, they were eventually confirmed by Meissner,' Schifi, Danilewsky,' and Kiihne,* the latter particularly contributing greatly to the development of our knowledge concerning this phase of digestive proteolysis. The proteolytic power was proved to be due to a specific ferment or enzyme, now universally called trypsin, which digests proteid foods to the best advantage in the presence of sodium carbonate. Digestive proteolysis in the human body was thus shown to be due mainly to the presence of two distinct enzymes, the one active in an acid fluid, the gastric juice, the other in an alkaline-reacting fluid, the pancreatic juice, but both endowed with the power of digesting all varieties of proteid foods, with the formation of a large number of more or less closely related products. ' Sur Tine Fonction pen connue du Pancreas : La digestion des aliments azotes. Paris, 1857-58. '^ Verdauung der Eiweisskorper dnrch den pankreatischen Saft. Zeit- schr. f. rat. Med., 3d ser.. Band 7, p. 17. 1859. sVirchow's Archiv, Band 25, p. 267. 1862. ^Ibid., Band 39, p. 130. 1867. O DIGESTIVE PEOTEOLYSIS. So much for tlie early history of our subject, and now, without attempting any exhaustive sketch of its gradual development during the last decade and a half, allow me to present to you digestive proteolysis as it stands to-day, developed somewhat, I trust, by the results I have been able to contribute to it during the last twelve years. THE GENEEAL NATUEE OF PEOTEOLYTIC ENZYMES. These peculiar bodies owe their origin to the constructive power of the gland-cells from which the respective secre- tions are derived. During fasting, the epithelial cells of the gastric glands and of the pancreas manufacture from the cell-protoplasm a specific zymogen or ferment-antece- dent, which is stored up in the cell in the form of granules. These granules of either pepsinogen or trypsinogen, as the case may be, are during secretion apparently drawn uj^on for the production of the ferment, and it is an easy matter to verify Langley's' observation that the amount of pepsin, for example, obtainable from a definite weight of the gland- bearing mucous membrane is proportionate to the number of granules contained in the gland-cells. During ordinary secretion, however, these granules of zymogen do not entirely disappear from the cell. When secretion com- mences and the granules are drawn upon for the produc- tion of ferment, fresh granules are formed, and inasmuch as these latter are produced through the katabolism of the cell-protoplasm it follows that anabolic processes must be simultaneously going on in the cell, by which new cell- protoplasm is constructed. Hence, as Heidenhain, Lang- ley, and others have pointed out, during digestion there are at least three distinct processes going on side by side in ' Proceedings of tlie Eoyal Society, vol. 32, p. 20 ; On the Histology and Physiology of the Pepsin-forming Glands. ORIGIN OF PEOTEOLYTIC ENZYMES. 9 the gland-cell, viz., the conversion of the zymogen stored up in the cell into the active ferment, or other secretory products, the growth of new cell-protoplasm, and the attendant formation of fresh zymogen to replace, or par- tially replace, that used up in the production of the fer- ment. Consequently, we are to understand that in the liv- ing mucous membrane of the stomach there is little or no preformed pepsin present. Similarly, the cells of the pan- creatic gland are practically free from the ferment trypsin. In both cases the cell-protoplasm stores up zymogen and not the active ferment, but at the moment of secretion the zymogen is transformed into ferment and possibly other organic substances characteristic of the fluid secreted. Absorption of the products of digestion tends to increase the activity of the secreting cells, but we have no tangible proof that any particular kinds of food are directly pep- togenous, i.e., that they lead to a storing up in the gastric cells, for example, of pepsinogen, although it may be that the so-called peptogenous foods give rise to a more active conversion of pepsinogen into pepsin,' As already stated, the zymogen is manufactured directly from the cell-proto- plasm, and the constructive power is certainly not directly controlled by the character of the food ingested. All this in one sense is to-day ancient history, but I recall it to your minds in order to emphasize the fact that these two energetic ferments or enzymes stand in close relation to the protoplasm of the cell from which they originate. So far as we can measure the transformations involved, there are only two distinct steps in the process, viz., the formation of the inactive zymogen stored up in the cell, and the conversion of the antecedent body into the soluble and active ferment. In this connection Pod- ^ Langley and Edkins : Pepsinogen and Pepsin. Journal of Phys- iology, vol. 7, p. 394. 10 DIGESTIVE PROTEOLYSIS. wyssozki ' has reported that the mucous membrane of the stomach exposed to the action of oxygen gas show-s a marked mcrease in the amount of pepsin, from which he infers that the natural conversion of pepsinogen into pepsin is an oxidation process. Further, he claims the existence of at least two forms of pepsinogen in the stomach mucosa, one closely akin to the ferment itself and very easily soluble in glycerin, while the other is more insoluble in this menstruum. Langley and Edkins,^ however, find that oxygen has no effect whatever on the pepsinogen of the frog's mucous membrane, thus throwing doubt on the above conclusion. Still, Podolinski' claims that tryp- sin originates from its particular zymogen through a process of oxidation, and Herzen* has proved that the ferment can be reconverted into trypsinogen under the influence of carbon-monoxide and again transformed into the ferment by contact with oxygen gas. This latter observer^ has also noticed a connection between the amount of trypsin obtainable from the pancreas and the dilatation of the spleen, from which he was eventually led to conclude that the spleen during its dilatation gives birth to a zymogen-transforming ferment which thus leads to the production of trypsin, presumably from the already manu- factured zymogen. In any event, their peculiar origin lends favor to the view that these two enzymes are closely allied to proteid bodies, and that they are directly derived from the albuminous portion of the cell-protoplasm. Analysis shows that they always contain nitrogen in fairly 1 Pflxiger's ArcMv f. Physiol., Band 39, p. 68. ^ Journal of Physiol., vol. 7, p. 400. s Pfliiger's Archiv f. Physiol., Band 13, p. 422. 4 Ueber den Euckschlag des Trypsins zu Zymogen unter dem Einfliiss der Kohlenoxydvergiftungen. Pfliiger's Archiv f. Physiol., Band 80, p. 308. ^ Ueber den Einfluss der Milz auf die Bildnng des Trypsins. Pfliiger's Archiv f. Physiol., Band 30, p. 295. PEEPAKATION OF PEPSIN. 11 large amount, althougli the percentage is sometimes less than that f omid in a typical proteid body. It must be remembered, however, that in spite of oft- repeated attempts to obtain more definite knowledge regarding the composition of these 23roteolytic enzymes our efforts have been more or less baffled. We are confronted at the outset with the fact that no criterion of chemical purity exists, either in the way of chemical composition or of chemical reactions. The only standard of purity avail- able is the intensity of proteolytic action, but this is so dependent upon attendant circumstances that it is only partially helpful in forming an estimate of chemical purity. My own experiments in this direction, and they have been quite numerous, have convinced me that it is practically impossible to obtain a preparation of either j)epsin or trypsin at all active which does not show at least some proteid reactions. Furthermore, such samples of these two enzymes as I have analyzed have shown a composition closely akin to that of proteid bodies. I will not take time to go into all the details of my work in this direction, contenting myself here with the statement that the purest specimens of pepsin and trypsin I have been able to pre- pare have always shown their relationship to the proteid ■ bodies by responding to many of the typical proteid reac- tions, and their comjDOsition, though somewhat variable, has in the main substantiated this evident relationshijD. The most satisfactory method I have found for obtaining a comparatively pure preparation of pepsin, and one at the same time strongly active, is a modification of the method published some years ago by Kiihne and myself.^ The mucous membrane from the cardiac portion of a pig's stomach is dissected off and washed with water. The upper surface of the mucosa is then scraped with a knife ' Zeitschr. f. Biol., Band 22, p. 428. 12 DIGESTIVE PROTEOLYSIS. until at least half of the membrane is removed. These scrapings, containing the fragments of the peptic glands; are warmed at 40°C. with an abundance of 0.2 per cent, hydrochloric acid for ten to twelve days in order to trans- form all of the convertible albuminous matter into pep- tone. The solution is then freed from insoluble matter by filtration and immediately saturated with ammonium sul- phate, by which the pepsin, with some albumose, is pre- cipitated in the form of a more or less gummy, or semi-adherent mass. This is filtered off, washed with a saturated solution of ammonium sulphate and then dis- solved in 0.2 per cent, hydrochloric acid. The resultant solution is next dialyzed in running water until the ammo- nium salt is entirely removed, thymol being added to prevent putrefaction, after which the fluid is mixed with an equal volume of 0.4 per cent, hydrochloric acid and again warmed at 40° C. for several days. The ferment is then once more precipitated by saturation of the fluid with ammonium sulj^hate, the precipitate strained off, dissolved in 0.2 per cent, acid and again dialyzed in running water until the solution is entirely free from sulphate. The clear solution of the ferment obtained in this manner can then be concentrated at 40° C. in shallow dishes, and if desired the ferment obtained as a scaly residue. So pre- pared, the pepsin is certainly quite pure, that is compara- tively, and although it may contain some albumose, the latter must be very resistant to the action of the ferment ; indeed, pepsin is in many respects an albumose-like body itself. In any event, the enzyme prepared in this manner shows decided proteid reactions, and contains nitrogen corre- sponding more or less closely to the recognized composition of an albumose. My own belief, therefore, is that these enzymes, both pepsin and trypsin, are proteid bodies REACTIONS OF PEOTEOLYTIO ENZYMES. 13 closely related to the albumoses. Tliey are soluble in water and more or less soluble in glycerin ; at least glycerin will dissolve them from moist tissues, or from moist pre- cipitates containing them. Langley/ however, states, and perhaps justly, that we have no positive proof that either ferments or zymogens are soluble in pure strong glycerin, and that if they are soluble, it is extremely slowly. In dilute glycerin, however, these ferments dissolve readily, as we very well know. Furthermore, they are practically non-diffusible, and, like many albumoses, are precipitated in part by saturation with sodium chloride and completely on saturation with ammonium sulphate. When dissolved in water and heated above 80° C, these enzymes are decom]30sed to such an extent that their pro- teolytic power is totally destroyed. The amount of coagu- lum produced by heat, however, is comparatively small, though variable with different preparations. Thus with trypsin, Kiihne originally considered that boiling an aque- ous solution of the ferment would give rise to about twenty per cent, of coagulated proteid and eighty per cent, of peptone-like matter. With the purer preparations now obtainable there is apparently less coagulable matter present, and Loew ^ has succeeded in preparing from the pancreas of the ox a sample of trypsin containing 52.75 per cent, of carbon and 16.55 per cent, of nitrogen, and yield- ing only a small coagulum by heat. Loew considered the ferment to be a true peptone, but in view of our present knowledge regarding the albumoses, I think we are justi- fied in assuming it to be an albumose-like body rather than a true peptone. At the same time it may be well to again emphasize the fact that our only " means of determining ' Gamgee's Physiological Chemistry of the Animal Body, vol. 2, p. 4. 1893. ^ Ueber die chem.ische Natur der ungeformten Fermente. Pfliiger's Archiv f. Physiol., Band 27, p. 203. 14 DIGESTIVE PEOTEOLYSIS. the presence of an enzyme is that of ascertaining the change which it is able to bring about in other substances', and since the activity of the enzymes is extraordinarily great, a minute trace suffices to produce a marked effect. From this it follows that the purified enzymes which give distinct proteid reactions might merely consist of very small quantities of a true non-proteid enzyme, adherent to or mixed with a residue of inert proteid material." ^ This quotation gives expression to a possibility which we cer- tainly cannot ignore, but my own experiments lead me to believe firmly in the proteid nature of these two enzymes. Further, we find partial substantiation of this view in the results obtained by Wurtz ^ in his study of the vegetable proteolytic ferment papain, and in my own results from the study of the proteolytic ferment of pineapple juice/ Thus, Wurtz prepared from the juice of Carica papaya an. active sample of papain, and found it to contain on analysis about 16.Y per cent, of nitrogen and 52.6 per cent, of carbon, while the reactions of the product likewise testified to the proteid nature of the enzyme. Martin, too, has concluded from his study of papain that the ferment is at least associated with an albumose.* With the proteolytic ferment of pineapple juice my observations have led me to the following conclusions, viz., that the ferment is at least associated with a proteid body, more or less completely precipitable from a neutral solution by saturation with ammonium sulphate, sodium chloride, and magnesium sulphate. This body is soluble in water, and consequently is not precipitated by dialysis. It is further non-coagulable by long contact with strong ' Sheridan Lea : Chemical Basis of the Animal Body, p. 55. ^ Sur la papaine : Contribution k I'histoire des ferments solubles, Comptes Eendus, Tome 90, p. 1379. Ibid., Tome 91, p. 787. 3 On the Proteolytic Action of Bromelin, the Ferment of Pineapple Juice. Journal of Physiol., vol. 15, p. 349. * The Nature of Papain, etc. Journal of Physiol., vol. 6, p. 336. PKOTEID NATURE OF ENZYMES, 15 alcohol, and its aqueous solution is very incompletely pre- cipitated by lieat. Placing it in line with the known forms of albuminous bodies it is not far removed from proto- albumose or heteroalbumose, differing, however, from the latter in that it is soluble in water without the addition of sodium chloride. At the same time, it fails to show some of the typical albumose reactions, and verges toward the group of globulins. In any event, it shows many charac- teristic proteid reactions, and contains considerable nitro- gen, viz., 10.46 per cent., with 50.7 per cent, of carbon. Consequently, we may conclude that the chemical reactions and composition of the more typical proteolytic enzymes, both of animal and vegetable origin, all favor the view that they are proteid bodies not far removed from the albuminous matter of the cell-protoplasm. Further, the very nature of these substances and their mode of action strengthen the idea that they are not only derived from the albumin of the cell-protoplasm, but that they are closely related to it. One cannot fail to be impressed with the resemblance in functional power between the unformed ferments as a class and cell-proto- plasm. To what can we ascribe the particular functional power of each individual f ei'ment 1 Why, for example, does pepsin act on proteid matter only in the presence of acid, and trypsin to advantage only in the jDresence of alkalies ? Why does pepsin act only on joroteid matter, and ptyalin only on starch and dextrins ? Why does trypsin produce a different set of soluble products in the digestion of albumin than pepsin does ? Similarly, why is it that the cell-protoplasm of one class of cells gives rise to one variety of katabolic products, while the protoplasm of another class of cells, as in a different tissue or organ, man- ifests its activity along totally different lines ? The answer to both sets of questions is, I think, to be found in the 16 DIGESTIVE PROTEOLYSIS. chemical constitution of the cell-protoplasm on the one hand, and in the constitution of the individual enzymes oh the other. The varied functional power of the ferment is a heritage from the cell-protoplasm, and, as I have said, is suggestive of a close relationship between the enzjines and the living protoplasm from which they originate. We might, on purely theoretical grounds, consider that these unformed ferments are isomeric bodies all derived from different modifications of albumin and with a common gen- eral structure, but with individual differences due to the extent of the hypothetical polymerization which attends their formation. Whenever, owing to any cause, the activity of the fer- ment is destroyed, as when it is altered by heat, strong acids, or alkalies, then the death of the ferment is to be attributed to a change in its constitution ; the atoms in the molecule are rearranged, and as a result the peculiar fer- ment power is lost forever. The proteolytic power of these enzymes is therefore bound up in the chemical con- stitution of the bodies, and anything which tends to alter the latter immediately interferes with their proteolytic action. But how shall we explain the normal action of these peculiar bodies ? Intensely active, capable in them- selves of producing changes in large quantities of material without being destroyed, their mere presence under suita- ble conditions being all powerful to produce profound alterations, these enzymes play a peculiar part. Present in mere traces, they are able to transform many thousand times their weight of proteid matter into soluble and diffusible products. All that is essential is then- mere presence under suitable conditions, and strangely enough the causative agent itself appears to suffer no marked change from the reactions set up between the other sub- stances. ACTION OF ENZYMES. lY There are many theories extant to explain this peculiar method of chemical change, but few of them help us to any real understanding of the matter. These enzymes are typical catalytic or contact agents, and by their presence render possible marked changes in the character of the proteid or albuminoid matter with which they happen to be in contact. But the conditions under which the contact takes place exercise an important control over the activity of the ferment. Temperature, reaction, concentration of the fluid, presence or absence of various foreign substances, etc., all play a very important part in regulating and con- trolling the activity of these two proteolytic enzymes. In fact, as one looks over the large number of data which have gradually accumulated bearing upon this point, one is impressed with the great sensitiveness of these ferments toward even so-called indifferent substances. Their &-pe- cific activity appears to hinge primarily upon the existence of a certain s|)ecial environment, alterations of which may be attended with an utter loss of proteolytic j)Ower, or, in some less common cases, with a decided increase in the rate of digestive action. This constitutes one of the peculiar features of these proteolytic enzymes ; powerful to produce great changes, they are nevertheless subject to the influence of their surroundings in a way which testifles to their utter lack of stability. Furthermore, as you well know, conditions favorable for the action of the one fer- ment are absolutely unfavorable for the activity of the other, and indeed may even lead to its destruction. Thus, while pepsin requires for its activity the pres- ence of an acid, as 0.2 per cent. HCl, trypsin is com- pletely destroyed in such a medium. Again, trypsin exhibits its peculiar proteolytic power in the presence of sodium carbonate, a salt which has an immediate destruc- tive action upon pepsin. Hence, a medium which is favor- 3 18 DIGESTIVE PROTEOLYSIS. able for the action of tlie one ferment may be directly antagonistic to the action of the other. Another factor of great moment in determining the activity of these two enzymes is temperature. That which is most favorable for their action is 38° to 40° C, and any marked deviation from this temperature is attended by an immediate effect upon the proteolysis. Exposure to a low temperature simply retards proteolytic action, doubtless in the same manner that cold checks or retards other chem- ical changes. There is no destruction of the ferment, even on exposure to extreme cold, the enzyme being simply inactive for the time being. Exposure of either pepsin or trypsin to a high temperature, say 80° C, is quickly followed by a complete loss of j)roteolytic power, i. e., the ferment is destroyed. It is to be noticed, how- ever, that the destructive action of heat is greatly modified by the attendant circumstances. Thus, fairly pure trypsin, dissolved in 0.3 per cent, sodium carbonate, is completely destroyed on exposure to a temperature of 50° C. for five to six minutes, while a neutral or slightly acid solution of the pure enzyme is destroyed in five minutes by exposure to a temperature of 45° C. On the other hand, the pres- ence of inorganic salts and the products of digestion, such as albumoses, amjDhopeptone, and antipeptone, all tend to protect the trypsin somewhat from the destructive effects of high temperatures, so that in their presence the enzyme may be warmed to 60° C. before it shows any diminution in proteolytic power. Alkaline reaction, combined with the presence of salts and proteid, viz., just the conditions existent in the natural pancreatic secretion, constitute the best safeguard against the destructive action of heat, and under such conditions trypsin may be warmed to about 60° C. before it begins to suffer harm. But all this testi- fies in no uncertain way to the extreme sensitiveness of the DESTEUCTIVE ACTION OF HEAT, 19 ferment to changes in temperature ; a sensitiveness whicli manifests itself not only in diminished or retarded proteo- lytic action, but terminates in destruction of the ferment when the temperature rises beyond a certain point. Similarly, pepsin dissolved in 0.2 per cent, hydrochloric acid feels the destructive effect of heat when a temperature of 60° C. is reached. In a neutral solution, on the other hand, destruction of the ferment may be complete at 55° C. Here, too, peptone retards very noticeably the destructive action of heat, especially in an acid solution of pepsin, so that under such circumstances the ferment may not be affected until the temperature reaches Y0° C. I have tried many experiments along this line, not only with pepsin and trypsin, but also with many other ferments. We may briefly summarize, however, all that is necessary for us to consider here in the statement that the |)ure isolated ferments are far more sensitive to the destructive action of heat than when they are present in their natural secretions. This, as stated, is due not only to the reaction of the respective fluids but also to the protective or inliibitory action of the inorganic salts and various proteids naturally present. We may thus say with Biernacki ' that the purer the ferment the less resistant it is to the effects of heat. It is thus plain that these enzymes, capable though they are of accomplishing great tasks, are nevertheless exceed- ingly unstable and prone to lose their proteolytic power under the slightest provocation. When, however, they are surrounded by their natural environment, the acid or alkali of the respective secretion, together with salts and proteids, they then appear more stable ; their natural labil- ity becomes for the time being transformed into semi- stability, and the temperature, for example, at which they ' Das Verhalten der Verdauungsenzyme bei Temperaturerlioliuiigen. Zeitschr. f. Biol., Band 28, p. 49. 20 DIGESTIVE PEOTEOLYSIS. lose tlieir peculiar power, is raised ten degrees or more. I have also found the same to be true of the vegetable pro- teolytic ferments, and also of the amylolytic ferment of saliva. The above facts furnish us, I think, a good illustration of how dependent these proteolytic enzymes are upon the proper conditions of temperature, to say nothing of other conditions, for the full exercise of their peculiar power. Toward acids, alkalies, metallic salts, and many otlier com- pounds they are even more sensitive than toward heat, and much might be said regarding the effects, inhibitory or otherwise, produced by a large number of common drugs or medicinal agents on these two ferments. Any lengthy discussion of this matter, however, would be foreign to our subject, and I will only call your attention in passing to one or two points which have a special bearing upon the general nature of the enzymes. Take, for example, the influence of such substances as urethan, paraldehyde, and thallin sulphate on the proteolytic action of pepsin- hydrochloric acid ^ and we find that small quantities, 0.1 to 0.3 per cent, tend to increase the rate of proteolysis, while larger amounts, say one per cent,, decidedly check proteo- lysis. Similarly, among inorganic compounds, arsenious oxide, arsenic oxide, boracic acid, and potassium bromide * in small amounts increase the proteolytic power of pepsin in hydrochloric acid solution, while larger quantities check the action of the ferment in proportion to the amounts added. Again, with the enzyme trypsin, similar results with such salts as potassium cyanide, sodium tetraborate, potassium bromide and iodide' may be quoted as showing ^ Chittenden and Stewart : Studies in Physiol. Chem., Yale University. Vol. 3, p. 64. ^ Chittenden and Allen, Ibid., vol. 1, p. 76. ^ Chittenden and Cummins, Ibid., vol. 1, p. 112. INFLIJENCE OF FOEEIGN SUBSTANCES. 21 not only tlie sensitiveness of the ferment toward foreign substances, but likewise its peculiar behavior, viz., stimula- tion in the presence of small amounts and inhibition in the presence of larger quantities. Furthermore, we have found that even gases, as carbonic acid and hydrogen sulphide, exert a marked retarding influence on the proteid-digesting power of trypsin. Moreover, while it is generally stated that proteolytic and other enzymes are practically indifferent to the presence of chloroform, thymol, and other like substances that quickly interfere with the processes of the so-called organized ferments, pepsin and trypsin certainly do show a certain degree of sensitiveness to chloroform, and indeed even to a current of air passed through their solutions. Thus, very recently, Bertels ' and Dubs,^ working under Salkowski's direction, have called attention to the peculiar behavior of pepsin to chloroform ; their results showing, first, that small amounts of this agent tend to increase the proteolytic power of the enzyme, while larger amounts decrease its digestive power. Another interesting point brought out especially by Dub's experiments is the fact that an impure solution of the ferment, viz., an acid extract, for example, of the mucous membrane of the stomach containing more or less albuminous matter, is far less sensitive to chloro- form than an acid solution of the purified ferment, thus showing again the protective influence of proteids and other extraneous matters ; the latter guarding the enzyme to a certain extent from both the stimulating and inhibitory action of various agents. Another point to be emphasized just here is that any chemical substance, such as a metallic salt, having a spe- ' Ueber den Einfluss des Chloroforms auf die Pepsinverdauung, Virchow's Archiv, Band 130, p. 497. - Der Einflnss des Chloroforms auf die kiinstliche Pepsinverdauung, Ibid., Band 134, p. 519. 22 DIGESTIVE PEOTEOLYSIS. cific action upon proteid matter, will almost invariably interfere more or less witli the proteolytic action of tliese enzymes, both through a direct action upon the soluble ferment itself, and also through an indirect action in mod- ifying or inhibiting the digestibility of the proteid exposed to proteolysis. All of these facts emphasize more or less the proteid-like nature of the enzymes, or at least the carriers of the ferments. It is further very suggestive that the destruction of these enzymes by heat happens to occur at approximately those temperatures which are gen- erally recognized as the coagulation points of ordinary proteids. Moreover, the apparent lack of stability so characteristic of these ferments, their inherent proneness to alteration, their marked susceptibility to every change in environment, all point to large complex molecules, such as we have in proteids and are familiar with in living protoplasm. Whatever the exact nature of these ^proteolytic enzymes, they are certainly endowed with the power of transforming relatively large amounts of proteid matter into soluble products, even though they themselves are present in very small quantity. They are derived, as we have seen, from living protoplasmic cells, and we might perhaps, with V. ]*^ageli ' and Mayer,^ consider them as retaining a por- tion of that molecular motion so characteristic of living protoplasm, by which the equilibrium of the dead food- proteid may be disturbed and thus changes started which result in what we call proteolysis. However this may be, we must look to some phase of catalytic or contact action as the true explanation of this power of proteolysis. At first glance, any explanation or theory involving the use of catalysis seems exceedingly vague and indefinite, and yet ' Theorie der Gahrung. Miinclieii, 1879. ^ Die Lehre von den chem. Fermenten. Heidelberg, 1882. THEORIES OF ENZYME ACTION. 23 many illustrations can be given of chemical reactions where the dominating agent evidently acts in this manner. " We call a force catalytic," says the philosopher of Heilbron, " when it holds no communicable proportion to the assumed results of its action. An avalanche is hurled into the valley. ... A puff of wind or the fluttering of a bird's wing is the catalytic force which has given the signal for, and which is the cause of the wide-spread disaster." ' In the older theories of catalytic action, tlie catalytic agent was supposed to remain passive, but not so in the more modern conception of catalysis. The ferment by its presence makes possible certain changes and combi- nations which could not occur in its absence, at least under the existing circumstances, although all the other condi- tions might be favorable. The proteids, for examjDle, have a natural tendency to undergo hydration ; thus, simple boiling with dilute acid or exposure to the action of super- heated water alone,'^ will produce many if not all of the products formed in natural digestive proteolysis. To be sure, they are not formed as readily as in artificial or natural digestion, and there may be some minor points of difference, but still proteolysis can be imitated in this manner. The proteolytic enzymes simply help on this natural tendency of proteid bodies to undergo hydration, and by their presence and action enable it to occur at lower temperatures than it otherwise could, and at the same time render it more rapid and comj)lete. This is hot accomplished, however, by simple contact. The enzymes, we may assume, combine in some manner with the proteid undergoing digestion, starting thereby a train of reactions ' Quoted from Gamgee's Physiological Chemistry of the Animal Body, vol. 2, p. 7. '^ Chittenden and Meara. A study of the primary products resulting from the action of superheated water on coagulated egg-albumin. Journal of Physiol., vol. 15, p. 501. 24 DIGESTIVE PROTEOLYSIS. in whicli the proteid and the water present are the main actors, they being, however, perfectly passive in the absence of the inciting agent, the enzyme. As expressed by Gamgee, "the ferment phenomena resemble those in which there is apparently a periodic synthesis and dissocia- tion of the catalyzing agent, which acts in a similar manner to the agent which exj)lodes a train of gunpowder," We can find many illustrations among chemical phenom- ena where one body, even thongh present in small quantity, acts as a go-between and makes possible an almost indef- inite exchange of matter and energy. Take as an illustra- tion, the part played by water in determining the explosion of oxygen and carbon-monoxide gas. Some years ago, Dixon * called attention to the fact that a mixture of these two gases when perfectly dry would not ex23lode even by contact with red hot platinum wire. The presence, how- ever, of a small amount of aqueous vapor would at once cause an exjDlosion to occur. In confirmation of this obser- vation, Traube' has reported that a flame of carbon- monoxide gas introduced into a perfectly dry atmosphere is at once extinguished. In a moist atniosj)here, on the other hand, the flame will continue to burn indefinitely, that is as long as the CO gas is supplied. In these cases, the water, which is so necessary for the appearance of the reaction, and which furnishes a striking illustration of the action of a contact or catalytic substance is not purely passive. To be sure, only a mmimal amount is necessary for the combustion of an indefinite amount of carbonic oxide, but the water enters into the reaction itself. It is to be noticed that carbonic oxide and water alone, even at high temperatures, will not react, but in the presence of ^ Cliemical News, vol. 46, p. 151. ^ Bericlit. d. deutsch. chem. Gesellsch., Band 18, p. 1890. THEOKT OF CATALYSIS. 25 oxygen the water is decomjjosed with formation of hydro- gen-peroxide, thns : CO + 2 H2O + O, = C0(0H)2 + H2O2. The hydrogen-peroxide thus formed combines with carbonic oxide to form carbonic acid, which in turn is decomposed into the anhydride CO,, with regeneration of water, the latter being available for further action of the same order : H2O2 + CO = C0(0H)2 2 C0(0H)2 = -2 CO2 + 2 H2O. Indeed, as can be readily seen from the equations, this may be kept up indefinitely, a small amount of water, i. e., the go-between, the catalytic agent, sufficing to accomplish the transformation of almost any amount of carbon- monoxide. This, I think, furnishes an excellent illustra- tion of the way in which catalytic agents, such as the proteolytic enzymes, may be supposed to act. It is truly contact action, but the agent is not purely passive; the enzyme combines with the substance undergoing prote- olysis, and the resultant compound thus formed is enabled now to combine with water and undergo hydrolysis, some- thing which could not be accomplished by the proteid and water alone, that is at body temperature. This new and more complex compound is naturally less stable and soon undergoes dissociation or cleavage with a S23litting o££ of the original enzyme for one product, which is thus avail- able for further action of the same order ; while, as other products, we find the hydrated and otherwise altered sub- stances coming from the proteid, and whose formation is the ultimate object of the whole process. The parallelism between this hypothetical action of the proteolytic enzymes and the known reactions in the above combustion of carbonic oxide is certainly very close, and 26 DiaESTIVE PEOTEOLTSIS. leaves little doubt tliat this explanation of enzyme action is, in a general way at least, correct. Tims the carbonic oxide, CO, brought in contact with pure, dry oxygen gas (apparently all that is necessary for its direct oxidation into carbonic acid, CO^), undergoes no change ; the burning CO gas is at once extinguished. Evidently, something more is necessary in order to start the process of oxidation. So, too, in proteolysis ; the process, as we shall see later on, is essentially one of hydration, but bring the proteid and the water, or acid-water, together and although all the condi- tions are apparently favorable for hydration there is, as you know, little or no change. But introduce the catalytic agent and immediately the reaction commences. In the case of the burning CO gas in contact with oxygen, the water acting as contact agent makes oxidation possible, enabling the main actors in the transformation to react upon each other. But, as we have seen, the contact agent is something more than a mere looker-on, it becomes for the time being an integral j)art of the molecule, undergoing change, combining with it and thus making possible the subsequent alterations characteristic of the specific trans- formation, in which, however, the regeneration of the contact agent is a prominent feature. So, too, with the proteolytic enzymes, pepsin and trypsin, they are the go- betweens, making possible the union of the proteids with water by combining with the proteid molecule and thus paving the way for both hydration and cleavage. In the cleavage of the complex molecule, we have the regenera- tion of the ferment as a prominent feature, and in prote- olysis we understand that the regenerated ferment may act not only upon more of the original proteid, but likewise upon the primary products of its action, thus giving rise eventually to a row of more or less closely related cleavage products. Finally, we can conceive that the enzyme may NATURE OF PEOTEIDS. 27 gradually be affected by the process, that its regeneration may become less complete, and thus digestive power be eventually diminished. Much more might be said in support of the above hypothesis. On the other hand, some objections might be raised against it, but I know of no more reasonable expla- nation of enzyme action than that here presented, or one which so well accords with all of the known facts concern- ing the conditions wdiich modify proteolytic action.' Thus, the influence of heat, of the products of proteolysis, of acids, alkalies, and various organic and inorganic salts on the action of these digestive enzymes is such as lends favor to the above view rather than opposes it. THE GENEEAL NATUEE OF PEOTEIDS. Peoteids are confessedly among the most complex bodies the physiologist has to deal with, while at the same time they are perhaps the most important, not only in view of their wide-spread distribution through animal and vegetable tissues, but because of the prominent part they take in the nutrition of the body. The more our knowl- edge is broadened concerning these varied substances, the more we are impressed with their complexity, and at the same time with the necessity for a more accurate study of both their composition and constitution. Concerning the latter, full fruition of our hopes is probably in the distant future, but every step of advance in this direction adds greatly to our resources in the interpretation of the varied and complex changes characteristic of proteid metabolism. ^ Compare L. de Jager, Erklaningsversuch iiber die Wirkungsart der ungeformten Fermente, Virchow's Archiv, Band 131, p. 183. Also Chandelon : Bulletin de 1' Academic Eoyale de Med. de Belgique, 1887, 1, p. 389. 28 DIGESTIVE PROTEOLYSIS. Every study of proteid decomposition adds something to our store of knowledge, and gives perhaps an added fact available for broadening our deductions.' Moreover, the comjDosition and general reactions of the proteids may be investigated with full confidence of obtaining many useful results, which must necessarily be an aid in interpreting the changes accompanying digestive proteolysis. Take, for example, the single question of peptonization by gastric digestion. What is the nature of the process ? Is the proteid transformed into a soluble and diffusible peptone as a result of hydration and cleavage, or is it a transformation which results from a simple depolymeriza- tion of the proteid molecule, i.e., are we to consider albumin and peptone as isomeric bodies ? These questions, on which physiologists seem loath to agree, can certainly be answered definitely ; not, however, by arguments but by careful ex23erimentation, in which the composition of the proteid undergoing digestion must be a necessary prelim- inary factor, and the composition of the resultant product, or products, a secondary factor of equal importance. Turther, the question needs to be answered not with reference to one proteid merely, but with reference to every proteid capable of digestion by either gastric or pancreatic juice. When these questions have been fully answered in this manner, we shall have positive data to deal with, and our conclusions will rest upon a foundation of fact not easily set aside. This is one of the problems upon which I have been at work for some years, and although progress may in one sense be slow, yet it is sure and gives results of no uncertain character. First, then, let us consider briefly the nature of the pro- teids whose proteolysis we may be interested in ; remem- ^ See Drechsel : Der Abbau der EiweisstofEe. Dn Bois Eeymond's Archiv f. Physiol., 1891, p. 248. CLASSIFICATION OF PEOTEIDS. 29 bering, however, that in so domg we can merely touch upon the points essential for our purpose. Allow me to say in parenthesis that there is being published in Moscow a work on proteids alone of five volumes, 900 pages each, which it is supposed will constitute an exhaustive treatise of the subject.^ If we attempt to classify all of the proteid bodies hitherto discovered and studied we are at once confronted with a problem of no small proportions. So varied are they in their reactions, solubilities, and behavior toward general reagents, so inclined to merge into each other by almost insensible gradations that it becomes an extremely difficult matter to make an arrangement that will satisfy all the requirements of the case. I have to suggest, however, the following classification, which is merely a modifica- tion of several existing ones, based primarily upon chem- ical composition, and solubility in the more common menstruums. Proteids may first be divided into three main groups as follows : I. Simple Proteids. — Composed of carbon, hydrogen, nitrogen, sulphur, and oxygen, and yielding by decomposi- tion aromatic bodies such as tyrosin, phenol, indol, etc. II. Compound Proteids. — Composed of a simple pro- teid united to some non-proteid body. III. Albuminoids. — A class of nitrogenous bodies related to and derived from proteids, but differing espe- cially from the latter by great resistance to the ordinary solvents of true proteids. The individual members of these three groups may be arranged as follows on the basis of solubility, coagulability, etc.: ^ Die Einlieit der ProteinstofEe. Historisclie u. experimentelle Unter- suclmngeii, by L. Morokhowetz. 30 DIGESTIVE PEOTEOLYSIS. I. Simple Peoteids. — A. /Soluble in water. — a. Coag- ulable by heat, and by long contact with alcohol. Albumins : serum-albumin, egg-albumin, lacto-albumin, myo-albumin, vegetable albumins, h. ]^on-coagulable by heat and by long contact with alcohol. Proteoses : ' proto- proteoses, deuteroproteoses. Peptones : * amphopeptones, antipeptones, hemipeptones. B. Insoluble in water., but soluble in salt solutions. — a. More or less coagulable by heat. Globulins. 1. Soluble in dilute and saturated ISTaCl solutions. Yitellins. 2. Soluble in dilute ISTaCl solutions, but precipitated by saturation with JSTaCl. Myosins, paraglobulin^ or serum- globulin, fibrinogen, myo-globulin, paramyosinogen, cell- globulins, b. Non-coagulable by heat, soluble in dilute ^aCl solution and precipitated by saturation with ISTaCl. Heteroproteoses. C. Insoluble in water and salt solutions, soluble in dilute alcohol — Zein, gliadins. D. Insoluble in water, salt solutions and alcohol', soluble in diUite acids or alkalies. — a. Coagulable by heat when suspended in a neutral fluid. Acid-albumins, alkali- albumins or albuminates, b. Non-coagulable by heat when suspended in a neutral fluid. Antialbumids, dysproteoses, glutenins. E. Insoluble in water, salt solutions, alcohol, dilute acids and alkalies y soluble in strong acids, alkalies, and in jpe]) sin-hydrochloric acid and alkaline solutions of trypsin. — Coagulated proteids, fibrin.^ ^ Used in the generic sense ; the proteoses including albumoses, glob- uloses, myosinoses, elastoses, etc., and the peptones the peptone-products formed in the digestion of any or all proteids. 2 Not completely insoluble in saturated NaCl solution. ^ In blood-fibrin we have a good illustration of the fact that these divisions are not absolutely exact, since this form of proteid matter, for example, is somewhat soluble in dilute acids and in salt solutions, although requiring a long time for marked solution. CLASSIFICATION OF PEOTEIDS. 31 II. Compound Peoteids. — A. Compounds of a proteid {glohuliri) with an It on-containing pigment^ soluble in water and coagulahle by heat and alcohol. Hemoglobin, oxylisemoglobin, methsemoglobin, etc. £. Compounds of jproteids with members of the carbo- hydrate group. Insoluble in water / soluble in 'very weak alkalies. — a. True mucins, b. Mucoids or mucinoids. C. Compounds of proteids with nucleic acid. Phos- phorized bodies yielding by decomposition metaphosphoric acid. Insoluble in water and in pepsin-hydrochloric acid, but more or less soluble in alkalies. — IN^ucleins. D. Compotmds of proteids with nucleins. Very soluble in dilute alkalies. — jSTucleoalbumins, as casein of milk, and nucleoalbumins of cell-protoplasm and cell-nuclei, etc. III. Albuminoids. — A. Soluble in boiling water with formation of gelatin and yielding by decomposition leucin and glycocoll — Collagen (gelatin). B. Insoluble in boiling water .^ and yielding by decom- position much leucin and some tyrosin, together with glyco- coll and ly satin. Slowly hydrated by boiling dilute acids and by treatment with pepsin-hydrochloric acid. — Elastin. C. Insoluble in water., dilute acids and alkalies., also in gastric and pancreatic juice. Yield leucin and ty rosin by decomposition. — Keratin, neurokeratin. We may now advantageously consider tbe composition of a few of the more prominent representatives of tbe individual groups, taking for illustration those bodies which have been most thoroughly studied, and which we may have occasion to refer to in our discussion of prote- olysis. 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