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 CORNELL UNIVERSITY. 
 
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 The method of enzyme acllon, 
 
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 ■^ Iv J. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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The original of tliis book is in 
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 http://www.archive.org/details/cu31924000911002 
 
THE METHOD 
 
 OF 
 
 ENZYME ACTION 
 
 JAMES BEATTY, M.A., M.D., D.P.H. 
 
 WITH INTRODUCTION BY 
 
 Professor E. H. STARLING, M.D., Sc.D., F.R.S. 
 
 PHILADELPHIA 
 
 P. BLAKISTON'S SON & CO. 
 
 I0I2 WALNUT STREET 
 
 1917 
 5 
 
IYb.5133 
 
 TO M.B. 
 (^TAT. 86) 
 
 (2^ 
 
 3 3^ 
 
PREFACE 
 
 A THEORETICAL treatment of a scientific subject can 
 lead only to a working hypothesis, and it is from this 
 point of view that the theory of the mode of action of 
 enzymes given at the end of this book should be judged. 
 Owing to the circumstances in which the following pages 
 were written, I have had neither the time nor the oppor- 
 tunity to verify the hypothesis by experiment. It is my 
 hope, however, that the conception of enzyme action 
 given will prove sufficiently interesting to induce some 
 worker to test it and either prove or disprove it. From 
 either result something of value will, I hope, be learnt. 
 
 The spinning of hypotheses without producing new 
 facts to render them more plausible may seem a useless 
 expenditure of energy, but it seems to me that if the 
 subject has been carefully reviewed before the hypothesis 
 is evolved in its final form — as I have tried to do in this 
 book — at least two advantages are gained. In the first 
 place the facts have been looked at from a new angle, and 
 this may result in the observation of new relationships ; 
 and in the second place a working hypothesis may be 
 reached, and this is the necessary prelude of fruitful 
 experimental work. There is therefore, I think, a field 
 open to anyone interested in a scientific subject who has 
 not the capacity or opportunity to carry out experimental 
 investigations, a field in which he can do useful work by 
 
iv PREFACE 
 
 suggesting ideas which may lead a research worker to 
 fresh lines of investigation. 
 
 In offering this working hypothesis for consideration I 
 do not claim that the central idea is in any respect new. 
 All I can say is that I have not met anywhere with the 
 conception of the essential unity of the action of both 
 hydrolysing and oxidising enzymes, nor an account of the 
 mechanism similar to that given. It is quite possible, of 
 course, that both have been previously suggested. 
 
 The bqoks of reference used are as follows, the first 
 three being those most frequently referred to and quoted 
 from : 
 
 Bayliss, 'The Nature of Enzyme Action,' 3rd edition. 
 Do ' The Principles of General Physiology.' 
 
 Euler, ' General Chemistry of the Enzymes.' Transla- 
 tion by Pope. 
 
 Werner, ' New Ideas on Inorganic Chemistry.' Trans- 
 lation by Hedley (191 1). 
 
 Kastle, ' The Oxidases and other Oxygen-catalysts 
 Concerned in Biological Oxidations.' 
 
 Dakin, ' Oxidations and Reductions in the Animal 
 Body.' 
 
 Mellor, ' Modern Inorganic Chfemistry.' 
 
 Senter, ' Outlines of Physical Chemistry,' 4th edition. 
 
 HoUeman, 'Textbook of Organic Chemistry.' Trans- 
 lation by Jameson. 
 
 Plimmer, ' Practical Organic and Bio-chemistry.' 
 
 Hatschek, ' An Introduction to the Physics and 
 Chemistry of Colloids,' 2nd edition. 
 
 Willows and Hatschek, ' Surface Tension and Surface 
 Energy.' 
 
 Wells, ' Chemical Pathology.' 
 
 Moore, ' The Origin and Nature of Life.' 
 
 Access to original papers was impossible in the circum- 
 
PREFACE V 
 
 stances in which the book was written. The deep blue 
 waters of the Mediterranean, now smooth as oil, now 
 tossing the ship like a toy, the gorgeous sunsets, the 
 brilliant stars of night, the glamour of Eastern ports, the 
 unclouded sun, the sands seeming to quiver in the heat, 
 and all the spiritual upheaval of this unprecedented time 
 certainly stimulated imagination and effort, but made it 
 impossible to quote from original sources. 
 
 My warmest thanks are due to Prof, (now Lieut.-Col.) 
 Starling for his great kindness in reading the manuscript, 
 for his advice as to publishing it, and above all for writing 
 an introduction and thus multiplying many times the 
 chances of the book being read. 
 
 March, 19 17. 
 
INTRODUCTION 
 
 Prof. E. H. STARLING, M.D., Sc.D,, F.R.S. 
 
 In this monograph Captain Beatty deals with a subject 
 far removed from the more primitive problems now 
 engaging the activities of this half of the civilised world, 
 though it is one that has attracted and will continue for 
 many years to attract the interest of a large body of 
 workers in science. Like so many ot^hers of the riddles 
 with which life presents us, the mode of action of ferments 
 fascinates both chemists and physiologists. In this 
 riddle lies hidden the mechanism of most vital pheno- 
 mena, and until it is solved we cannot hope to penetrate 
 further into the workshop of the cell itself, or to under- 
 stand the various intracellular activities which in their 
 sum make up the life of the individual. 
 
 When Captain Beatty gave me his manuscript to read, 
 written in the intervals of active service, I was struck 
 with the lucid and concise manner in which he had 
 brought together the chief results of modern research on 
 enzyme and catalytic actions, and felt that his presenta- 
 tion ought to be made available to all workers in this 
 field, as well as to the many others interested in the 
 fundamental problems of life. 
 
viii INTRODUCTION 
 
 His survey, however, leads him to formulate — I think 
 with more precision than has been done hitherto — a 
 hypothesis of ferment action. 
 
 Life can be regarded as a development in the presence 
 of the two essentials, air and water, probably under the 
 influence of the energy of the sun's rays. Since tlie 
 majority of vital processes are associated with some form 
 of ferment action, which in turn is always connected with 
 the mobilisation and shifting of OH and H ions, life 
 itself might be regarded as the dynamical history of 
 water. 
 
 The author tries to explain this mobilisation. He 
 shows that for the directed reactions which are the dis- 
 tinguishing feature of living things and the condition of 
 their evolution, two processes are required : 
 (a) Control of rate of reaction. 
 {b) Control of sphere of reaction. 
 
 These processes are achieved by the development of 
 ferments, in the form of colloids or attached to colloid 
 moleculesT which are able to act only after adsorption, or 
 attraction to the surface, of other colloidal molecules. 
 
 He shows that any enzyme displays two qualities : 
 
 (a) A power, common to all ferments, of attracting 
 
 the H or OH groups in water. 
 
 (b) A power, which is specific to the ferment in 
 
 question, of adsorbing some particular substrate. 
 He suggests that these two properties, general and 
 specific, find their explanation in a dual composition of 
 the enzyme — which in every case must be compounded of 
 one substance exercising the specific and another the 
 general function. This is certainly true of some of the 
 oxidases. These, however, according to the author, fall 
 into the general category of ferments as mobilisers of H 
 or OH ions. Their function is not as direct carriers of 
 
INTRODUCTION ix 
 
 oxygen, but as activating the H or OH group of the water 
 molecule. 
 
 If the hydrolytic enzymes are built up in the same way 
 the author points out that it should be possible actually 
 to manufacture some of them, and boldly stakes the truth 
 of his hypothesis on this possibility. The challenge is 
 worth taking up. 
 
 Salonica: 
 January, 1917. 
 
CONTENTS 
 
 
 
 
 PAGE 
 
 Preface ...... 
 
 III 
 
 Introduction 
 
 
 
 VII 
 
 Catalysis .... 
 
 
 
 I 
 
 Summary 
 
 
 
 7 
 
 General Considerations on Enzymes 
 
 
 
 7 
 
 Colloids .... 
 
 
 
 9 
 
 Summary 
 
 
 
 20 
 
 Adsorption 
 
 
 
 22 
 
 Summary 
 
 
 
 32 
 
 The Properties of Enzymes 
 
 
 
 34 
 
 Summary 
 
 
 
 57 
 
 Chemical Action. Hydrolysis . 
 
 
 
 60 
 
 Summary 
 
 
 
 81 
 
 Chemical Action, Oxidation and the Oxidases 
 
 84 
 
 Reduction and the Reducases 
 
 99 
 
 Summary ..... 
 
 102 
 
 The Method of Enzyme Action. A Hypothesis 
 
 105 
 
 Summary ..... 
 
 128 
 
 Deductions ..... 
 
 131 
 
 Conclusion ..... 
 
 137 
 
 Index .... 
 
 
 
 139 
 
THE METHOD OF ACTION OF 
 
 ENZYMES. 
 
 The numerous changes in chemical substances produced 
 by the living organism can be imitated in the laboratory, 
 but in many cases only by the help of powerful agents 
 and with the application of a considerable amount of 
 energy in the form of heat, electricity, etc. What is done 
 with difficulty in this way outside the body is often done 
 inside it with apparent ease, and with the help of trifling 
 amounts of energy. The mechanism of these changes 
 appears to be connected with the presence of enzymes, 
 such as pepsin, trypsin, etc., and the nearest approach in 
 ordinary chemistry to their action is that of catalysts. 
 
 Catalysis. 
 
 The characteristic action of a catalyst is to affect the 
 rate of a reaction. Most catalysts increase this rate, but 
 retardation is also known, as when the slow oxidation 
 of phosphorus in air is stopped by a trace of ether vapour. 
 Examples of the more usual type are : the power of finely 
 divided platinum to bring about the combination of 
 hydrogen and oxygen when the mixed gases are exposed 
 to it ; the influence of platinised asbestos on the oxidation 
 of sulphur dioxide — if air and sulphur dioxide mixed 
 
 I 
 
2 ACTION OF ENZYMES 
 
 are passed through a porcelain tube heated to 400° C. 
 then in the absence of platinised asbestos about 10 per 
 cent, of sulphur trioxide is formed, but in its presence 
 nearly 100 per cent. 
 
 While catalysts affect the rate of a reaction they do not 
 affect the final result, and they themselves can be recovered 
 unchanged at the end of the reaction. It is extraordinary 
 what minute amounts of the catalyst can act. It is stated 
 that one form of platinum can act on 1,000,000 times its 
 weight of hydrogen peroxide, and the influence of molybdic 
 acid on the decomposition of hydriodic acid by hydrogen 
 peroxide is still greater. 
 
 A reaction which illustrates the qualities of a catalyst, 
 and one which has been closely studied, is that of the 
 evolution of oxygen from heated potassium chlorate when 
 catalysed by manganese dioxide. Heat alone can cause 
 the evolution of oxygen at a temperature of 356° C. After 
 this has continued for a time the previously melted mass 
 solidifies and now contains potassium perchlorate and 
 potassium chloride. If the temperature is now raised 
 over 600° C. the potassium perchlorate decomposes and 
 more oxygen is given off; finally nothing is left but 
 potassium chloride. The reaction follows a different 
 course if manganese is first mixed with the potassium 
 chlorate. Neither substance heated alone gives off 
 oxygen at a low temperature, manganese dioxide requir- 
 ing a temperature of at least 400° C. and potassium 
 chlorate at least 340° C, but the mixture gives off oxygen 
 at about 200° C. ; when the reaction is complete potassium 
 chloride alone is left except for the manganese dioxide, 
 which can be entirely recovered. Other oxides can be 
 used in place of manganese dioxide, though they do not act 
 so vigorously, e. g. ferric, copper, cobalt, or nickel oxide. 
 Now on close examination of the reaction it has been 
 
CATALYSIS 3 
 
 found that the manganese is not quite unchanged at the 
 close oijhe reaction ; it is not changed in its amount or its 
 chemical composition, but it is changed physically. Added 
 in the crystalline form it has become amorphous. It 
 appears also that it does enter into the reaction in the 
 sense that the potassium chlorate does not give off its 
 oxygen directly but instead oxidises the manganese dioxide, 
 forming, it is believed, an unstable higher oxide of man- 
 ganese. This higher oxide breaks up almost as soon as 
 it is formed, liberating free oxygen and regenerating the 
 dioxide. The regenerated dioxide is again oxidised and 
 decomposed, and so the reaction proceeds to its end. 
 Such a reaction, where intermediate products are produced 
 which do not necessarily appear in the final products, is 
 described as a " consecutive " reaction. Obviously at least 
 two reactions occur, and it will depend on their relative 
 speed whether the intermediate products can be detected 
 or not. In the case where manganese dioxide was not 
 used, potassium perchlorate was formed as a side reaction ; 
 but this cannot be detected when manganese dioxide is 
 used, and probably none is formed. The manganese 
 dioxide is said to catalyse or accelerate the main reaction, 
 that of the evolution of oxygen, and in so doing it renders 
 the high temperature at which potassium perchlorate 
 would be produced unnecessary. A still closer study of 
 the reaction shows that traces of other products are present, 
 R. g. ozone, chlorine, and potassium permanganate, indeed 
 reactions in general are rarely entirely confined to one or 
 two products. 
 
 The formation of intermediate products, however, as 
 an explanation of catalysis, is a hypothesis difficult to 
 sustain when such catalysers as finely divided platinum, 
 platinised asbestos, charcoail, etc., are in question, and if 
 it is to be maintained an enlarged conception of the term 
 
4 ACTION OF ENZYMES 
 
 " intermediate product " must be formed. This meaning 
 will be treated more fully when the conception of " adsorp- 
 tion " is considered, but meantime it may be pointed out 
 that these substances appear to have the power of con- 
 densing certain gases and other substances on their 
 particles. To such an extent is this possible that it has 
 been estimated that gaseous carbon dioxide condenses on 
 wood charcoal in layers about 0*005 ^^- thick, and the 
 gaseous layer is nearly as dense as liquid carbon dioxide. 
 If two substances capable of inter-reaction are thus con- 
 densed together it must follow that their molecules have 
 much greater opportunities of reacting than when not so 
 condensed, and consequently the speed of the reaction 
 must be enormously increased. Intermediate products in 
 this sense are the physical unions of the reacting sub- 
 stances with the charcoal, etc., and the term " inter- 
 mediate product " connotes not merely chemical but also 
 what are called physical combinations. 
 
 Another objection which has been raised to the inter- 
 mediate product explanation of catalysis is that in certain 
 cases the obvious intermediate product which ought to be 
 formed cannot replace the original catalyst in the reaction. 
 For example, the catalytic action of hydrochloric acid in 
 the formation of esters from methyl alcohol and acids is 
 not shown by methyl chloride. This, however, merely 
 proves that if an intermediate product is formed it is not 
 methyl chloride. It will appear later that other inter- 
 mediate products are possible when the solvent is taken 
 into consideration. 
 
 While a catalyst can alter the speed of a reaction it 
 cannot alter the final result. This is proved by a con- 
 sideration of those cases where the products of the reaction 
 remain in solution and are not removed from the sphere 
 of action either by precipitation, as when silver nitrate 
 
CATALYSIS 5 
 
 reacts with chlorides, or by evaporation, as when evolution 
 of gas occurs. For example, when ethyl alcohol and 
 acetic acid in molecular proportions react according to 
 the equation : 
 
 CjHsOH + CHsCOOH = CHsCOOCjHe + H^O 
 it is found that when the reaction is complete the mixture 
 has the composition 1/3 mol. alcohol, 1/3 mol. acid, 
 2/3 mol. ethyl acetate, and 2/3 mol. water, and these sub- 
 stances are described as being in equilibrium with each 
 other. This reaction, when the pure substances are mixed 
 with each other, takes weeks to reach completion, but in 
 the presence of a mineral acid it is completed in a few 
 hours. In spite of the difference of speed the final result, 
 with or without the acid, is the same. 
 
 Again, it is found that the same result follows if instead 
 of a mixture of ethyl alcohol and acetic acid, ethyl acetate 
 and water are used in molecular proportions, and this 
 occurs whether a mineral acid, such as hydrochloric acid, 
 is used or not, though the speed of the reaction is 
 enormously raised by the acid. This introduces the con- 
 ception of " reversible " equations where the reaction can 
 be started from either end ; such an equation is usually 
 indicated by the sign '^ instead of the sign = . The first 
 form of the equation may be considered as that represent- 
 ing the direct action, the second, the reverse action ; and 
 it follows from the direct experiments made that the 
 catalyst accelerates the speed of both the direct and the 
 reverse actions. Apart from direct experiment, however, 
 this conclusion follows from the fact that the position of 
 equilibrium is unchanged, for had the catalyst accelerated 
 only the direct action while the speed of the reverse action — 
 which must be coming into operation as soon as any mole- 
 cules of ethyl acetate and water are formed — was un- 
 changed the final result must have been an almost complete 
 
6 ACTION OF ENZYMES 
 
 transformation of the acid and alcohol into acetate and 
 water. As this does not occur and the equilibrium is 
 unchanged, the speed of the reverse reaction must have 
 been accelerated equally with that of the direct. 
 
 Another result which follows from a consideration of 
 these equilibrium equations is that where the products of 
 any reaction are present in such a form as to be capable 
 of reaction, reactions between them take place with 
 resulting reformation of the original substances ; in other 
 words, the final position of equilibrium is due to a balance 
 between two opposing reactions. In addition to this it is 
 clearly possible for these products to alter the equilibrium 
 which would occur in the absence of the catalyst if they 
 enter into combination with it. Mere removal of the 
 catalyst in this way from the operation would not affect 
 the equilibrium, as the reaction slowed by this removal 
 would still proceed to its normal end ; but the position of 
 equilibrium would be affected if the new substance pro- 
 duced by the combination of catalyst and product was 
 capable itself of reacting with the bodies present in the 
 mixture. In enzymes' action it will be found that there 
 is evidence that these do combine with the products. 
 
 The amount of the catalyst added is of importance as 
 regards the velocity of the reaction, for it is found usually 
 that this is in direct proportion to the amount. At this 
 point, again, enzymes are found to differ from inorganic 
 catalysts. 
 
 Lastly, some catalysts are very specific in their action, 
 failing to act in one of two closely allied reactions though 
 acting on the other. As an instance, hydriodic acid can 
 be oxidised with separation of iodine by hydrogen peroxide. 
 This reaction is catalysed by tungstic acid, but the closely 
 allied reactions of the oxidation of hydriodic acid by per- 
 sulphates or bromic acid are not affected by tungstic acid. 
 
GENERAL CONSIDERATIONS 7 
 
 Summary. 
 
 (i) Catalysts are believed to be chemical substances. 
 
 (2) Their action is to affect the rate of a reaction. 
 
 (3) They do not affect the final result except in the rare 
 cases where, perhaps owing to their complexity, new 
 reacting bodies are formed with the products of the 
 reaction. 
 
 (4) They exist unchanged in amount at the end of the 
 reaction. 
 
 (5) They can act in very minute quantities. 
 
 (6) In many cases intermediate products are formed — 
 sometimes chemical combinations, sometimes physical 
 unions. 
 
 (7) The rate of the catalysed reaction varies with the 
 amount of the catalyst. 
 
 (8) In a few cases their action is specific, reactions 
 nearly related to that which they affect not being 
 catalysed. 
 
 Enzymes. General Considerations. 
 
 The action of enzymes closely resembles that of the 
 inorganic catalysts, but there are certain differences, 
 depending apparently on their constitution and on their 
 surroundings. In living organisms from the lowest to 
 the highest they appear to be of universal occurrence, so 
 much so that it is a question whether all the reactions of 
 life do not depend on the presence of enzymes. It is true 
 that there are many changes produced by living proto- 
 plasm which no enzyme as yet known is able to effect, 
 but even these reactions may be carried out by protoplasm 
 through the agency of enzymes having only a temporary 
 existence. They are, however, plainly of simpler constitu- 
 
8 ACTION OF ENZYMES 
 
 tion than protoplasm, for the life of protoplasm is impos- 
 sible in the presence of an amount of antiseptic which has 
 little or no effect on enzymes. This may be due merely 
 to the physical conditions of the cell, the antiseptic inter- 
 fering with diffusion so that the exchange of cleavage pro- 
 ducts between different parts of the cell becomes impossible. 
 Moreover, the distinction does not apply to all antiseptics, 
 for certain enzymes are very sensitive to formaldehyde. 
 
 Provisionally, enzymes or ferments may be defined as 
 the catalysts of living organisms. They may be divided 
 according as they are usually excreted from cells into the 
 surrounding medium, where they can be detected by their 
 characteristic properties apart from the cells — ecto- 
 enzymes — or they are contained only in the cells — endo- 
 enzymes — from which they have to be extracted by special 
 methods. 
 
 A distinction used to be drawn between " organised " 
 ferments, such as yeast, and "unorganised," such as 
 pepsin; but it has been shown by Buchner that by 
 grinding up yeast cells in a mortar with sand and 
 kieselguhr and then exposing the mass to a pressure of 
 300 atmospheres in a hydraulic press, a tissue juice can 
 be extracted which, on purification from structural 
 cellular elements, possesses the fermentative powers of 
 yeast. It is believed, therefore, that yeast owes its powers 
 to its content of unorganised ferments, and the distinction 
 is no longer drawn. 
 
 Ectoenzymes may be obtained in an impure state by 
 filtering the fluid in which the cells have been grown free 
 from the cells, or in the case of glands by collecting the 
 secretions; but the preparation of endoenzymes is more 
 difficult, as the cells must first be ruptured before their 
 contents can be obtained. This is accomplished by the 
 following methods : 
 
COLLOIDS 9 
 
 (i) Drying the cells at a low temperature, and some- 
 times warming subsequently to 50-60° C. 
 
 (2) Drying by short exposure to alcohol or acetone. 
 
 In both cases the material obtained is extracted with 
 water; the aqueous solution is filtered and precipitated 
 with alcohol. 
 
 (3) Permitting autolysis — self-digestion — to occur in 
 the presence of toluene or other antiseptic. The enzymes 
 pass into the solution. 
 
 (4) Mechanical disintegration, extraction with water, or 
 by high pressure. 
 
 In both cases the material obtained is filtered and 
 precipitated by alcohol. 
 
 Purification is difficult, and the methods used often 
 affect the activity of the enzymes. The methods are : 
 
 (i) Precipitation and reprecipitation. Alcohol and 
 acetone are generally used. 
 
 (2) Dialysis. In some cases co-enzymes are lost, or 
 the enzyme passes through the membrane. 
 
 (3) Biological. This is possible in a few cases ; e. g. in 
 the case of amylase, which is not a food for yeast, the 
 protein and carbohydrate in its solution can be used up 
 by growing yeast in it. 
 
 In all cases the precipitate obtained can be dried only 
 at a low temperature and at a reduced pressure. 
 
 Colloids. 
 
 Systems containing enzymes are heterogeneous — that 
 is, they consist of more than one phase. The separate 
 portions of matter of a system in equilibrium are termed 
 phases, each phase being in itself homogeneous and 
 separated by bounding surfaces from the other phases. 
 This is implied in the statement that enzymes are colloids. 
 
10 ACTION OF ENZYMES 
 
 The character of colloids from which the name is derived 
 is that they do not dialyse through parchment paper or 
 other membranes. Examples are: solutions of gum 
 arabic, albumin, and Bredig's colloidal metals formed by 
 producing an electric arc between two terminals of the 
 metal it is desired to bring into colloidal solution under 
 water. Graham, who first studied the subject systemati- 
 cally, divided all substances into colloids and crystalloids, 
 dividing them according to their capacity to pass through 
 membranes or not. It fs now known that a colloid, or 
 rather the colloidal state, is only a form of matter, and 
 that practically all substances can be brought into this 
 state by appropriate methods. Even such a crystalloid 
 as sodium chloride has been obtained in colloidal condition 
 in petroleum ether. 
 
 Colloids are composed of one form of matter in a very 
 finely divided condition distributed through a second, 
 which may be a gas, a liquid, or a solid. The finely 
 divided phase is called the dispersed or internal phase, 
 the other containing it is called the continuous or external 
 phase. An example of a mixture of gas in liquid is seen 
 in foam, of liquid in gas in fog, of liquid in liquid in an 
 emulsion {e. g. milk), of liquid in solid in jelly, of solid in 
 gas in smoke, of sohd in liquid in colloid suspensions, of 
 solid in solid in some kinds of coloured glass. 
 
 The proof that these systems are really heterogeneous 
 is furnished by an application of the Tyndall-Faraday 
 phenomenon. A beam of sunlight or other powerful 
 light, e. g. the electric arc, when passed through a darkened 
 room makes visible the particles of dust floating in the 
 air. This principle has been used in the ultramicroscope, 
 where the rays from an electric arc are concentrated on a 
 cell containing the liquid to be examined and viewed by a 
 microscope placed at right angles to the path of the rays. 
 
COLLOIDS II 
 
 Two cones of light are seen, one converging to the focus 
 of the rays, the other diverging. The fluid is strongly 
 illuminated, and any particles in it are rendered visible. 
 This visibility is due to the diffraction of light where it 
 strikes each particle. The particle appears surrounded 
 with a luminous zone, and the combination of particle 
 and zone brings the whole within the range of microscopic 
 visibility. There is a limit to microscopic vision con- 
 ditioned by the wave-lengths of the rays forming the 
 visible spectrum. These vary from 400 to 700 fxfi, and 
 the limit of microscopic vision is reached at particles with 
 diameters between 800 and 200 fifx. The light diffracted 
 by the particles is polarised owing to the small size of 
 the particles (in a colloid solution), and this means that 
 their diameters are less than the mean wave-length of the 
 light forming the beam. Not all colloids examined 
 showed separate particles ; in many these are so fine that 
 only a haze can be made out, but this is in itself proof of 
 the existence of particles, for a perfectly pure, true solu- 
 tion is quite transparent. It must, of course, be predicated 
 that the particles are either opaque or at any rate possess 
 a different refractive index from that of the fluid containing 
 them, or else particles, though present, would not be 
 rendered visible. 
 
 The effect of this extremely minute subdivision of 
 matter is to produce a great extension of the surface of 
 the substance forming the disperse phase. An illustration 
 will make this clear. A cubic centimetre in the form of a 
 cube possesses a surface of 6 sq. cm. ; if divided into 
 1,000 cubes the surface becomes 60 sq. cm. ; if the sub- 
 division is carried further till the edge of each cube 
 measures only 10 mx, the aggregate surface is 60 sq. metres. 
 The characteristic properties of colloids depend on their 
 enormous surfaces. Specific surface is defined as the 
 
12 ACTION OF ENZYMES 
 
 surface which unit volume assumes through subdivision 
 in any given system. Disperse systems begin to show 
 their characteristic properties when the specific surface 
 reaches the order of lo^ sq. cm. Although the subdivision 
 is so minute the particles are still very much larger than 
 molecules, and that they differ essentially from molecules 
 is indicated by the fact that the electric charge which 
 they usually bear is variable both in sign and amount, 
 while it is invariable in both respects in molecules when 
 these possess a charge. 
 
 Colloid solutions are permanent; though composed of 
 particles they do not gradually precipitate on standing. A 
 collpid solution of gold prepared by Faraday fifty years ago 
 is still preserved. There is more than one reason for this. 
 
 (i) It can be shown that the velocity of a small body 
 falling in a liquid is, other things being equal, proportional 
 to the square of the radius. Hence, if the particles are 
 small and not much heavier than the liquid, a suspension 
 will take a long time to show any marked clearing. But 
 the fact that mere shaking up of irreversibly precipitated 
 colloids will not reform the suspension indicates that 
 other factors are at work. 
 
 (2) The electric charge on the particles. Most sub- 
 stances suspended in water have a negative charge. The 
 origin of the charge is in doubt ; it may be present 
 naturally, or conferred on the particles by electrolytes. 
 Surface ionisation, whether due to dissociation of the 
 substance of the particle itself or to adsorption of outside 
 ions, accounts for the majority, if not all the cases. From 
 the point of view of the permanency of colloids, however, 
 the existence of the charge is important, for it is clear 
 that the particles, being all similarly charged, will repel 
 each other, and the tendency to aggregation and precipi- 
 tation is obviated. 
 
COLLOIDS 13 
 
 (3) Probably the most powerfully acting cause at work 
 is the constant motion of the particles. Even particles 
 well within the range of microscopic vision exhibit con- 
 stant motion, or what is known as Brownian movement 
 from its discoverer. This is composed of an oscillating 
 motion of particles round a central position, and an erratic 
 translatory motion; The movements are seen in greatly 
 intensified form in particles seen only by the ultra- 
 microscope ; indeed so active are the motions that they 
 were at first thought to be something different from 
 Brownian movement. The cause at root is probably the 
 molecular movements which the kinetic theory of gases 
 assumes, and indeed they furnish a direct proof of the 
 theory. The constant bombardment which each particle 
 receives from others must prevent its precipitation. 
 
 (4) The density of the medium ; for the greater the 
 density the less the effective weight of the particles. 
 
 (5) The viscosity of the medium. The greater the 
 internal friction of the medium the longer will the par- 
 ticles take to fall under the influence of gravity. 
 
 In virtue of the large size, compared with molecules, of 
 the particles which compose a colloid solution, colloids 
 exert very little osmotic pressure, i. e. the pressure which 
 a solute exerts on a solvent, and which is comparable to 
 the pressure of a gas on its container, as is proved by the 
 increase in the amount of solvent when put in a dialyser 
 where the membrane is impervious to the solute but 
 allows the solvent to pass. Diffusion to some extent, 
 however, is possible in certain cases ; for example, hasmo- 
 globin diffuses slowly into gelatine, and some membranes 
 are pervious even to protein if given time enough. 
 
 Colloids are divided into two main classes depending on 
 the constitution of the disperse phase. Where this is a 
 solid, the colloid has affinities with suspensions and is 
 
14 ACTION OF ENZYMES 
 
 described as a suspensoid ; where it is liquid the affinity 
 is with emulsions and the colloid is an emulsoid. The 
 characteristics of suspensoids relate them to the macro-^ 
 scopic suspensions, but the emulsoids approximate much 
 more closely to true solutions. This is indicated 
 especially by their respective viscosities. The viscosity 
 of a suspensoid is that of the continuous phase, while that 
 of an emulsoid depends on the amount of the disperse 
 phase and may be very great. 
 
 A closer examination of the emulsoids shows that the 
 two phases present are similar in constitution, the con- 
 tinuous phase being a dilute solution while the disperse 
 phase is a concentrated one. This is the case in those 
 emulsoids where the same solvent is present in both. 
 These are described as lyophile emulsoids. It may be 
 asked why the two do not blend — why the phases remain 
 separate. This appears to be due to the rigidity of 
 particles of liquid when these are very small ; thus the oil 
 particles of a fine oil-water emulsion can be retained by 
 some filters. Each liquid particle possesses so great an 
 internal pressure preserving its shape that it cannot be 
 distorted sufficiently to pass through the filter pores. 
 
 The two varieties differ greatly in their stability^ 
 Suspensoids are easily precipitated by electrolytes, emul- 
 soids with much more difficulty. If to a suspensoid a 
 trace of emulsoid is added, the suspensoid acquires in this 
 respect the properties of an emulsoid, the cause appearing 
 to be the deposit of a film of the emulsoid on each suspen- 
 soid particle. 
 
 Emulsoids are the more important class from the point 
 of view of enzymes, as the colloids of the organism are 
 lyophile emulsoids. 
 
 Emulsoids vary in their reaction to heat and in the 
 influence of the amount of the solvent. There is the agar 
 
COLLOIDS 15 
 
 and gelatine class which melt on heating and gelatinise on 
 cooling, the liquid stage being termed a sol, and the solid 
 a gel. The condition is reversible. Another case is seen 
 in the only important inorganic emulsoid, silicic acid. 
 This, when first formed, is in the sol condition, but on 
 standing it is transformed into a gel, and the gel cannot 
 be changed again into a sol. The change is irreversible, 
 though no water separates. The course of the change is 
 not like that of the solidification of molten metal, where 
 the transformation is sudden and the course therefore 
 discontinuous ; it is continuous, the viscosity steadily 
 increasing. Another form is illustrated by albumin, which 
 does not form a gel on cooling or on increasing concen- 
 tration, but which, when the temperature is raised to about 
 60° C, sets irreversibly. Previous to this denaturation 
 occurs, so that the albumin comes to behave as a suspen- 
 soid and not as an emulsoid. Lastly, there are emulsoids 
 like gum arable solution, whose viscosity increases 
 steadily on cooling or additional concentration, but which 
 does not gelatinise nor does it set on heating. 
 
 The process of gelatinisation appears to be a change of 
 distribution of the solvent between the two phases. It 
 has been stated previously that the ordinary lyophile 
 emulsoid colloid consists of a dilute continuous and a 
 concentrated disperse phase. When gelatinisation occurs 
 it is found that the continuous phase has become the 
 more concentrated, and the disperse phase the more 
 dilute. The sol of such a substance as gelatine therefore 
 consists of a continuous phase of a dilute solution of gela- 
 tine containing liquid particles of a more concentrated 
 solution. The gel, on the other hand, consists of a con- 
 centrated solution of gelatine as a continuous phase 
 containing liquid particles of a dilute. Owing to the con- 
 centration of the gelatine in the continuous phase it has 
 
l6 ACTION OF ENZYMES 
 
 some of the characteristics of a solid, and a gel is a solid 
 containing liquid particles everywhere separated' from 
 each other by the solid phase. Under these conditions 
 the liquid cannot be squeezed out without great pressure, 
 and the condition is reversible. If gelatinisation is pro- 
 duced by formaldehyde the liquid phase is the continuous 
 one, and the solid exists as a meshwork of threads. The 
 first is an example of the vesicular type of gel, the second 
 of the sponge-like. Obviously it is easy to squeeze the 
 fluid out of the second form. 
 
 Electrolytes have a powerful action on colloids ; in 
 many cases precipitation or coagulation occurs. The 
 suspensoid and the emulsoid colloids differ markedly in 
 their sensitiveness, the suspensoids being easily precipi- 
 tated, while the emulsoids require much stronger solutions 
 of electrolytes to affect them. The difference, however, is 
 only one of degree. Since suspensoids are kept in the 
 permanent form largely by the electrical repulsion of their 
 particles it is clear that any substance altering the charge 
 must affect their stability. The charge can be affected by 
 ions possessing a charge opposite to that of the particles 
 of the colloid. As a rule, univalent ions, except hydrion, 
 H, and hydroxidion, OH, added in the form of acid or 
 alkali, have little effect, but the effects are marked if a 
 divalent, and still more if a trivalent ion is added. The 
 effect is apparently due to the alteration of the charge on 
 some of the particles, so that they cease to repel and 
 begin to attract those whose charge has not yet been 
 altered, and as a result aggregation takes place, which, 
 beyond a certain degree, becomes visible precipitation. If 
 the precipitating power of a univalent ion be called x, 
 then that of a divalent ion will be x^, and that of a triva- 
 lent ion x'. It is true that ions cannot be added sepa- 
 rately; with a positive ion there must be added an 
 
COLLOIDS 17 
 
 equivalent negative ion, but it is found that only the ion 
 possessing a charge of opposite sign to that of the colloid 
 particles acts — Hardy's rule. Emulsoid colloids, while 
 largely insensitive to univalent ions, are readily acted on 
 by trivalent ions. On precipitation the ions causing the 
 coagulation are carried down in the precipitate ; they 
 can sometimes be removed by washing, sometimes 
 directly, sometimes after prior treatment with a univalent 
 ion, when the colloid solution is reformed, indicating 
 that the ion has a physical, not a chemical, action on the 
 colloid. 
 
 The effect of acids and alkalies is largely due to the 
 power of their ions, H and OH, to alter the electric 
 charge, for these ions, though univalent, have, as already 
 stated, a powerful action, but they have other effects as 
 well. It is well known that gelatine and other colloids 
 can absorb great quantities of water, the process being 
 known as imbibition. The amount of water which can 
 thus be taken up is greatly increased by acid or alkali. 
 
 Imbibition is probably due to condensation of water on 
 the particles of the colloid, leading to filling up of the 
 capillary spaces between them, the particles themselves 
 being unchanged in size. The forces developed are enor- 
 mous; under a pressure of 41 atmospheres gelatine can 
 still take up 16 per cent, of its volume of water, and at 
 atmospheric pressure it can take up 330 per cent. (The 
 swelling of a Laminaria tent is a familiar use to which 
 this power is put.) It is found, besides, that though the 
 gelatine has swollen, the bulk of the combination of water 
 and gelatine is less than the bulk ot the two taken sepa- 
 rately ; in the case of tragacanth the contraction is almost 
 2 per cent. This implies the compression of water, and 
 to compress water to such an extent would require a 
 pressure of about 400 atmospheres. 
 
i8 ACTION OF ENZYMES 
 
 What applies to other emulsoid colloids applies also to 
 the proteins, which are emulsoid colloids. In solutions 
 which contain no electrolytes these are amphoterous, 
 moving to either pole in an electric field, but any electro- 
 lyte, and especially acids and alkalies, confers a charge on 
 the protein. As the amino-acids, whose combinations 
 form proteins, are both acid and basic, this is due to com- 
 bination of the electrolyte with the protein and subsequent 
 ionisation of the combination. The absorption of water 
 has been found to be increased greatly when this ionisa- 
 tion occurs, and this increase goes with other properties 
 such as viscosity, solubility, osmotic pressure, coagula- 
 bility by alcohol and heat, surface tension, and rotation of 
 polarised light. Hence these effects of adding acid and 
 alkali are really due to increase of water content, and this 
 to the production of protein ions. 
 
 Neutral salts are in some respects similar to acids and 
 alkalies in their action on emulsoid colloids. The effect 
 is similar to that which occurs with suspensoid colloids, 
 though less marked. The presence of an electric charge 
 is obviously the cause, and therefore the effect of neutral 
 salts is more marked when a charge has been conferred 
 on the protein by the previous addition of acid or alkali. 
 
 Neutral salts have special actions on emulsoid colloids 
 as well. Taking albumin and the effect, first, of cations, 
 it is found that the salts of the alkalies produce coagula- 
 tion or " salting out " only in great concentrations, and 
 the process is reversible. The salts of the alkaline earths 
 salt out in similar concentration, but the precipitate 
 becomes insoluble on standing even for a short time. 
 Finally, the salts of the heavy metals salt out irreversibly 
 even in low concentrations. The anions, secondly, follow 
 the order of the Hofmeister series. Beginning with the 
 most powerful precipitant the series is : citrate, tartrate, 
 
COLLOIDS 19 
 
 sulphate, acetate, chloride, nitrate, chlorate, while iodide 
 and thiocyanate do not salt out even in saturated solu- 
 tion. The addition of thiocyanate to albumin can prevent 
 its coagulation on heating even to 100° C. This order is 
 followed when the reaction of the albumin is neutral ; 
 when it is faintly acid the order of the series is reversed. 
 Now it is found that the salts of the Hofmeister series 
 affect the properties of water such as viscosity, compressi- 
 bility, etc., in the same order as in the salting out of 
 albumin ; it is clear, therefore, that the effect on albumin 
 is due to the removal of water from the internal phase. 
 When their water is removed the particles become solid 
 and the emulsoid becomes a suspensoid, and so is easily 
 precipitated by electrolytes, etc. 
 
 In addition to this effect of salting out, neutral salts 
 combine with emulsoid colloids, but not apparently in the 
 same way as do acids and alkalies, for while the latter 
 frequently combine chemically as with the proteins, the 
 combination with neutral salts appears to be of the nature 
 of a physical union. They are, in fact, adsorbed. This 
 accounts for the fact that the apparent solubility of com- 
 paratively insoluble salts is much greater in a solution of 
 protein than in water. If, for example, phosphoric acid 
 is added to a solution of casein in lime water, calcium 
 phosphate is not precipitated, though it is formed. 
 
 It may be asked whether the heterogeneity of emulsoid 
 colloids affects the chemical reactions taking place in 
 them. It appears that in dilute solutions the effect is 
 practically nil, the reactions taking place as rapidly as in 
 water. In concentrated gels, however, diffusion is much 
 slower than in water, and can be varied by the addition of 
 various substances. The substances are those which 
 affect the distribution of water between the phases, and it 
 is hence believed that diffusion takes place in the liquid 
 
20 ACTION OF ENZYMES 
 
 phase. Again, reactions in gels do not proceed continu- 
 ously, but the product when insoluble is deposited in 
 strata separated by apparently clear intervals (Liese- 
 gang's phenomenon). This has not been satisfactorily 
 explained. 
 
 The formation of colloids appears to be connected with 
 complexity of constitution. Certain dyes like Congo-red 
 possess such large molecules that their solutions in water 
 take on the colloid form ; in other cases increase in 
 molecular weight is accompanied by a tendency to poly- 
 merisation and thus to pass into the colloid state. So the 
 lower fatty acids ionise normally and show normal osmotic 
 pressure ; sodium oleate, on the other hand, in 20 per cent, 
 solution has no osmotic effect, and therefore behaves as if 
 it had infinitely large molecules. In more dilute solution 
 it behaves more like a true solution. 
 
 Finally, colloid complexes can occur where the disperse 
 phase consists of two or more constituents each of which 
 is capable by itself of forming with the solvent a colloid 
 solution. 
 
 Summary. 
 
 (i) Enzymes are divided into ecto- and endo-enzymes, 
 of which the latter can be obtained only by rupture of the 
 cells containing them. 
 
 (2) Enzymes are colloids. They do not diffuse through 
 membranes. 
 
 (3) Colloids are heterogeneous systems consisting of 
 matter in a finely divided condition — the disperse or 
 internal phase — scattered through a homogeneous con- 
 tinuous or external phase. In many cases this can be 
 proved directly by the ultramicroscope. 
 
 (4) The effect of the fine division of the disperse phase 
 
COLLOIDS 21 
 
 is to extend its surface greatly. Many of the peculiarities 
 of colloid action are due to surface action. 
 
 (5) Colloid solutions are stable, their stability depending 
 partly on the electric charge of the particles, but mainly on 
 their continual motion. 
 
 (6) Their osmotic pressure is very small, but in some 
 cases measurable. 
 
 (7) Colloids are divided into suspensoids where the 
 disperse phase consists of solid particles, and emulsoids 
 where the particles are liquid. 
 
 (8) By their reaction to heat and other agents emulsoids 
 can be divided into types, such as the gelatine, the silicic 
 acid, the albumin, and the gum arable types. 
 
 (9) The process of gelatinisation is due to the trans- 
 ference of the solvent from the continuous phase to the 
 disperse phase. 
 
 (10) The action of electrolytes is in part due to their 
 alteration of the electric charge on the particles. Acids 
 and alkalies, in virtue of possessing H and OH ions, are 
 very powerful in this direction. Acids and alkalies also 
 increase the power of imbibition. 
 
 (11) Imbibition is a deposit of the solvent, water usually, 
 on the particles, filling up the capillary spaces between 
 them. The process is accompanied by compression of 
 the water. 
 
 (12) Proteins are emulsoid colloids and show their 
 properties. Acids and alkalies can combine with them 
 chemically with later ionisation of the combination. 
 This protein ionisation leads to increased imbibition. 
 
 (13) Neutral salts have several actions. They affect 
 the electric charge ; they can extract water from the 
 internal phase — salting out; they can unite with the 
 particles by a physical union — adsorption. 
 
 (14) Reactions in dilute colloids are not affected, but 
 
23 ACTION OF ENZYMES 
 
 diffusion is slowed in concentrated gels. Reactions 
 accompanied by precipitation show the precipitate in 
 layers in the gel. 
 
 (15) Tendency to colloid formation is favoured by 
 complexity of constitution, owing both to the size of the 
 individual molecules and the tendency of complex mole- 
 cules to polymerise. 
 
 (16) The disperse phase of a colloid may consist of 
 more than one form of matter each capable by itself of 
 forming the disperse phase of a colloid — a colloidal 
 complex. 
 
 Adsorption. 
 
 There are certain compounds known which do not 
 resemble chemical compounds in constancy of composi- 
 tion and more or less resistance to decomposition by 
 physical agencies. These are the adsorption compounds. 
 Examples are the compounds of charcoal and caramel, or 
 charcoal and litmus formed when finely divided charcoal 
 is shaken up with a solution of either of these colouring 
 matters. The charcoal on settling carries the colouring 
 matter with it, leaving the solution practically clear. 
 Charcoal has also the power of taking up gases, especially 
 those which are easily liquefied, such as ammonia or 
 sulphur dioxide. 
 
 An examination of the conditions in which such com- 
 pounds are formed shows that surfaces are always con- 
 cerned, whether these are the obvious surfaces at the 
 junction of liquids and gases, or the similar junction 
 between a precipitate and a supernatant fluid, or the 
 innumerable interfaces between colloid particles and the 
 solvent in which they float. That many substances have 
 a tendenc)' to come to the surface is indicated by the 
 
ADSORPTION 23 
 
 frothing which occurs when solutions containing soap, 
 albumin, saponin, etc., are shaken. Each bubble repre- 
 sents an interface between the liquid and air, and if the 
 whole be analysed it is found that the quantity of soap, 
 saponin, or albumin in the froth is proportionately much 
 greater than in the body of the solution. So condensed 
 may albumin and saponin be that they form solid pellicles 
 which are no longer soluble in the solution. 
 
 Adsorption compounds then are compounds formed at 
 surfaces between the body possessing the surface and 
 other substances which are in such a form, molecular or 
 atomic, as not to possess a surface. Colloidal complexes 
 have already been distinguished from these. 
 
 As is to be expected from the site of action, viz. surfaces, 
 the quantity of any substance adsorbed is proportional to 
 the active surface. Hence if the surfaces^ say, of a colloid 
 are already partly occupied by the same or another adsorbed 
 material, less available or active surface is left for further 
 adsorption. 
 
 It may be thought that the facts would be equally 
 accounted for by supposing that there is a solution of the 
 adsorbed body in the adsorbent, an absorption in fact, but 
 the arguments against this seem conclusive. In the first 
 place such a process would naturally be slow, as time 
 would be required for the diffusion of the substance 
 adsorbed into the adsorbent, whereas it is very rapid. 
 Secondly, the ratio of the distribution of the substance 
 between the disperse and continuous phase, if solid solu- 
 tion were the cause, would be independent of the con- 
 centration, provided the molecular weight of the solute 
 was the same in both solvents. Applying this to a 
 mixture of charcoal and water as representing the two 
 phases, with acetic acid as the substance adsorbed it is 
 found that the adsorption is not independent of the con- 
 
24 ACTION OF ENZYMES 
 
 centration, and hence either the molecular weight is not 
 the same in the two solvents, or the explanation of a solu- 
 tion in the charcoal is incorrect. A calculation based on 
 the assumption that the molecular weight is not the same 
 shows that the molecular weight in the charcoal must be 
 one-fourth only of that in water. Now acetic acid is 
 known to exist in water as single molecules, hence it must 
 exist in charcoal as quarter molecules, and this is impos- 
 sible. The explanation of solution in the disperse phase 
 is, therefore, incorrect. 
 
 It might also be thought that the compounds are 
 chemical in their nature, but substances like argon can 
 combine with charcoal which are incapable of entering 
 into any reaction ; also it is found that the composition of 
 the compound of acetic acid and charcoal varies with the 
 concentration of the acetic acid in the water, whereas a 
 chemical compound should be constant in composition 
 and independent of the concentration, at least over a 
 certain range of concentration, assuming that more than 
 one compound of the substances was possible. 
 
 It would, however, be a mistake to imagine that adsorp- 
 tion compounds are loose combinations which can be of 
 any proportions in a given set of circumstances. In given 
 circumstances of temperature and concentration these 
 compounds are perfectly definite. 
 
 While in a case of partition between two solvents where 
 a solute always distributes itself in proportion to its solu- 
 bility in each, doubling the concentration doubles the 
 concentration in both phases, and while again a chemical 
 compound is not altered in amount by increasing the con- 
 centration provided the total amount of the reacting 
 phase has been used up by the solute reacting with it, in 
 the case of adsorption compounds increasing the con- 
 centration does increase the amount of adsorption com- 
 
ADSORPTION 25 
 
 pound, but not in proportion to the amount of increase of 
 concentration. Doubling the concentration does not 
 multiply the amount of adsorption compound by 2, but 
 by some root of 2, or, expressing it algebraically, by 
 
 n t 
 
 V 
 
 2, or 2» 
 
 Freundlich's general formula is : 
 
 — = aC" . 
 m 
 
 In words: if x be the amount adsorbed by m grams of 
 adsorbent, and C the final concentration after all adsorp- 
 tion has taken place, a and n being constants for a 
 particular surface and solution, then the amount adsorbed 
 by m grams of adsorbent is equal to a constant multiplied 
 by the »"' root of the concentration. The law is not strictly 
 accurate for extremes of concentration, for in these satura- 
 tion of the adsorbent, if present in limited quantity, is 
 possible, after which no increase in the concentration of 
 the surrounding medium adds to the amount adsorbed, 
 but it is in satisfactory agreement with the facts through 
 a wide range of adsorption processes. 
 
 Since the amount adsorbed is related to the concentra- 
 tion, it follows that at a given concentration, the tempera- 
 ture remaining constant, an equilibrium is established 
 between the adsorbed material and the surrounding solu- 
 tion, and that the equilibrium is completely reversible — 
 diminishing the concentration diminishes the amount 
 adsorbed. In actual fact, however, fixing of the adsorbed 
 substance is often observed. This may be brought about 
 by a physical change in the adsorbed material (which may 
 become insoluble, for instance) or by a chemical reaction 
 with the adsorbent, leading in many cases to the destruc- 
 tion or denaturation of the adsorbed material. This fixa- 
 
a6 ACTION OF ENZYMES 
 
 tion is a relatively slow process and is non-reversible ; it 
 accounts for the fixing of various enzymes by charcoal, 
 e. g, trypsin. In this special case the fixation is probably 
 not due to a chemical reaction, for though water cannot 
 remove the enzyme casein can. 
 
 It follows also from the law connecting adsorption with 
 concentration that relatively niore adsorption takes place 
 in dilute solution. 
 
 An adsorbent is by no means indifferent in its action. 
 Some substances are more strongly adsorbed than others. 
 As will appear later, this is often due to the second reducing 
 the surface tension more strongly than the first, and just 
 as the components of chemical substances can be replaced 
 by others, so in adsorption compounds one substance can 
 replace another if it is more strongly adsorbed. The 
 action of casein in breaking up the combination between 
 enzyme and charcoal is an instance. Another is the effect 
 of saponin on a charcoal-rennet combination. The action 
 of rennet on milk is prevented by its adsorption to charcoal, 
 but if saponin be added to such an inactive mixture the 
 milk sets because the rennet has been displaced from its 
 combination with the charcoal by the saponin. 
 
 The actual process of adsorption is rapid once the 
 surface of the adsorbent has been reached. Cases which 
 seem to show a time intei'val are probably really due to 
 slowness of diffusion, not of adsorption, and consequently 
 adsorption is hastened by shaking. 
 
 Heat has a double effect on adsorption. The actual 
 rate of adsorption is increased, but the amount is 
 diminished, for heat dissociates an adsorption compound. 
 The explanation of this is found in the fact that surface 
 tension has a negative temperature coefficient, i. e. it 
 deer-eases with rise of temperature. 
 
 The condition of the adsorbed material from the point 
 
ADSORPTION a/ 
 
 of view of its reactivity is of importance. For the time 
 being it adheres so strongly to the adsorbent that it 
 cannot show its characteristic reactions. This applies to 
 salts which are in the "masked" condition. Not merely 
 can they not react chemically, but they cannot affect the 
 osmotic pressure of the solution. 
 
 The causes of the formation of adsorption compounds 
 seem to be mainly three — mechanical surface tension, the 
 electrical conditions, and chemical attraction ; but the 
 surface film of any solution, including in this the films at 
 the interfaces where there is a particulate phase, has 
 properties which differentiate it from the remainder of 
 the solution, e.g. potassium sulphate is 60 per cent, more 
 soluble in the surface film. Also there are evidences of 
 great powers of compression at the interface, as was men- 
 tioned in connection with carbon dioxide under the head 
 of colloids. The tendency to adsorption of the easily 
 liquefiable gases, sulphur dioxide and ammonia, comes 
 under this head. 
 
 The action of the three main causes given is on the 
 energy of the system, and is in accordance with the 
 principle that the energy content of a system always tends 
 to decrease. The Gibbs theorem states that if a body in 
 solution by concentration on the surface of another 
 phase lowers the surface energy, the process will tend to 
 take place. Surface energy, however, is only one of the 
 factors concerned ; electrical, chemical, and thermal 
 forces also play their parts. Hence Ostwald has general- 
 ised the theorem into a statement that increase of con- 
 centration at a surface will always occur when the 
 potential of any form of energy at this surface can be 
 diminished by the process. 
 
 That the surface of fluids behaves as if covered by a 
 stretched film can be demonstrated by a loop of string in 
 
28 ACTION OF ENZYMES 
 
 a soap film. If the film be punctured through the loop, 
 the loop will be drawn into a circular form, i. e. it sur- 
 rounds as large an area as possible. The spherical shape 
 of drops is another indication, as this shape possesses the 
 smallest surface possible. This fact has been made the 
 basis of measurement of surface tension by the stalagmo- 
 meter. The cause of surface tension appears to be the 
 unbalanced attraction of the molecules in the surface film 
 by those at a greater depth from the surface. 
 
 Surface tension in fluids can be measured at the face 
 between them and a gas or vapour, but the surface 
 tension of solids in contact with a liquid cannot be 
 measured directly ; by indirect methods of measurement 
 it appears to be much larger than the former ; hence the 
 extreme condensation which may occur or charcoal 
 particles. 
 
 Most substances in solution lower the surface tension 
 at a liquid-air interface ; inorganic salts raise it, but not 
 to any great extent. At the interface between liquids all 
 substances, including inorganic salts, lower the surface 
 tension. Since these bodies reduce the surface energy 
 when at an interface, by the principle enunciated they 
 must inevitably be forced to that interface, and hence 
 adsorption takes place. Very great variations, however, 
 are to be found in the power of bodies to lower surface 
 tension ; some, such as the bile salts, have a great effect ; 
 others, such as sugar, have very little. 
 
 The second force at work aiding adsorption is electrical. 
 Electrolytes are adsorbed in much larger or smaller 
 amounts than can be accounted for by the action of 
 mechanical surface tension. Most substances in water 
 have a negative charge, as mentioned under the head of 
 " Colloids," therefore a positively charged body will be 
 attracted to them. But if the adsorbent has a charge of 
 
ADSORPTION 29 
 
 the same sign little adsorption will take place. It has 
 been mentioned under the head of " Colloids " also that 
 electrolytes can diminish, annul, or reverse the charge ; 
 if, therefore, a neutral salt be added the charge on the 
 adsorbent will be altered and adsorption can take place. 
 For the same reason adsorption is hindered by a neutral 
 salt if the adsorbent and body adsorbed already have 
 charges of opposite sign. With emulsoid colloids, how- 
 ever, their comparative insensitiveness to electrolytes tells 
 against this reversal of the charge. 
 
 The relation of the electric charge to the surface tension 
 is interesting, as its effect is to diminish the latter. Sur- 
 face tension tends to pull the molecules of the surface 
 film together ; electric charge to force them apart. As 
 applied to the disperse phase of a colloid the electrical 
 forces are usually much stronger than mechanical surface 
 tension. Lewis has shown that the amount of pure sodium 
 oleate adsorbed by a water-oil interface is one hundred 
 times greater than that calculated to be due to diminution 
 of surface tension. Bayliss speaks of this as an example of 
 the power of electrical adsorption, but Willows and 
 Hatschek think that such substances as sodium oleate, 
 methyl orange, Congo-red, etc., form colloidal and not 
 true solutions, and that when they are adsorbed they 
 form gelatinous or semi-solid skins on the adsorbing 
 surface in which the concentration may be very great. 
 
 The third cause of adsorption is chemical attraction. 
 While adsorption is generally the prelude to chemical 
 action, and in the organism practically always precedes it, 
 it does not follow that chemical action must follow 
 adsorption. The two bodies attached may be incapable 
 of reacting chemically, e. g. in the case of the adsorption 
 of aniline to mercury. The attraction of an acid dis- 
 solved in the medium to a base forming, or on the disperse 
 
30 ACTION OF ENZYMES 
 
 phase of a colloid may be put down to this cause, or may 
 be looked at as an electrical phenomenon. That the 
 latter is the main cause is said to be proved in such cases 
 as the union of Congo-red with thorium hydroxide, where 
 a change of colour takes place on chemical union, but 
 precipitation takes place before the change of colour. 
 Nevertheless it is impossible to deny that chemical attrac- 
 tion may be at the root of such cases, for it is clear that 
 chemical action cannot take place between two substances 
 till their molecules come into contact, and it may be that 
 in colloids the adsorption, which is a preliminary stage to 
 chemical action, where this occurs, is caused by chemical 
 attraction, and that the electrical charges have either 
 only a secondary effect or are so intimately bound up with 
 chemical attraction as to be inseparable from it. 
 
 There are a series of cases of adsorption which are not 
 adequately accounted for by the effects of mechanical 
 surface tension or by electrical attraction. These are 
 spoken of as due to " specific " adsorption where a par- 
 ticular kind of surface takes up preferentially a particular 
 substance. It appears that in these cases at any rate the 
 chemical configuration of the surface must be taken into 
 account. For example, carbon and red oxide of iron 
 adsorb benzoic acid ten times as strongly as they do 
 acetic acid, chromic acid adsorbs both acids equally, 
 while platinum black adsorbs acetic acid slightly more 
 strongly than benzoic acid, but neither to any very marked 
 extent. Crystalline substances, such as barium carbonate, 
 only adsorb crystalloids when these are isomorphous, i. e. 
 crystallise in a similar form. Gelatine only adsorbs sugar 
 after treatment with formaldehyde. The action of enzymes 
 on optical isomers, an enzyme affecting one isomer and 
 not, or only slightly, another, comes under this head. 
 Indeed a great deal of enzyme action is bound up with 
 
ADSORPTION 31 
 
 specific adsorption, and it is on this account unfortunate 
 that the subject has not been more studied. Tentatively 
 I suggest that chemical attraction lies at the root of these 
 cases. Where chemical action is well marked it occurs 
 so rapidly that the preceding adsorption is masked, or is 
 practically simultaneous with it in colloid solutions. 
 Where it is weak in the given conditions adsorption is 
 separable from chemical action as in the case of Congo- 
 red and thorium hydroxide, where the chemical reaction 
 does not take place till the temperature is raised. The 
 very different affinities of the elements for oxygen are 
 cases in point. The gradations run from the intense 
 reaction of metallic sodium or potassium in water to the 
 practical absence of action with the " noble " metals. If 
 a tendency to chemical action exists which may be 
 fulfilled if the experimental conditions are altered, as in 
 the case of charcoal in oxygen before heat is applied, then 
 it is probable that an attraction which may be called 
 specific exists between such bodies, and if one of these 
 bodies possesses surfaces the phenomenon of specific 
 adsorption will show itself. It is true that the examples 
 given of specific adsorption do not look as if they were 
 the preliminaries of chemical action, but Willows and 
 Hatschek state that chemical combination cannot be 
 rejected a priori even where it appears extremely improb- 
 able. Thus it is fairly definitely established that oxidation 
 of carbon takes place when potassium permanganate is 
 adsorbed by charcoal. The slow increase in the taking 
 up of iodine by charcoal after the initial rapid adsorption 
 may be due to a passage of iodine into the mass of the 
 soHd, but it is suggested by Freundlich that chemical 
 combination is more probable, since iodine is a very 
 reactive substance. The fact that the second part of the 
 process is irreversible and does not vary with the kind of 
 
32 ACTION OF ENZYMES 
 
 charcoal used, while the first does and is reversible, is in 
 favour of a slow chemical action. 
 
 Certain other facts bearing on the question of specific 
 adsorption may be mentioned, though their explanation is 
 still to seek. Gelatine takes up more acid fuchsin than 
 Congo-red, while filter paper takes up the same amount 
 of both. Gelatine takes up calcium salts more readily 
 than potassium salts. If strips of filter paper are immersed 
 in solutions of various salts it will be found that potassium 
 salts rise higher than those of calcium and barium, though 
 the water rises to the same height in all three cases. 
 Mastic precipitates as adsorption compounds some of the 
 proteoses in Witte's peptone, but not others. Kieselguhr 
 takes from a mixture of the two proteolytic enzymes of 
 the spleen large quantities of the a-protease, leaving the 
 6-protease almost untouched. Possibly the electric charge 
 of one protease differs from that of the other in sign. 
 Invertase is adsorbed by certain inert powders but not by 
 others. In the case of the proteases of the spleen charcoal 
 adsorbs the same amount of both. 
 
 Summary. 
 
 (i) Adsorption compounds are formed at suriaces. The 
 tendency of many substances to come to the surface is 
 seen in froth. 
 
 (2) The quantity of substance adsorbed is proportional 
 to the active surface. 
 
 (3) Solution of the adsorbed substance in the adsorbent 
 does not account for the facts. 
 
 (4) There is an essential difference between adsorption 
 and chemical compounds, especially indicated by the 
 want of constancy of composition when the conditions are 
 altered. 
 
ADSORPTION 33 
 
 (5) In given conditions of concentration and tempera- 
 ture adsorption compounds are perfectly definite. 
 
 (6) The law relating the concentration of an adsorption 
 compound to the concentration of the solvent shows that 
 while the concentration increases with that of the solvent 
 it does not do so in direct proportion. 
 
 (7) A reversible equilibrium exists between the adsorp- 
 tion compound and the surrounding solution. Fixation 
 of the compound, however, sometimes occurs. 
 
 (8) Relatively more adsorption takes place in dilute 
 solution. 
 
 (9) A substance which is more strongly adsorbed can 
 replace one which is less strongly adsorbed. 
 
 (10) The actual process of adsorption is rapid. 
 
 (11) Increase of heat increases the rate of adsorption 
 but diminishes the amount adsorbed. 
 
 (12) The adsorbed substance is so firmly bound to the 
 adsorbent that it cannot give the chemical or physical 
 reactions which it would give if not adsorbed. 
 
 (13) The causes of the formation of adsorption com- 
 pounds are, mainly, mechanical surface tension, the 
 electrical conditions, and chemical attraction, but changes 
 in solubility and compressibility at the surface also play a 
 part. 
 
 (14) These causes act by diminishing the energy of the 
 system. 
 
 (15) The behaviour of a soap film and the shape of 
 drops of water are proofs of the existence of surface 
 tension. 
 
 (16) Electrical adsorption d( peiids on the >if;ns of the 
 charges on the bodies affected, .nuJ these cm be altered 
 by electrolytes. 
 
 (17) Electric charf^es tend t dimini.sh the effect of 
 mechanical surface tension. 
 
34 ACTION OF ENZYMES 
 
 (i8) Chemical attraction has probably a great influence, 
 but this has not been fully studied. Specific adsorption 
 may be due to chemical attraction. 
 
 The Properties of Enzymes. 
 
 Before discussing the method of action of enzymes it is 
 necessary to understand clearly what their action is. The 
 ground has been to some extent cleared by the discussion 
 of colloids and the adsorption compounds which they 
 form, and it will appear that enzyme action as compared 
 with that of inorganic catalysts is largely modified by 
 their peculiar physical form. Their resemblances to, and 
 their differences from inorganic catalysts will require con- 
 sideration. 
 
 (i) Unlike inorganic catalysts no enzyme has been 
 isolated, so far as is known, in a pure condition, and their 
 existence is recognised in a solution by their action. It 
 is found that in reactions which can be brought about 
 either by an enzyme or by an inorganic catalyst, the 
 enzyme action is the more powerful of the two. Lactase 
 has been found to hydrolyse about one-fourth of the milk- 
 sugar in a 5 per cent, solution in one hour at 35° C, whereas 
 even so strong an acid solution as twice normal hydro- 
 chloric acid required about five weeks to do the same. 
 Another proof of the great activity of enzymes is furnished 
 by invertase, which can hydrolyse 200,000 times its weight 
 of cane-sugar. 
 
 The action of an enzyme, which alone furnishes the 
 proof of its existence, can be investigated by discovering 
 the chemical nature of the bodies that are formed, and 
 when these are known by studying the rate of change and 
 the various conditions which affect it. The chemical 
 nature of the products is discovered by the usual chemical 
 
PROPERTIES OF ENZYMES 35 
 
 methods, while for the rate of change various physical 
 methods are available. These are mentioned in some 
 detail by Bayliss. An enumeration of these methods 
 may be of interest. The rate of change is related to the 
 optical activity of the solution, its copper-reducing power, 
 its refractive index, its viscosity, its molecular concentra- 
 tion as measured by its effect on the freezing point, and 
 its electrical conductivity, the last being a method in 
 frequent use. Where gas is evolved this can be measured, 
 and the time during which it occurs. The spectro-photo- 
 meter is used in colour changes, and the dilatometer in 
 changes of volume. The difficulties of investigation are 
 considerable in the later stages when the action is very 
 slow, or when very small amounts of enzyme are present, 
 for these are liable to destruction. The possibility that 
 what is being measured in these cases is the slow non- 
 enzymic rate of change has to be kept in mind. 
 
 (2) The action of enzymes is largely specific ; they act 
 on only one compound, e. g. invertase only on cane-sugar, 
 or on only one group of compounds, e. g. maltase only on 
 the a-glucosides, emulsin (from almonds) only on the 
 /3-glucosides. This differential action on optical isomers, 
 which do not differ in chemical formula but only in the 
 arrangement of one or more of their component radicles 
 in space, so impressed Fischer that he compared the 
 action of enzymes to that of a lock and key where only 
 one key will open a particular lock, 'no other key will fit 
 it, nor will its key fit other locks. This specificity is not a 
 unique condition in chemistry. Euler points out that the 
 reactivity of the simple and substituted mono-, di-, and 
 tri-phenols is dependent on their constitution, whether 
 oxidases or the inorganic manganese compounds as cata- 
 lysts are used. For example, taking the dihydric phenols 
 where in hydroquinone the OH radicles are in the para 
 
36 ACTION OF ENZYMES 
 
 position, in pyrocatechol in the ortho position, and in 
 resorcinol in the meta position, the first is oxidised 
 rapidly, the second more slowly, while the last is 
 extremely resistant to oxidation. Again, lipase shows 
 great differences in its power of hydrolysing such closely 
 allied chemical individuals as ethyl acetate and butyrate, 
 but hydrochloric acid also in relation to ethyl formate and 
 methyl benzoate shows constants of hydrolysis in the 
 ratio of I'l to 0*0003. The exact chemical constitution 
 of the substrate, or substance on which the enzyme or 
 inorganic catalyst acts, is therefore of great importance, 
 though it may be that a similar chemical constitution is 
 not incompatible with great physical differences. Even 
 the specificity as regards optical isomers has chemical 
 analogies, for if an optically active alcohol esterifies two 
 opposed optically active acids it is found that the veloci- 
 ties of esterification are not equal. In general it may be 
 said that two optical antipodes when combining with the 
 same asymmetric substance do so with unequal velocities. 
 An interesting consequence follows. Lipase is found to 
 hydrolyse inactive menthyl mandelate, but forms the 
 dextro component more rapidly than the Isevo. This is 
 exactly what occurs in the hydrolysis of the ester formed 
 by an optically active alcohol with two opposed optically 
 active acids, and hence it follows that lipase is optically 
 active. Optical activity is widespread among the enzymes 
 and appears to account for the great specificity shown in 
 their action on optical isomers. Optically active catalysts 
 of known composition act in the same way on optical 
 isomers. 
 
 While a well-marked specificity is characteristic of 
 enzyme action it would be a mistake to suppose that it is 
 absolute. Lipase, for example, certainly affects the 
 dextro-component of menthyl mandelate most, but it can 
 
PROPERTIES OF ENZYMES 37 
 
 also affect the Isevo-component. Taking the cases of 
 maltase and emulsin as representing specific action in its 
 most marked form, it is found that if these act together on 
 glucose and alcohol, by synthetic action a mixture of the 
 «- and j3-glucosides is formed. If, however, they are 
 added in sequence, so that the first has had time to have 
 its full effect and therefore only one glucoside is present, 
 the addition of the second, when time is given for its 
 action, brings the position of equilibrium to the same 
 position as if the two had been added simultaneously. In 
 this case one glucoside must have been converted into the 
 other, and the conversion has been brought about by an 
 ■ enzyme supposed to be incapable of acting on it. In 
 fact, Fajans shows that the various experimental data as 
 to the action of different enzymes acting on optical 
 isomers, including synthetic action, can be explained by 
 the hypothesis of both isomers being hydrolysed by the 
 same enzyme but at different rates. 
 
 Nevertheless, when all deductions are made, the almost 
 complete specificity of enzymes is an outstanding fact 
 which has to be accounted for. The enzymes which 
 hydrolyse the fats do not hydrolyse the proteins, and the 
 same is true of the enzymes of carbohydrates. It is, 
 however, difficult to admit the assumption that every 
 substance found to be affected by an enzyme is affected by 
 an enzyme which acts on it alone or on the small group 
 of substances to which it belongs. Enzymes are found, 
 like lactase in almonds, which have never had their 
 specific substrate exposed to their action ; if the assump- 
 tion then were to be admitted, not merely would enzymes 
 be numbered by hundreds, but it would be necessary to 
 admit that enzymes are formed as by-products where 
 they are never likely to be wanted. Probably the 
 specificity is a group specificity, and any substance con- 
 
38 ACTION OF ENZYMES 
 
 forming in any of its parts to the physical or chemical 
 type characteristic of the group can be affected by the 
 group enzyme. 
 
 (3) It has already been pointed out in considering 
 inorganic catalysts that in a reversible equation equi- 
 librium can be reached from either side of the equation, 
 and that if a catalyst catalyses the direct action it must 
 also catalyse the reverse. As the changes produced by 
 enzymes are generally of the reversible order, an equi- 
 librium being produced, it follows that, given the appro- 
 priate conditions, the reverse action must be accelerated 
 by the enzyme. Most enzymes have a hydrolytic action, 
 one substance becoming two by the addition of the 
 elements of water, hence the two substances can becorne 
 one by the subtraction of water. The first definite proof 
 of a synthetic action was brought forward by Croft Hill, 
 who synthesised a form of maltose from glucose under the 
 influence of maltase. Since then^ " so many other cases 
 have been discovered that the impression is distinctly 
 given that it is merely a question of finding the proper 
 conditions to be able to obtain synthesis from all 
 enzymes" (Bayliss). 
 
 Most enzyme reactions are carried out in the presence 
 of excess of water, and hence it follows that hydrolysis 
 predominates over synthesis. In synthetic reactions the 
 amount of water must be limited. A certain amount is 
 necessary, for in the synthesis of a true fat from glycerine 
 a certain small amount of water must be present, since 
 if pure glycerine is used there is scarcely any synthesis. 
 
 There are many evidences of synthesis in the organism : 
 the deposit of glycogen in the liver though glucose 
 iilone is brought to it, and the formation of proteins in the 
 body though the proteins of the food are broken down to 
 their amino-acid constituents by the erepsin of the intes- 
 
PROPERTIES OF ENZYMES 39 
 
 tinal wall. Since water must be limited for synthesis to 
 take place, special arrangements must exist for securing 
 this, for there is excess of water in all the fluids of the 
 body. Possibly there is actual solution in the disperse 
 phase of the body colloids where water is nearly absent, 
 or the protoplasmic sols everywhere present contain much 
 less water generally than the body fluids. 
 
 E. F. Armstrong has expressed the view that enzymes 
 do not build up the same molecules as those they break 
 down, founding his opinion on the fact that Croft Hill's 
 synthesis produced not maltose but isomaltose, and on 
 other analogous facts. There is, however, a special 
 explanation in Croft Hill's case given by Euler, and as 
 enzyme preparations are as yet of no high degree of purity 
 the usual view that the same molecules are synthesised as 
 those broken down has much to recommend it. 
 
 Even when concentrated solutions are used, and the 
 amount of water present severely limited, certain reactions 
 proceed almost to the point of complete hydrolysis. The 
 explanation probably is that the acceleration of the syn- 
 thetic reaction may be very slow as compared with the 
 hydrolytic. For chemical reasons there may be peculiar 
 difficulties in the synthesis as in the case of the tertiary 
 alcohol formed in the hydrolysis of salicin, which, like 
 other tertiary alcohols, is esterified with difficulty. 
 
 (4) " The supposition that combination of the catalyst 
 with the substrate yields the molecules which carry on the 
 reaction has already received general acceptance in 
 enzymology" (Euler). 
 
 " There is abundant evidence that a combination of 
 some kind is formed between the enzyme and the sub- 
 strate preparatory to the action of the former. There is 
 also similar combination between enzyme and products " 
 Bayliss). 
 
40 ACTION OF ENZYMES 
 
 All the facts bearing on the specificity of enzymes con- 
 firm this conclusion, for these can only be explained on 
 the hypothesis that enzymes form addition compounds 
 with their substrates. 
 
 Again, enzymes are much more sensitive to heat when 
 in a fairly pure condition than when either their substrates 
 or products are present. For example, invertase will 
 stand a temperature 25° higher in the presence of cane- 
 sugar than in its absence, and it is also protected from 
 the action of heat by its products. It is difficult to see 
 how this could occur unless the enzyme entered into some 
 kind of union with the sugar. 
 
 Another argument in favour of this combination is 
 derived from the reaction velocity in the initial stages of 
 certain enzyme reactions when low concentrations of the 
 enzyme are employed. The rate of change is directly 
 proportional to the time of action, i. e. equal amounts are 
 changed in equal times. When the amount of substrate 
 was varied, still keeping the enzyme low proportionally, 
 in the early stages the amount inverted in equal times was 
 nearly the same in all. The law of mass action was not 
 followed. This can only be explained by presuming a 
 compound between substrate and enzyme which persisted 
 for an appreciable time, so that at first the enzyme was 
 always in a saturated condition as regards the substrate 
 and could not take any more up till that already taken up 
 was hydrolysed. 
 
 Again, the enzyme may produce products which retard 
 its action, precipitate, or even destroy it, this altogether 
 apart from the slowing of the general reaction which 
 necessarily occurs as the reverse action gathers way. For 
 example, benzaldehyde is produced by emulsin and is 
 deleterious to it. It is possible that these bodies influence 
 the enzyme as a colloid and reduce the active surface. 
 
PROPERTIES OF ENZYMES 41 
 
 These bodies could scarcely exert their influence without 
 combining with the enzyme. 
 
 Besides the products proper the reaction of the medium 
 is of great importance, and this may be altered inevitably 
 by the course of the reaction. 
 
 The nature of the compound formed between the 
 enzyme and its substrate is at first at any rate a surface 
 condensation, an adsorption compound, for it is found 
 that the law of adsorption is obeyed, and there is also 
 definite evidence of the formation of such compounds. 
 Amylase which can pass a porous clay filter by itself 
 cannot do so when it is mixed with the calcium salt of 
 caseinogen, on which it has no action whatever. Pancreatic 
 lipase is entirely insoluble in fats, fatty acids, and solutions 
 of fats in ether, yet it acts on these bodies, hence the 
 reaction must take place in or on the enzyme, probably on 
 it, for in the case of emulsin, which is able to hydrolyse its 
 substrate in 90 per cent, alcohol (in which it is completely 
 insoluble), there was in these circumstances no sign of 
 imbibition. Again, a definite adsorption compound between 
 starch and amylase has been observed. A 2 per cent, 
 solution of starch remains unchanged alone, but if a small 
 amount of amylase is added a precipitation gradually 
 appears in half an hour or less. Hydrolysis takes place 
 subsequently. Starkenstein has shown that adsorption 
 may take place without chemical action in the case of 
 dialysed liver amylase, but, generally speaking, adsorption 
 is the preliminary stage of chemical action. Whether 
 the enzyme enters into the chemical action or not 
 will be discussed later when the method of action of 
 enzymes is considered. Bayliss states that it is not 
 known. He points out that mere concentration on the 
 surface will by mass action greatly accelerate any process 
 naturally occurring, but he does not think it possible 
 
4a ACTION OF ENZYMES 
 
 to explain all the various activities of enzymes in this 
 way. 
 
 (5) In spite of certain differences in the course of their 
 action the essential action of enzymes is catalytic. The 
 resemblances to inorganic catalysts are much greater than 
 their diiferences, especially if the fact is taken into con- 
 sideration that the catalysis takes place in a heterogeneous 
 system, not in a homogeneous. In a homogeneous system 
 where all the reacting bodies are in solution it may be 
 assumed that the reacting molecules are within each 
 other's spheres of influence. In a heterogeneous system 
 where one phase is particulate the facts, first, of diiFusion 
 to the particles by the dissolved substance, and, second, of 
 its adsorption to the particles, must be taken into account, 
 for these are the necessary conditions of chemical 
 action. Both are rapid, nevertheless the rate of the 
 reaction is necessarily to some extent conditioned by 
 them. These considerations apply to catalysis in any 
 heterogeneous system where the catalyst is in the par- 
 ticulate form, and, therefore, variations due to this 
 seen in the action of enzymes cannot be put down 
 to difference between enzymic and inorganic catalytic 
 action. 
 
 The resemblances may be classified as follows : 
 
 {a) They increase the rate of a normally very slow 
 reaction. Water alone at 100° C. can hydrolyse cane 
 sugar to glucose at a measurable rate; it is probable, 
 therefore, that the process takes place at room tempera- 
 ture at a very slow rate, and the action of invertase is to 
 increase this rate. 
 
 (b) In certain cases they can be recovered at the end 
 unchanged. It is here that, speaking generally, one of 
 the differences between enzymes and inorganic catalysts 
 is to be found. Nevertheless in certain cases the enzyme 
 
PROPERTIES OF ENZYMES 43 
 
 can be extracted after the reaction is complete to hydrolyse 
 fresh material. 
 
 (c) Minute quantities are capable of effecting a large 
 amount of change. Facts bearing on this have already been 
 mentioned. 
 
 {d) The curve of velocity of action resembles that of 
 inorganic catalysts. At this point again differences are to 
 be noticed. 
 
 (e) The enzyme has no effect on the final position of 
 equilibrium. In certain cases a false equilibrium is 
 reached owing to the destruction or inactivation of the 
 enzyme before it has done its work. It is not a real 
 equilibrium, for the normal uncatalysed reaction is still 
 progressing, but so slowly as to be practically non- 
 measurable. 
 
 Before classifying the differences it is desirable to point 
 out on what factors the velocity of the enzymic reaction 
 depends. It is clear that the velocity is determined by 
 the concentration of the dissolved body in the colloid. 
 Analysing this the factors are : 
 
 (i) The concentration of the substrate. The rate should 
 be by mass action directly proportional to this concentra- 
 tion, but this only occurs when the concentration of the 
 enzyme is not too low, else the enzyme will be saturated 
 with the substrate without reducing appreciably the con- 
 centration of the substrate. There is also an upper limit 
 to the concentration of the substrate beyond which action 
 is again slowed. This may depend on the colloidal con- 
 stitution of enzymes. 
 
 (ii) The concentration of the enzyme. If the substrate 
 is in excess the velocity will be in direct proportion to the 
 concentration of the enzyme, because the whole of it can 
 enter into combination with the substrate. Later, when the 
 substrate diminishes, the velocity will be a function of the 
 
44 ACTION OF ENZYMES 
 
 concentrations of both. At the end of the reaction, when 
 the enzyme is in excess, it will again be a function of one 
 only, but this time of the concentration of the substrate. 
 With inorganic non-particulate catalysts it is usually found 
 that the velocity of the reaction is in direct linear propor- 
 tion to the amount of the catalyst added. 
 
 (iii) The presence or absence of electrolytes. As colloids 
 enzymes are very sensitive to the action of electrolytes, 
 but the effects produced will be discussed in a separate 
 section. 
 
 (iv) The nature of the antiseptic added to prevent cell 
 or bacterial action. Toluene is the most generally satis- 
 factory as it is chemically inert. It is noteworthy that all 
 enzymes do not react in the same way to the same anti- 
 septic : to some it may be harmless, to others injurious. 
 Obviously injury to the enzyme by removing the whole or 
 part of it from the reaction interferes with the velocity of 
 the reaction. 
 
 The differences between the actions of inorganic catalysts 
 and enzymes may be thus classified : 
 
 (a) The velocity of reactions affected by inorganic cata- 
 lysts depends on the concentration of the catalyst ; of 
 enzymic reactions at first on that of the enzyme, later on 
 the concentration ratio between enzyme and substrate, 
 and last on that of the substrate only, as discussed under 
 (ii) above. 
 
 (6) The position of equilibrium is in some cases not the 
 same under enzymic action and under the action of an 
 inorganic catalyst catalysing the same reaction, e. g. an 
 acid. The factors at work here may be : 
 
 (i) When intermediate compounds are formed their 
 rates of formation and decomposition are not neces- 
 sarily identical, so that in equilibrium there may be 
 excess of one or other. 
 
PROPERTIES OF ENZYMES 45 
 
 (ii) The heterogeneity of the system. For example, 
 surface films have different powers of solubility from 
 those of the body of the solution. It is found that 
 the equilibrium of the hydrolysis of a salt of a fatty 
 acid is altered by the presence in its solution of 
 extensive surfaces. 
 
 (iii) A very small amount of energy is required to 
 change the equilibrium point in reactions which are 
 practically thermo-neutral. This energy may be 
 derived from an obscure surface action. 
 
 (iv) If the enzyme takes up water the concentra- 
 tion of water at the point of action will be greater 
 than in the body of the solution ; this excess of con- 
 centration of water would mean a shifting of the 
 equilibrium towards the point of greater hydrolysis. 
 
 (c) The products of the reaction directly iniiuence the 
 enzyme. The velocity of an enzymic reaction is altered 
 by the addition of the products of the reaction to the 
 system, not only as a condition of a reversible equation, 
 as would occur with acid as a catalyst, but also by 
 combination with and inactivation of the enzyme. 
 
 (d) Whereas great specificity of action is exceptional 
 among inorganic catalysts it is the rule with enzymes. 
 
 (6) Heat has a marked effect on enzymes : most are 
 destroyed by exposure to a temperature of 65-75° C., but 
 there are exceptions. Some of the oxidases, e. g. medicago 
 laccase, are not destroyed at 100° C, and trypsin in acid 
 solution can be boiled. Since coagulable protein is practi- 
 cally always present the enzyme is precipitated probably 
 because it is adsorbed to this, as occurs with fibrin ferment 
 though this is not really a ferment. The effect of the 
 presence of substrate or products is to diminish the 
 sensibility to a raided temperature, as previously men- 
 tioned. As a rule enzymes are coagulated by heat or 
 
46 ACTION OF ENZYMES 
 
 precipitated, but in some cases the enzymes seem to be 
 merely carried down by adsorption or changed in their 
 physical state reversibly. As a general rule chemical 
 reactions increase in speed by rise of temperature. Van't 
 HofPs rule is that for every rise of io° C. the rate of a 
 reaction is about doubled or trebled. This, the temperature 
 coefficient, is high for enzymes, and it has hence been 
 argued that the action of enzymes is chemical. But the 
 temperature coefficient of imbibition is also high, so that 
 the argument is of doubtful value. 
 
 If the action of heat on enzymes is similar to that of 
 the heat coagulation of proteins the cause is probably the 
 same, viz. a change in the state of solution. In many 
 cases salts increase the stability of enzymes; thus the 
 optimum temperature of pancreas amylase in a starch 
 solution containing 0'2 per cent, of sodium chloride is at 
 50° C, while in pure aqueous solution it is at 35° C. The 
 expression, optimum temperature, has no physical meaning 
 beyond being a statement of the fact that " at a certain 
 temperature the increased velocity of action due to this 
 raised temperature is more than sufficient, for a time 
 only, to counteract the rapid destruction of the enzyme " 
 (Bayliss). 
 
 Extreme cold has no effect. Enzymes are not 
 destroyed by exposure to the temperature of liquid air 
 for two days. 
 
 At this point the effect of light and other radiations may 
 be mentioned. The biological action of light on the 
 inactivation of enzymes is of two kinds, one requiring the 
 presence of oxygen and accelerated by fluorescent sub- 
 stances, the other produced only by the action of ultra- 
 violet rays where no part is played by oxygen or by 
 fluorescent substances. 
 
 Rontgen rays do not weaken enzymes. 
 
PROPERTIES OF ENZYMES 47 
 
 Radium rays influence some enzymes, but not all in the 
 same way. 
 
 (7) The action of electrolytes on enzyme reactions is 
 marked. An ideal description of this action would 
 divide the subject into (i) the action on enzymes them- 
 selves, and (2) the action on their substrates and products, 
 and each of these would have two subdivisions according 
 as the action was (a) chemical or (b) physical. It is not 
 possible in the present state of knowledge to follow such 
 an ideal treatment of the subject, as the exact method of 
 action of an electrolyte is often unknown. On this 
 account the subject will be divided according to the 
 general action of electrolytes as adjuvants or paralysers, 
 and a distinction will be drawn between acids and alkalies 
 on the one hand and salts on the other. 
 
 A distinction must also be drawn at the outset between 
 the more general action of electrolytes and the specific 
 action of certain electrolytes on certain enzymes. These 
 latter will be treated under the heading of " Co-enzymes." 
 
 As regards acids and alkalies it is found that while 
 most enzymes are assisted by acids in low concentration 
 others are inhibited and are assisted by alkalies, while 
 others again act best in a neutral medium. Among the 
 acid-assisted are pepsin, invertase, lactase, nuclease, and 
 the autolytic enzymes. Those accelerated in action by 
 alkalies are mainly the enzymes of the pancreas and 
 intestine, and also the peroxidases. Malt diastase, blood 
 lipase, and emulsin are best suited by a neutral medium. 
 In all cases the concentration of the acid and alkali must 
 be low : higher concentrations inhibit the enzyme action. 
 
 The action of acids and alkalies may be classified as 
 follows : 
 
 (a) Conversion of proenzymes, or zymogens, into the 
 active enzymes. This is especially well seen with pep- 
 
48 ACTION OF ENZYMES 
 
 sinogen, which is converted very rapidly by hydrochloric 
 acid. 
 
 (b) They change the velocity of the reaction, which 
 reaches a well-defined maximum for a certain concentra- 
 tion of the H ions. 
 
 (c) They influence the stability or decomposition of the 
 enzyme itself, this stability exhibiting a maximum for a 
 certain concentration of the H and OH ions, which is not 
 necessarily the same as that for maximum action. 
 
 The sensitiveness of enzymes to acids and alkalies is 
 very great, has been explained by the excessively small 
 concentration of the enzyme itself. As to their method of 
 action certain suggestions have been made. 
 
 (a) In many cases the concentration determining the 
 optimum of acid or alkali must correspond exactly with 
 the quantity of acid or alkali necessary for the neutralisa- 
 tion of the solution. This means that their action is on 
 the H ion concentration. 
 
 (b) In some cases they act as activators of zymogens. 
 Possibly the zymof^en exists as a salt and the enzyme is 
 set free by the acid or alkali. 
 
 (c) The acid or alkali may form a complex compound 
 with the enzyme, which increases its activity, but this is 
 pure hypothesis. 
 
 {d) In certain cases their action is on the substrate. In 
 peptic digestion Euler believes that the hydrochlorides of 
 the proteins are the molecules acted on, and in pancreatic 
 the alkali salts. Analogies to this exist. In alkaUne 
 solution polyphenols are oxidised by the oxygen of the air 
 with great readiness, but in acid solution only with 
 difficulty. The action of lactic acid on the action of 
 ricinus lipase seems due to this, as it has been shown 
 that it has no action on the enzyme apart from the 
 substrate. 
 
PROPERTIES OF ENZYMES 49 
 
 (e) The acid or alkali may act as the common solvent, 
 i. e. the common link between enzyme and substrate. 
 This applies also to the action of salts. If instead of 
 expressing the relation as that of a common solvent the 
 action is considered as a facilitation of adsorption, Bayliss 
 view of many cases is stated. 
 
 The action of salts hardly admits of classification. 
 
 Calcium and magnesium salts increase the adsorptioH 
 of trypsin by paper (cellulose) . As substances in aqueous 
 solution generally have a negative charge, it is probable 
 that this action of calcium is that of a divalent ion 
 reversing the charge on one or other of the colloids, and 
 so favouring adsorption. Calcium is said to be able to 
 activate trypsinogen slowly, but its action can hardly be 
 specific as it also accelerates the action of pancreas 
 lipase. On the other hand, it has no influence on stomach 
 lipase. 
 
 The action of ptyalin is diminished by the salts of weak 
 acids. As these would hydrolyse to a small extent and 
 the hydroxide of the metal, if an alkali metal, would 
 dissociate practically completely while the weak acid 
 would not, I suggest that the action on ptyalin is due to 
 the occurrence of OH ions, for it is found that sma I 
 amounts of acid favour the action of ptyalin, while 
 alkalies in similar concentration inhibit. 
 
 By dialysis, which removes electrolytes, certain enzymes 
 can be rendered inactive, e.g. pancreas amylase. The 
 addition of chlorides, nitrates, sulphates, phosphates, etc., 
 of the alkali metals, but especially chlorides, restores the 
 activity. So dialysed liver amylase is restored by sodium 
 chloride. In a sense these salts are co-enzymes ; but, on 
 the other hand, they may be essential parts of the enzyme 
 which is not an enzyme without them, but at best a 
 zymogen. It has been stated that they enable adsorption 
 
 4 
 
50 ACTION OF ENZYMES 
 
 to take place, but adsorption can occur in their absence. 
 Dialysed liver amylase is inactive; shaken with soluble 
 starch at 40° C. and afterwards with rice starch in powder 
 diffused through the solution, centrifuged, and filtered, 
 the filtrate was found to contain no enzyme, for on addi- 
 tion of sodium chloride no sugar was produced. When 
 the residue is similarly treated sugar is produced. I 
 think it is clear that the residue must have adsorbed the 
 enzyme, for, if not, a percentage of the latter would have 
 been found in the filtrate, yet though adsorbed it is inac- 
 tive without sodium chloride ; therefore the action of the 
 sodium chloride is on the chemical process and not on the 
 adsorption. 
 
 Manganese is an essential constituent of most oxidases. 
 Its action will be considered in connection with the 
 oxidising enzymes. 
 
 Alkali phosphates accelerate the action of zymase and 
 - amylase, and they are absolutely necessary for the action 
 of ptyalin and liver amylase. 
 
 Sodium chloride facilitates the action of amylase, 
 maltase, and ptyalin, but retards the majority of enzymes. 
 
 Chlorates, nitrates, and hydrogen peroxide are dele- 
 terious to catalase, and hydrocyanic acid is especially so. 
 The oxidising powers of the first three are clearly con- 
 nected with this; the action of the last appears to be 
 specific, as a strength of i in 1,000,000 reduces the velocity 
 of reaction by one half (Euler). 
 
 In general, like acids and alkalies, salts must be used 
 in low concentrations. In such concentrations possibly 
 double decomposition occurs with alteration in the con- 
 centration of the H ions, these being apparently the real 
 agents. In higher concentrations by their dissociation 
 they hinder the dissociation of the protein salts, and it is 
 possibly these which enter the reaction when dissociated. 
 
PROPERTIES OF ENZYMES 51 
 
 Irreversible changes in the enzymes may also be caused 
 by salts. The enzymes do not act apart from salts, and 
 it appears to me that the active bodies formed by the 
 combination, if not provided with their proper substrates, 
 turn on themselves, so to say, and so the enzyme is 
 gradually destroyed. 
 
 The special action of acid salts may be mentioned. 
 These are vi^ell known to act as " buffers," i. e. they keep 
 the H ion concentration steady, so that excess of acid, 
 base, or salt, in the proportions likely to be found in life, 
 can be absorbed so far as their influence on this concen- 
 tration goes. 
 
 Non-electrolytes have little effect on enzymes, but the 
 influence of narcotics may be mentioned. According to 
 Euler the activity of those enzymes which are combined 
 with the plasma in the living cell, e. g. zymase, is annulled 
 by narcotics, while the enzymes occurring free in the cells, 
 e.g. invertase, are not influenced. Meyerhof finds that 
 the inhibitory effect of indifferent narcotics on enzymes is 
 completely reversible, and interprets it as being due to 
 the driving off of adsorbed substrate by the more strongly 
 adsorbed narcotic. 
 
 In contact with collodion membranes most enzymes 
 lose their activity and then exhibit a retarding action on 
 fresh enzyme solutions. In seems to me that this might 
 be accounted for on the same lines as for certain pheno- 
 mena of dialysis, the collodion adsorbing some essential 
 constituent of the enzyme, presumably a salt. 
 
 Some electrolytes have a poisonous or paralysing effect 
 on enzymes. Some of these have been mentioned. 
 Others are : perchloride of mercury is harmful to 
 .catalase especially, but also to amylase, urease, and 
 trypsin, less so to erepsin and invertase ; the strengths 
 of the harmful solutions vary, but are always very small. 
 
52 ACTION OF ENZYMES 
 
 Cyanide of mercury has the same, but not so marked an 
 effect. Calcium chloride weakens the effect of invertase, 
 possibly owing to alteration of the electric charge. Iron 
 salts are injurious to pepsin, and potassium permanganate 
 strongly inhibits lipase. 
 
 (8) As described under the head of "Electrolytes" 
 some enzymes require the presence of particular salts or 
 other substances in order to manifest their action. Where 
 one specific substance is required for a particular enzyme 
 it is described as a co-enzyme. A distinction has been 
 drawn between co-enzymes and the activators of zymogens 
 on the ground that the action of co-enzymes is reversible 
 — the co-enzyme can be extracted from the enzyme thus 
 rendered inactive — while the relation of zymogen to enzyme 
 is irreversible (Bayliss). Euler evidently thinks that the 
 non-reversibility of the latter is not quite settled, as he 
 says : " It is highly desirable that a more extended series 
 of experiments should be made to decide if the conversion 
 of pro-enzyme into enzyme is a reversible process." He 
 considers that " there are no grounds for making am 
 essential distinction between the activation of the zymogen 
 with initiation of the reaction and the action of acids, 
 alkalies, and many salts in accelerating the reaction." 
 However this may be, it is usual to consider co-enzymes 
 as reversible activators of enzymes. In some cases the 
 co-enzyme is known ; in others it is known to exist, but 
 its composition is unknown. An example of the latter is 
 found in connection with liver lipase. This is found in 
 extract of liver. If the extract is dialysed it loses its 
 power to hydrolyse lipoids. Add the dialysate and the 
 power is regained. Boiled liver extract acts as well as 
 the dialysate, as does liver extract from which the proteins 
 have been precipitated by uranyl acetate. The activating 
 body is precipitable by ether, but is soluble in alcohol ; it 
 
PROPERTIES OF ENZYMES 53 
 
 is not present in the ash of liver. It follows that it is 
 thermostable, at any rate at the temperature of boiling 
 water, that it is not a protein or adsorbed to proteins 
 precipitated by uranyl acetate, and that it is decomposed 
 on incineration. It is therefore not an ordinary mineral 
 salt which would appear in the ash, but it might be an 
 ammonium salt ; the fact that it stands boiling is opposed 
 to this. It might also be an organic body of comparatively 
 simple constitution as it can be dialysed. 
 
 Another example is found in yeast juice obtained by 
 Buchner's method. If this is filtered under pressure 
 through a Martin's gelatine filter the colloids left behind 
 are inactive, but mixed with the filtrate become active. 
 This co-enzyme can be removed from the filtrate by 
 dialysis and is not destroyed when the dialysate is boiled, 
 but it disappears during fermentation or autolysis. 
 Soluble inorganic phosphates greatly increase the activity 
 of yeast juice, but they do not activate the inactive 
 residue. It has, however, been proved that the presence 
 of phosphates is necessary. Buchner has, therefore, 
 inferred that the co-en2yme is an ester of phosphoric 
 acid (Bayliss). Euler finds that lecithin and other 
 organic compounds of phosphorus activate zymase, and 
 states that these compounds constitute the active con- 
 stituent in boiled pressed yeast juice. 
 
 An example of a known co-enzyme is furnished by bile- 
 salts, as these are the co-enzyme of pancreas lipase. 
 Sodium chelate is as active as the glycocholate, and the 
 synthetic bile-salts have the same action as the natural 
 bodies. It is not a question entirely of facilitating emul- 
 sion of insoluble fats, since the hydrolysis of esters which 
 are soluble in water is also accelerated. The action is on 
 the enzyme itself. Like other rapid activators, prolonged 
 action causes destruction of the lipase. Bayliss suggests 
 
54 ACTION OF ENZYMES 
 
 that the action is physical and is due to increased sub- 
 division of the internal phase of the lipase colloid pro- 
 ducing a larger active surface, for bile-salts greatly lower 
 surface tension. Other views are mentioned by Euler, 
 viz. that the condition of solution of the substrate is influ- 
 enced, or that a lipasogen is converted into lipase. It is 
 to be noted that the lipase of the stomach and that of the 
 intestines are not affected by bile-salts. 
 
 An interesting observation has been made connecting 
 lipase and haemolysis. Substances which exert a haemo- 
 lytic action, such as alcohol, soaps, saponin, digitoxin, 
 increase the action of pancreas lipase, while this is 
 annulled by cholesterol. Pancreas lipase itself becomes 
 hsemolytic if activated by fat. As the hsemolytic sub- 
 stances can produce haemolysis without the presence of 
 lipase it is clear that there is some element common to 
 cells, or rather to cell membranes, and to pancreatic 
 lipase. 
 
 Other substances which have been described as co- 
 enzymes are sodium chloride, which can activate dialysed 
 liver amylase, and phosphates, which are absolutely 
 necessary for ptyalin and greatly assist the action of 
 zymase and amylase. These have been mentioned under 
 the head of " Electrolytes." 
 
 Other substances which probably act in a different 
 manner have been found in connection with the hydro- 
 lysis of starch. This is favoured (as well as by phosphates) 
 by asparagine, amino-acids, proteins, and picric acid. An 
 addition of 0*05 grm. of asparagine to 100 c.c. of a starch 
 solution containing amylase increases the velocity seven- 
 fold. 
 
 (9) Whether anti'enzymes can be obtained in the way 
 that other antibodies can is a matter of dispute. Anti- 
 emulsin was the first obtained, but Bayliss describes its 
 
PROPERTIES OF ENZYMES 55 
 
 inhibitory action as entirely due to the change of H ion' 
 concentration, and if this change be imitated in a solution 
 containing emulsin by the use of acid or alkaline phos- 
 phates the same inhibition is obtained. An antibody is 
 obtained, he says, but only to the protein inevitably 
 present in the impure solution of the enzyme. It is 
 certainly remarkable that injection of an enzyme does not 
 inevitably produce an antibody, e. g. none has been 
 obtained for papain, and others are of very slight activity. 
 
 Another possible cause of anti-enzymic activity may be 
 adsorption. The antitryptic activity of the serum has 
 been found to be connected with the albumin fraction 
 and not with the globulin as is usual with antibodies. 
 Bayliss suggests that trypsin is adsorbed by the albumin 
 and thus its action annulled. Serum albumin is difficult 
 of digestion and is therefore little affected by the digestive 
 action of the adsorbed enzyme. 
 
 On the other hand, Wells states that anti-enzymes 
 obtained by injection are highly specific, e. g. the serum 
 of an animal immunised against dog trypsin shows a much 
 greater effect on dog trypsin than it does on trypsin 
 derived from other animals. 
 
 The question, however, is not of great importance from 
 the point of view of the method of action of enzymes. It 
 arises chiefly in the discussion of the protein character or 
 not of enzymes, as antibodies are apparently not formed 
 against bodies other than proteins, with some doubtful 
 exceptions. 
 
 It should be mentioned that some investigators have 
 attributed a synthesising action to these anti-enzymes, but 
 this is very doubtful. 
 
 (lo) The existence of zymogens has already been 
 frequently referred to. That the enzymes in the case of 
 the secreting glands do not exist preformed but in a con- 
 
56 ACTION OF ENZYMES 
 
 dition from which the enzyme can be liberated at need 
 may be a means of regulating the rate of enzyme action, 
 also a means of defence of the body against its own 
 enzymes, and, lastly, a method of storage. 
 
 The best-known zymogens are trypsinogen and pep- 
 sinogen. Trypsinogen is actually secreted as such, and 
 only on meeting with the enterokinase of the intestine is 
 the active trypsin formed. There is evidence that calcium 
 salts also can activate trypsinogen, but the method of 
 their action is disputed. It is suggested that traces of 
 enterokinase are present in the juice as secreted, which, 
 on precipitation of calcium salts already present, can act. 
 That some other factor than calcium is necessary seems 
 proved by the fact that calcium salts will not activate a 
 juice which has been previously decalcified. 
 
 There is also a difference of opinion as to enterokinase. 
 Bayliss and Starling think it an enzyme, stating that it 
 can activate indefinite amounts of trypsinogen, others 
 claim that a given quantity can activate only a certain 
 amount of trypsinogen. 
 
 Pepsinogen can be obtained from the gastric mucous 
 membrane after autolysis by special methods in a nearly 
 pure condition. As the solution so obtained gives only 
 two of the protein reactions, and those but faintly, pep- 
 sinogen must be of a non-protein nature. It is much less 
 sensitive to alkali than pepsin, which is rapidly destroyed. 
 Its relation to hydrochloric and other acids is of great 
 interest, as nearly all the pepsinogen present in the 
 aqueous extract of a cat's gastric mucous membrane may 
 be converted into pepsin by o'l per cent. HCl in thirty 
 seconds. As Glaessner remarks, the conversion by very 
 dilute mineral acids with such smoothness, rapidity, and 
 completeness is only explicable on the assumption of a 
 very simple chemical process. 
 
PROPERTIES OF ENZYMES 57 
 
 Summary. 
 
 The properties of enzymes have been described under 
 the following heads : 1. Recognition only by activity. 
 2. Specificity. 3. Synthetic or reverse action. 4. Com- 
 bination with substrates and products. 5. As catalysts, 
 with their resemblances to and differences from inorganic 
 catalysts. 6. The effect of temperature. 7. The effects 
 of electrolytes. 8. Co-enzymes. 9. Anti-enzymes, lo. 
 Zymogens. 
 
 The following are the chief conclusions : 
 
 (1) No enzyme has yet been obtained in a pure con- 
 dition, so that their existence is recognisable only by their 
 activity. 
 
 (2) The methods of investigation of the activity of 
 enzymes are : (a) chemical — the study of the nature of 
 their products ; (6) physical — the study of the rate of the 
 reaction and the influences which act on it. Various 
 methods are available for both. 
 
 (3) The action of enzymes is largely specific. This is 
 especially seen in their relation to optical isomers. Analo- 
 gies in general chemistry are to be found. 
 
 (4) Enzymes in general appear to be optically active 
 bodies. 
 
 (5) The specificity, though great, is not absolute ; it is 
 rather a group-specificity than a substance- specificity. 
 
 (6) While the action of enzymes in aqueous solution is 
 as a rule hydrolytic, with limitation of the amount of 
 water the opposite reaction, that of synthesis of the 
 products into the usual substrate, appears to be possible 
 in all cases. Water must not be completely absent if the 
 synthetic reaction is to succeed. 
 
 (7) This synthetic power of enzymes is of great import- 
 
38 ACTION OF ENZYMES 
 
 ance in the animal economy. Arrangements for the limi- 
 tation of water are possible. 
 
 (8) The view that any enzyme can synthesise those 
 molecules which it breaks down is not universally held, 
 but seems the more probable view. 
 
 (9) " There is abundant evidence that a combination of 
 some kind is formed between the enzyme and substrate 
 preparatory to the action of the former." 
 
 (10) The enzyme also combines with the products of 
 the reaction, and in some cases these retard, injure, or 
 even destroy it. 
 
 (11) The compound formed between enzyme and sub- 
 strate is, at first at any rate, an adsorption compound. 
 Whether the enzyme enters into the subsequent chemical 
 action or not is a matter for discussion ; it is at the 
 root of the whole question of the method of action of 
 enzymes. 
 
 (12) The essential action of enzymes is catalytic. The 
 differences between them and inorganic catalysts are 
 partly due to the heterogeneous character of the system in 
 which they act. 
 
 (13) The velocity of an enzyme reaction depends on (i) 
 the concentration of the substrate, (2) the concentration 
 of the enzyme, (3) the presence or absence of electro- 
 lytes, (4) the influence of the antiseptic used to exclude 
 cell and bacterial action. 
 
 (14) The differences between the action of enzymes and 
 that of inorganic catalysts are : 
 
 (a) The dependence of the action of the enzyme 
 not merely on its own concentration, but also on the 
 ratio of the concentrations of enzyme and substrate 
 except at the beginning and the end of the reaction. 
 
 (b) The position of equilibrium is in many cases 
 not the same when the same substances are cata- 
 
PROPERTIES OF ENZYMES 59 
 
 lysed. The probable causes of this are enume- 
 rated. 
 
 (c) The products of the reaction influence the 
 enzyme. 
 
 [d) Specificity is the rule with enzymes, the excep- 
 tion with inorganic catalysts. 
 
 (15) Most enzymes are destroyed by a temperature of 
 65-73° C. The effect of the presence of substrate or 
 products is to diminish this sensibility to heat. Salts 
 may also increase the stability of enzymes to heat. 
 
 (16) Light has considerable action, but this varies with 
 the character of the light ; the same applies to the effects 
 of other radiations. 
 
 (17) The action of electrolytes on enzymes is marked. 
 Acids and alkalies are especially powerful, but enzymes 
 vary in their reaction to these. 
 
 (18) The action of acids and alkalies is shown (a) in 
 the conversion of zymogens into enzymes, (6) on the 
 velocity of the reaction, (c) on the stability of the enzyme. 
 Various explanations are given, and it is especially neces- 
 sary to keep in mind that the action may be on the substrate. 
 
 (19) Salts may act by changing the electric charges of 
 the reacting bodies. This may occur through hydrolysis 
 of the salt owing to increase of the H or OH ions. 
 
 (20) Some salts are absolutely necessary to the action 
 of certain enzymes. It may be that they are essential 
 parts of the complete enzymes. 
 
 (21) All electrolytes must be used in low concentrations 
 when studying their effects; in higher concentrations all 
 inhibit the action of enzymes. 
 
 (32) Narcotics may act through adsorption by the 
 enzymes with displacement of the usual substrate. 
 
 (23) Some salts paralyse enzyme action, especially those 
 of mercury. 
 
6o ACTION OF ENZYMES 
 
 (24) When a specific electrolyte (or non-electrolyte) is 
 required by a particular enzyme before the latter can act, 
 it is described as a co-enzyme. Bile-salts are the co- 
 enzyme of pancreatic lipase, an organic phosphorus 
 compound of zymase. 
 
 (25) Co-enzymes are reversible activators of their 
 enzymes, and are thus distinguished from the activators 
 of zymogens, but this view? is not fully accepted. 
 
 (26) Besides electrolytes, protein, amino-acids, and 
 other complex bodies can act as stimulators of certain 
 enzyme actions. 
 
 (27) The question of the existence of anti-enzymes is in 
 dispute. Their apparent eifects have been put down to 
 changes in the H ion concentration and to adsorption. 
 
 (28) Zymogens, or the precursors of enzymes, are 
 probably of value to the body as a means of regulating 
 enzyme action, and as a means of protection against 
 enzyme action when the action is not wanted. 
 
 (29) The best-known zymogens are trypsinogen and 
 pepsinogen. The activation of trypsinogen by entero- 
 kinase may be an enzyme action, but the view is disputed. 
 The rapidity of the conversion of pepsinogen by hydro- 
 chloric acid indicates a very simple chemical reaction. 
 
 Chemical Action. Hydrolysis. 
 
 Euler gives a list of forty enzymes whose individuality 
 has been established, but he states that there are many 
 more, though in some cases, he thinks, their existence is 
 not proved. At first sight, therefore, it would seem impos- 
 sible to detect any chemical action common even to a 
 large number, and certainly not to all. Nevertheless on 
 examination it is found that, however complicated the 
 constitution of enzymes may be, and however much they 
 
CHEMICAL. ACTION. HYDROLYSIS 6i 
 
 may differ in composition, many of them act on their sub- 
 strates in precisely the same way. Euler classifies them 
 according as they are concerned with (i) hydrolysis, 
 (2) decompositions, (3) syntheses, (4) transference of oxy- 
 gen, (5) unknown processes accompanied by coagulation, 
 (6) fermentations, (7) oxidations. More than half of the 
 forty mentioned, however, act as hydrolysers, and it will 
 be found on analysis of the action of some of the remainder 
 that the action is really that of hydrolysis. 
 
 A typical hydrolysis is an interaction between a salt and 
 water whereby free acid and free base, or an acid and a 
 basic salt, are formed. Hydrolysis is thus a kind of rever- 
 sion of the process of neutralisation of an acid by a base, 
 or of a base by an acid. Except both acid and basic 
 radicles of the salt are weak, hydrolysis is always small in 
 amount, and in the case of a salt of a strong acid and a 
 strong base, hydrolysis, except in special circumstances, 
 does not take place, or only to an insignificant extent. In 
 the case of magnesium chloride and water a certain 
 amount of the magnesium chloride is decomposed and 
 magnesium hydroxide and hydrochloric acid are formed. 
 
 MgCl^ + 2HOH = Mg(OH)a + 2HCI 
 or possibly 
 
 MgClss + HOH = Mg(OH)Cl 4- HCl. 
 Hydrolysis belongs to the class of reversible equations, 
 and an equilibrium is established when the speed of hydro- 
 lysis is equal to the speed of recombination of the pro- 
 ducts of the hydrolysis. If one or both of the products of 
 hydrolysis are removed from the sphere of the reaction, 
 either by the formation of a precipitate or the liberation 
 of a gas, the whole of the salt may be hydrolysed. If 
 "^ bismuth chloride, BiClg, be mixed with water, for example, 
 the basic salt BiCUOH)^ is quantitatively precipitated, 
 BiCIs + 2HOH -^ BiCI(0H)3 + 2HCI. In the case of 
 
62 ACTION OF ENZYMES 
 
 magnesium chloride simple solution of the salt in water is 
 sufficient to produce hydrolysis, but in other cases solution 
 in water, while sufficient to produce hydrolysis, does so 
 only very slowly, and the action can be hastened by a 
 catalyst. The hydrolysis of ethyl acetate is such a case. 
 It has been mentioned already that under the action of 
 HCl, acting as a catalyst, ethyl acetate takes up a 
 molecule of water and is transformed into ethyl alcohol 
 and acetic acid. 
 
 CHgCOOCaHj + HOH I^ CH3COOH + QHjOH. 
 
 Similarly enzymes acting as catalysts hydrolyse their 
 substrates when water is in excess, and synthesise the 
 products of such a reaction when it is in defect. In other 
 words, the action of these enzymes is either to add water 
 or to remove it. 
 
 Taking the enzymes which act on the three great 
 classes of food-stuffs, the fats, the carbohydrates, and the 
 proteins, it is found that their main action is to hydrolyse. 
 Thus the lipases hydrolyse fats into fatty acids and 
 glycerine. 
 
 CsHsCSt)^ + 3HOH = CsH5(OH)3 + 3HSt 
 Glyceryl stearate Glycerine Stearic acid, 
 
 or stearin. 
 Under the head of the lipases may be classed the esterases, 
 such as butyrase, which hydrolyse the esters of the lower 
 fatty acids. 
 
 The enzymes of the carbohydrates may be typified by 
 the inversion of cane sugar. 
 
 C13H23O11 + HOH = CeHiA + CeHiijOe 
 [Cane sugar Glucose Fructose, 
 
 dextro-rotatory. dextro. Isevo. 
 
 The laevo-rotation of fructose is stronger than the dextro- 
 rotation of glucose, and so the mixture of the two is Isevo- 
 
CHEMICAL ACTION. HYDROLYSIS 63 
 
 rotatory. The same action (the taking up of water) appears 
 in the hydrolysis of the glucosides. 
 
 The proteins and their derivatives are attacked in the 
 same way. The proteins are the most complicated of the 
 food-stuffs in chemical constitution, but at root they are 
 mainly built up of amino-acids of which glycine or 
 glycocoll is the simplest, CH5(NH2)COOH. If two 
 molecules of this are conjoined as in glycylglycine, 
 CH2(NHa)CO.NH.CH2COOH, and this is hydrolysed, the 
 equation is : 
 
 CHa(NHa)C0.NH.CH2C00H + HOH = 2CH2(NH2)COOH. 
 As a matter of fact, boiling with one of the mineral acids 
 splits up a protein into its constituent amino-acids. The 
 albumoses, peptones, and polypeptides are simply stages 
 on the path of the decomposition of the proteins, and 
 their hydrolyses are of the same type. 
 
 Of simpler bodies subject to hydrolysis, urea may be 
 
 mentioned. Under the influence of urease, ammonium 
 
 carbonate is formed, which breaks up into ammonia, 
 
 carbon dioxide, and water. 
 
 CO(NH3)2 + 2HOH = (NH^)sjC03 = 2NH3 -I- CO3 + HOH. 
 
 The amino-acids themselves are attacked by the des- 
 amidases, ammonia being split off and the amino-acid 
 converted into the hydroxy-acid. The type is : 
 
 CHs,(NH3)COOH -I- HOH = CH2(0H)C00H -1- NH3. 
 A similar change takes place in the conversion of adenine 
 into hypoxanthine, or guanine into xanthine, OH being 
 substituted for NHj, and H combining with NH^ to form 
 ammonia. 
 
 In general hydrolysis may be represented as the inter- 
 action of water and a substrate, with the result that one 
 of the radicles of water combines with one part of the 
 substrate and the other with the other : 
 
 XY + HOH = YOH + HX. 
 
64 ACTION OF ENZYMES 
 
 Of the other enzymes mentioned by Euler some, it is 
 likely, are not really such. Fibrin ferment is not an 
 enzyme. It is not destroyed by heat, and it enters 
 quantitatively into the products of the reaction (Bayliss). 
 Chymosin, or rennet, in clotting milk shows only a colloid, 
 not an enzyme reaction. Indeed, Euler describes such 
 actions as " unknown processes accompanied by coagula- 
 tion." 
 
 Under the head of decompositions he places that of 
 hydrogen peroxide into water and oxygen, and of hydroxy- 
 nitriles into aldehyde and hydrocyanic acid. The first will 
 be dealt' with in connection with the oxidising enzymes. 
 As regards the second, the action is usually repre- 
 sented as : 
 
 CjHjCH-^JJ = CgHjCHO + HCN. 
 
 Rosenthaler considers the action of the enzyme as hydro- 
 lytic, but there is no evidence of this in the above equa- 
 tion, which shows simply a decomposition. If, however, 
 an intermediate compound is formed with water, the 
 hydrolytic action is plain : 
 
 QHjCH-^^ + HOH = CeHsCH;^^ -I- HCN. 
 
 The intermediate compound shows two OH radicles 
 bound to one carbon atom. Such a combination is 
 
 OH 
 
 exceedingly unstable, chloral hydrate, CCIjCH'qjj 
 
 glyoxylic acid, CH(OH)aCOOH, mesoxalic acid, 
 HOOC.C(OH)3.COOH, being rare exceptions. That, 
 however, it can be formed, even if but momentarily, is 
 shown by the hydrolysis of ordinary nitriles. 
 
 .OH 
 CH,CN -1- 3H0H= CHjC.OH-f- NH3= CHgCGOH-f- HOH-1-NH3 
 
 .OH 
 
 It is true that such compounds as the intermediate one 
 
CHEMICAL ACTION. HYDROLYSIS 65 
 
 shown are not found, but their existence is by no means 
 improbable, as compounds containing three alkoxyl 
 groups exist, viz. the ortho esters, e. g. 
 
 CHa-COCaHs. 
 •OQHg 
 
 The full equation then would be : 
 
 CsHsCH;^^ + HOH = CsHjCHlgg +HCN 
 
 = CsHjCHO + HCN + HOH, 
 or an hydrolysis with subsequent loss of water by an 
 unstable product. 
 
 Of the remaining classes, syntheses are with great 
 probability considered to be merely the reverse action of 
 enzymes. Processes involving oxidation or the trans- 
 ference of oxygen require separate treatment. There 
 remains the class of fermentations — e. g. the transforma- 
 tion of sugar into alcohol and carbon dioxide. 
 
 Fermentation is not a simple process ; glucose does not 
 become alcohol and carbon dioxide by a single step. In 
 the animal organism alcohol is not formed; but apart 
 from this ihe steps of the process are probably the same as 
 with yeast, the formation of alcohol taking place under the 
 action of yeast owing to the absence of oxygen. The steps 
 are believed to be, with the chemical changes required : 
 Glucose. 
 
 CHgOH.CHOH.CH OH.CH OH.CHOH.CHO. 
 = Glyceric aldehyde, two molecules. 
 
 CH2OH.CHOH.CHO. 
 This appears to be a case of hydrolysis taking 
 place at the point underlined, thus : 
 CHOH.CHOH - CHO + HOH 
 
 = CH°2 + §COH - CHO 
 
 = CHO + CHjOH - CHO + HOH, 
 
 5 
 
66 ACTION OF ENZYMES 
 
 with subsequeilt elimination of water from the 
 
 aldehydrol, CH^tj. 
 
 = Pyruvic aldehyde. CHj.CO.CHO. 
 
 This appears to be due to loss of water, with 
 subsequent redistribution of the atoms in the 
 molecule. 
 CHgOH.CHOH.CHO = CH^ : C(OH).CHO + HOH. 
 CHg : C(OH).CHO = CHs.CO.CHO. 
 
 The change is exactly analogous to the forma- 
 tion of pyruvic acid from glyceric acid, on distil- 
 lation with potassium hydrogen sulphate, by loss 
 of one molecule of water. 
 
 Another analogy is found in the formation of 
 acrolein from glycerine after loss of two mole- 
 cules of water. 
 CHjOH.CHOH.CHaOH = CH^ : C : CHOH + 2HOH 
 CHa : C : CHOH = CHg : CH.CHO. 
 = Lactic acid. CH3.CHOH.COOH. 
 
 This is due to the hydration of the last, 
 CH3.CO.CHO + HOH = CH3.C(OH)2.CHO 
 = CH8.CH0H.C00H with redistribution of the 
 atoms. 
 
 = Pyruvic acid. CH3.CO.COOH. 
 
 This involves oxidation and elimination of 
 water. 
 
 CHs-Cgjj.COOH + O = CHj.Cq^COOH 
 
 = CH3.CO.COOH + HOH. 
 = Acetaldehyde. CH3.CHO. 
 
 This appears to be accounted for by hydrolysis 
 with formation of carbonic acid, which breaks up 
 into carbon dioxide and water. The hydrolysis 
 takes place between the CO and COOH radicles. 
 
CHEMICAL ACTION. HYDROLYSIS 67 
 CHs.CO.COOH + HOH = CHs.Cq + HO.COOH 
 
 COq§ = CO2 + HOH 
 
 =: Carbon dioxide and water. 
 
 This involves oxidation. 
 CH3.CHO + 50 = 2C0g + 2HOH. 
 
 Whether the explanations given of the actions represent 
 the facts accurately or not, they show at any rate that the 
 whole complex process can be represented as a mixture 
 of hydration, dehydration, hydrolysis, rearrangement of 
 atoms, and oxidation, and that these processes are the 
 only ones actually required, even though the changes 
 which really occur may differ to some extent from the 
 picture given. 
 
 All the classes so far examined, therefore, omitting the 
 coagulation processes as being colloidal, not chemical 
 reactions, are connected with hydrolysis through hydration 
 and its opposite. In order, therefore, to understand 
 enzyme action it is necessary to form as clear a conception 
 as possible of what occurs during hydrolysis apart from 
 catalytic action, such as happens, for example, when 
 magnesium chloride is dissolved in water. 
 
 It must first be premised that when a salt like potassium 
 chloride, KCl, is dissolved in water it exists in the solution 
 partly as molecules of KCl and partly as charged atoms 
 of K and CI, the metallic radicle bearing a positive charge 
 and the acid residue a negative. These charged atoms 
 are known as ions ; they may be atomic, as in the case 
 given, or compound radicles like NOg, SO^, etc. The 
 oppositely charged ions are not completely free from each 
 other ; there is an electrostatic attraction between them 
 which prevents their complete separation by mechanical 
 means. 
 
68 ACTION OF ENZYMES 
 
 Some of the facts which have led to this conclusion 
 may be briefly stated as follows. 
 
 The osmotic pressure of a solution of, for example, 
 sugar conforms to the laws which express the action of a 
 gas in a closed container, for in the case of a solution the 
 container is the solvent and the gas is represented by the 
 solute. It has, however, been found that the osmotic 
 pressure for bases, acids, and salts, calculated on the 
 assumption that they are present in solution as molecules, 
 does not agree with the observed facts, but that the pres- 
 sure is higher than it should be; in fact the solution 
 behaves as if there are more active particles present than 
 there are molecules. 
 
 It was next shown by Arrhenius that only those solutions 
 which have an abnormally high osmotic pressure conduct 
 the electric current. 
 
 Faraday had previously shown that when an electric 
 current is passed through such a solution chemical changes 
 occur like the development of acid round the positive 
 pole and alkali round the negative. It was clear, there- 
 fore, that matter travelled with the current, and to these 
 travelling parts of the solution Faraday gave the name of 
 ions ; those going to the anode or positive pole were 
 called anions, and those to the negative or cathode, 
 cations. 
 
 It was noticed that the chemical change took place 
 immediately the current was made, and as on measurement 
 it appeared that Ohm's law held for electrolytes as much 
 as for other conducting substances it was evident that 
 the electrical energy expended in electrolysis is entirely 
 used up in overcoming the resistance of the electrolyte, 
 and that no work is done in pulling apart the components 
 of the molecule. It followed that the ions existed in the 
 solution previous to the passage of the current, and 
 
CHEMICAL ACTION. HYDROLYSIS 69 
 
 hence the abnormal osmotic pressure of these conductors 
 of electricity in solution, or electrolytes, received an 
 explanation. 
 
 This ionisation of electrolytes is also described as 
 electrolytic dissociation. It has been found that the 
 order of the strengths of acids is the same as that of their 
 electric conductivities in equivalent solution, and this is 
 determined by the degree of dissociation. Since the 
 hydrogen ions of any acid take a predominant share in 
 conveying a current it is natural to suppose that the 
 activity of acids is due to their common constituent, 
 hydrogen ions. This assumption is in complete accord 
 with the experimental facts. A strong acid, then, is one 
 that dissociates largely, and a weak acid one that dissociates 
 slightly at a given moderate dilution. Similarly the 
 strengths of bases depend on the degree of their dissocia- 
 tion and the resulting concentration of the OH ions. 
 
 Now the common solvent, water, HOH, contains H 
 and OH in union, and the question arises whether any 
 dissociation of these occurs in pure water. A water abso- 
 lutely pure is exceedingly difficult to prepare, but the 
 purest so far obtained is found to conduct electricity 
 slightly, and from the measurement of this, and by other 
 indirect methods, it has been discovered that the concen- 
 tration of the H ions, which is necessarily the same as 
 that of the OH ions, is i"i x 10-' at 25° C. In other 
 words pure water contains by weight i gram of H ions 
 and 17 grams of OH ions (the actual number of each kind 
 of ions being the same) in 10,000,000 litres. 
 
 It follows from this that water can act both as a weak 
 base and a weak acid. Regarding it as a weak acid it has 
 been found that when two acids are allowed to compete 
 for the same base the latter distributes itself between the 
 acids in proportion to their avidities, and it has also been 
 
70 ACTION OF ENZYMES 
 
 shown that the ratio of their avidities is the ratio of the 
 extent to which they are electrolytically dissociated. In 
 aqueous solutions water, in virtue of its hydrogen ion 
 concentration, can be regarded as one of the competing 
 acids. In the case of a salt of a strong acid, such as 
 sodium chloride, so weak an acid as water can take no 
 appreciable amount of the base ; hence in an aqueous 
 solution of sodium chloride there are only Na and CI ions 
 and undissociated sodium chloride in appreciable amount, 
 and the solution is neutral. If, instead, a salt of a weak 
 acid and a strong base is taken, the conditions are different. 
 Water is comparable in strength to a weak acid like 
 hydrocyanic acid, and, therefore, a distribution of the 
 base takes place between the acid and water according to 
 the equation KCN + HOH ijl KOH + HCN. Since 
 KOH is much more highly dissociated than HCN there 
 are many more OH ions than H ions in the solution, and 
 it is therefore alkaline. A similar argument applies to 
 the salt of a weak base and a strong acid, where water as 
 a base competes for the acid, and the acid so formed, 
 being much more strongly dissociated than the hydroxide 
 of the base, makes the solution acid. The taking up of 
 the H or OH ions already existing in water destroys the 
 equilibrium between them and the undissociated water, 
 and, consequently, fresh ions must be formed by the 
 splitting of hitherto undissociated molecules. This pro- 
 cess goes on till a new equilibrium is established, taking 
 in the OH ions present in the case of the strong base and 
 weak acid, and the H ions in the case of the weak base 
 and strong acid. In both cases the amount of hydrolysis 
 is small, except the base or acid is very weak indeed. 
 Sodium carbonate in i in lo molar solution at 25° C. is 
 hydrolysed 3*17 per cent., KCN I'la per cent., and sodium 
 acetate, though acetic is a fairly weak acid, o'ooS per cent. 
 
CHEMICAL ACTION. HYDROLYSIS 71 
 
 Also ammonium chloride 0*005 P^^ cent., aniline hydro- 
 chloride i"5 per cent., glycocoll hydrochloride 20 per cent., 
 as glycocoll is a very weak base. The degree of hydrolysis 
 is in both cases dependent on the dilution. When, how- 
 ever, the salt is one where both acid and base are weak, 
 the degree of hydrolysis is independent of dilution and is 
 considerable, e.g. aniline acetate about 44 per cent. 
 Nevertheless hydrolysis does not always occur when it 
 might be expected, e.g. sodium stearate is considerably 
 hydrolysed, sodium palmitate is not. 
 
 With regard to the salts of strong acids and strong 
 bases where no appreciable hydrolysis occurs a certain 
 cautiousness of statement must be observed. It is true 
 that the salts of the alkalies and alkaline earths are 
 hydrolysed at ordinary temperatures to an inappreciable 
 extent, but that hydrolysis does occur is indicated by 
 Emich's experiment, where drops of water are applied to 
 sodium chloride heated to redness. The drops are found 
 to contain acid, and the salt left in the crucible after 
 cooling is found when dissolved to give an alkaline reac- 
 tion. This is in line with such facts as the appreciable 
 hydrolysis of cane sugar by water at 100° C. and of salicin 
 at 150° C. previously mentioned. 
 
 While the theory of hydrolysis just stated is the accepted 
 one, it must be mentioned that Werner gives strong 
 reasons for thinking that it does not explain the hydro- 
 lytic change in the aquometallic ammino salts, e.g. the 
 
 aquopentammincobalti salt r^°/vij| \ IXj, where Xis an 
 
 acid residue. He fiijds that it- is due primarily to the 
 stability of the water in the complex, and that this is the 
 same thing as saying that it depends on the degree of 
 strength with which the H ion, or hydrion, is linked. 
 Reasoning from these compounds he believes that an 
 
7a ACTION OF ENZYMES 
 
 ordinary simple salt first takes up water, e.g. KCN 
 
 rK n 
 becomes |_ttO.HJCN, and that this in neutral solution 
 
 splits up into ttO and HCN. Then the bodies so formed 
 
 ionise according to their strengths as acids or bases. 
 KOH is strongly dissociated, and hence OH ions are 
 present in the solution to a considerable extent, while 
 HCN is very little dissociated, and hence few H ions are 
 present ; as a result the solution is alkaline. 
 
 The difference between the two theories may be stated 
 as follows. Both theories put down hydrolysis to the 
 action of water ; but Arrhenius' theory makes hydrolysis 
 dependent 'on the existing ions of water ; Werner's theory 
 makes it dependent on the previous association of the salt 
 with undissociated molecules of water, which, when in 
 combination with the salt, split, the metallic portion of 
 the salt going with the OH half and the acid residue with 
 the H half. 
 
 Obviously Werner's theory is based on the assumption 
 that molecular combination between solute and solvent, 
 when the latter is water, occurs. Evidence for this will 
 now be considered. 
 
 (i) The solubility of gases in water is diminished both 
 by electrolytes and by some non-electrolytes. Now the 
 amount of gas taken up by a definite volume of liquid 
 depends on (a) the pressure of the gas, (6) the tempera- 
 ture, (c) the nature of the gas, and (d) the nature of the 
 liquid ; (a), (b), and (c) remaining constant, the effect of 
 solutes must be on the nature of the liquid. Were the 
 
 i 
 
 solute evenly distributed throughout the liquid without 
 combination there would be present the same amount of 
 liquid as before the solute was added, and the same 
 amount of gas should be taken up ; but if combination of 
 
CHEMICAL ACTION. HYDROLYSIS 73 
 
 some sort takes place between the solute and the water 
 molecules the amount of liquid available for dissolving the 
 gas would be diminished and the effect accounted for. 
 
 (2) The absorption of light by solutions of substances 
 is by Beer's law proportional to the number of molecules 
 through which the rays pass. If the absorption spectra of 
 solutions of copper chloride of different degrees of concen- 
 tration be examined, when the depth of the solution is 
 varied in inverse proportion to the concentration so that 
 the same total amount of solute Hes in the path of the 
 light, it will be found that more ultra-violet light is 
 absorbed the more concentrated the solution is, although 
 by Beer's law, the number of molecules remaining the 
 same, the absorption ought to be the same. This might 
 be accounted for by either of two assumptions : (a) that 
 in the more concentrated solutions aggregates of the 
 solute are formed which increase the absorbing power, or 
 (6) that in the more dilute solutions aggregations of water 
 and solute are formed, the amount of water taken up 
 being in proportion to the dilution, and that these 
 diminish the absorbing power. To test this, raise the 
 temperature. The effect of this is to break up aggregates, 
 whether of the solute molecules alone or of the solute 
 molecules and water molecules. If the effect is to diminish 
 the absorption, then probably the cause of increased 
 absorption with increased concentration is aggregation 
 of solute molecules ; if, on the other hand, the effect is to 
 increase the absorption, this is probably due to the breaking 
 up of the combination between solute and water, so that 
 the diminishing effect on the absorption of the combina- 
 tion is prevented. As a matter of fact, rise of temperature 
 increases the absorption, therefore the diminished absorp- 
 tion on dilution is probably due to combination of water 
 and solute. 
 
74 ACTION OF ENZYMES 
 
 (3) Kohlrausch has observed that the influence of 
 temperature on the electrolytic resistance of dilute 
 solutions of salts approximates to the temperature co- 
 efficient of the mechanical friction of water. This is 
 intelligible if it is assumed that the ions are surrounded 
 with water molecules, as the resistance to movement of 
 the water-coated ions would then be the frictional resist- 
 ance of water against water. 
 
 (4) The degree of dissociation of a solute can be deter- 
 mined by conductivity measurements, etc. This being 
 known, the total amount of ionised and of undissociated 
 salt is' known, and from this the osmotic pressure can be 
 calculated. Further, the depression of the freezing-point 
 which ought to be produced by the addition of such an 
 amount of the solute to pure water can be predicted. As 
 a matter of fact, however, the experimental depression of 
 the freezing-point is in many cases greater than it should 
 be. If it be assumed that association of part of the 
 solvent with the solute takes place, so that the amount of 
 solvent present in the free state is diminished, then the 
 solution will be more concentrated than was allowed for 
 and the abnormal depression of the freezing-point 
 accounted for. 
 
 (5) Kohlrausch found that the rates of migration of 
 different ions approached nearer to the same value as the 
 temperature was raised, and that at the critical tempera- 
 ture of water the mobilities would be identical. This can 
 be accounted for on the assumption that at this tempera- 
 ture the ions are completely free from water, but that at 
 lower temperatures some take up more molecules of water 
 than others, and the bulkier the combination is the slower 
 the movement. 
 
 (6) The behaviour of double salts in solutions appears 
 to be the strongest evidence available, and it is on this 
 
CHEMICAL ACTION. HYDROLYSIS 75 
 
 that Werner has founded his theory of valency of which 
 the combination of water and solute molecules is only 
 one section. Werner's theories are based on the fact that 
 it is impossible to represent the structure of many double 
 salts along the lines of ordinary valency formulae.-" For 
 example, while the structure of KjSO^ derived from the 
 reaction represented by the equation KaO + SO3 = K^SO^ 
 
 is thus shown o/'^\OK' ^* ^^ impossible to represent a 
 double halide in this form, yet there is clearly an analogy 
 between the two 
 
 KgO + SO3 = KaSO, 
 
 KCl + AuCls = KAuCl^. 
 To represent the structure of KAuCl^ in the ordinary 
 manner involves conceptions of changes of valency on the 
 part of Au or CI. Similarly for other double salts it is 
 necessary to assume so many changes of valency that it 
 would almost appear that an element can unite with an in- 
 definite number of atoms of another element. Now, it is true 
 that up to a certain point this is correct, e. g. four chlorides 
 of vanadium have been isolated with two, three, four, and 
 five atoms of combined chlorine respectively, but there 
 seems to be a maximum for each element. This maxi- 
 mum can be determined by a study of the compounds of 
 an element, but evidences of the variability of the valency 
 of some elements are either not to be found or the facts 
 can be explained in some other way. Chlorine can be 
 represented as a monad in practically every case. 
 
 Werner believes that there is one cause underlying all 
 such apparent difi&culties of the theory of valency, which, 
 being granted, an harmonious scheme becomes possible. 
 The cause assumed is, that in addition to the usual 
 principal valencies which unite atoms to atoms there also 
 exist other valencies, which he calls auxiliary valencies. 
 
76 ACTION OF ENZYMES 
 
 and which are capable of uniting molecules. For example, 
 to take the case which is of most immediate interest in 
 this connection, water combines with oxides, chlorides, 
 iodides, and many other classes of compounds, e. g. : 
 0\ O Cl\ Cl\ 
 
 0=S +OH2 = 0=S.OH3 Cl-Au + 0H„ = Cl-Au.OHj. 
 O^ O^ CI/ " CI/ 
 
 Werner does not denythat in the first of these compounds 
 a further transformation is possible to q^S^qjj' ^^^ ^^ 
 
 points out that such a transformation is impossible in 
 the second body. Similarly, PtCl^ behaves like SO3 in 
 forming addition compounds with 2HCI and 2OH2, in 
 both cases forming dibasic acids, and it also unites with 
 zNHs, yet in none of these cases does CI appear as an 
 ion. For this and other reasons he concludes that the 
 CI atoms are directly united to the Pt, i. e. they are, as 
 he phrases it, in the first or non-dissociable zone of the 
 attraction of the Pt atom, while the two H atoms, which 
 are dissociated (in the first two combinations) are in the 
 second or dissociable zone. He therefore formulates the 
 compounds thus : 
 
 —CI CI— —CI CI — —CI CI — 
 
 ClPtCl 
 -CI Cl- 
 
 H2 
 
 H, 
 
 ClPtNHg 
 —CI NHs- 
 
 ClPtOH 
 -CI OH- 
 
 or (PtCl6)H3, (Cl,Pt(OH)2)H2 and (Cl.PtCNHa)^), the 
 last not being dissociated in any way. 
 
 Applying these views to the question of the hydration 
 of salts {i.e. the taking up of molecules of water) it 
 appears that there is no reason why they should not form 
 compounds with water in all cases, and that there is 
 definite evidence that in many cases they do actually form 
 such compounds. For example, the following cobalt com- 
 pounds have been isolated : 
 
 [^^(NhJJ^^' [^"(NHs),]^"' L^°(NH8)8J'^'" 
 
CHEMICAL ACTION. HYDROLYSIS 77 
 
 X being a monobasic acid residue ; and many hydrates of 
 metallic salts are known, of which he mentions those of 
 chromium, stating that these have been thoroughly 
 investigated. 
 (Cr(OH)e)Cl3. 
 
 KiH,)JCL. + HOH. [Crg^H,)JCl + 2HOH. 
 
 Many metallic salts form hydrates with six molecules of 
 water, e.g. (Ca(0H2)e) Clj, (Fe(OH2)6)Cl3. Six appears 
 to be the maximum number of water molecules with 
 which a salt can unite, at any rate in the first zone of 
 attraction to the central atom ; when this occurs all the 
 acid residues in the salt are ionised. Water can also be 
 bound in the second zone, as in the case of the sulphates, 
 which can take up seven molecules of water, six in the 
 first zone, one in the second. 
 
 Acids, as well as salts, can form hydrates, thus the 
 following havebeen isolated : N030H.OHa,N030H.30H2, 
 HCI.2OH3, HCI.3OH2. To explain such a compound as 
 HCl.OHj (which has not actually been isolated, but to 
 which analogies exist in organic chemistry), the theory of 
 the quadrivalency of oxygen has been elaborated, thus 
 
 TT^O^Q, but it is impossible to apply this to compounds 
 
 with two or more molecules of water except on the 
 assumption of the existence of chain compounds, e.g. 
 
 vy^^ll/O^rv ^^'^ chain formulae for the hydrates of 
 
 metallic salts, for the metallic ammino-complexes, etc., 
 to-day are accepted universally as incorrect (Werner). It 
 is simpler to assume that hydrogen can act as a central 
 atom, and the formulae become : 
 
 CIH.OH2, ClH:°^^etc. 
 
 On these general grounds Werner comes to the con- 
 
78 ACTION OF ENZYMES 
 
 elusion that in solution in water* a salt (where Me = 
 metallic radicle and X = acid residue) should be repre- 
 sented by the formula [^g^O.H]x. If only electri- 
 cally dissociated this becomes { jf^CH) and X~, but 
 if hydrolysis precedes electrolytic dissociation the bodies 
 present will be H+, X~ and (^O) not or little dis- 
 sociated if a weak base, and the solution will be acid. 
 If HX is a weak acid and MeOH a strong base the bodies 
 present will be Me+, OH~ and HX not or little dis- 
 sociated, and the solution will be alkaline. 
 
 Werner's hypothesis that all salts in solution, being 
 combined with molecules of water, hydrolyse by separation 
 at the junction of the H and OH of the water, if correct, 
 afford? an explanation of the effect of precipitating a 
 negatively charged suspensoid colloid, such as arsenious 
 sulphide, by the adsorption of ions of opposite sign, e. g. 
 the barium ion of barium chloride. It has been shown in 
 this case that the Ba ions go down with the precipitate, 
 while the liquid becomes acid owing to free hydrochloric 
 acid. The difficulty is the disposal of the CI ions. Even 
 if we suppose that more water is dissociated to give the 
 increase of H ions shown by the acid reaction there still 
 remain the corresponding OH ions to be accounted for 
 (Bayliss). This difficulty arises from assuming that the 
 ions formed are Ba+"'", CV, and CF. But if it is assumed 
 in accordance with Werner's theory that the salt was 
 hydrated before it exerted its action, then it would have 
 
 the form rB'aQTTjjJr'i. and if this merely ionised the 
 
 ions would be rBaQTTTrj , Cl~, and Cl~, and no doubt 
 in this form it reaches the colloid. Now it is clear 
 
CHEMICAL ACTION. HYDROLYSIS 79 
 
 that adsorption exerts a chemical action on the salt, for 
 the solution becomes acid, and if this chemical action is 
 granted to be a hydrolysis of the Werner type, the whole 
 would split at the junction of the H and OH radicles of 
 water, and as a result BaCOH),, H+, H+, CF, CF, would 
 be present. What finally unites with the colloid is the 
 Ba(OH)g, which in virtue of its union does not dissociate, 
 at any rate in the solvent, while hydrochloric acid is set 
 free. Whether this explanation is accepted or not, one 
 thing is clear, that hydrolysis of the salt has taken place, 
 and this is much more easily explained as a result of 
 adsorption of the salt than as due to the ions of water 
 already existing in the solvent. 
 
 Werner's theory deals only with chemical combination 
 of water in other molecules, although of an unusual kind, 
 he leaves untouched the question whether association of 
 still another kind occurs between the solute and the sol- 
 vent, and whether also the ions themselves are associated 
 with particles of the solvent. The evidence previously 
 given is in favour of this additional association. 
 
 There is, therefore, reasonable ground for believing 
 that, in the case of water at any rate, molecular combina- 
 tion between solute and solvent occurs, though it must be 
 stated that Nernst considers the hypothesis by no means 
 proven. 
 
 Apart from all theories, however, since in any case of 
 hydrolysis the result is brought about by the union of 
 an H radicle with one of the parts into which the solute 
 divides, and of an OH with the other, it is clear that the 
 hydrogen ion concentration must be a matter of great 
 importance. (The H ion concentration gives also the 
 OH concentration, since the product of the two is a con- 
 stant.) In fact the rate at which the esters, methyl and 
 ethyl acetate, are hydrolysed in water is found to be pro- 
 
8o ACTION OF ENZYMES 
 
 portional to the H ion concentration present. Since both 
 H and OH ions cannot be present in equal concentration 
 at the same time except at the point of neutrality when 
 the concentration of either is extremely low at ordinary 
 temperatures, and since the increase of one means the 
 proportionate decrease of the other, it follows that any 
 hydrolytic process can be carried out in connection with 
 one of the two only, the other coming into existence 
 secondarily as required. That one ion is sufficient appears 
 from the following scheme. Assume the existence of a 
 free OH ion. This meeting with a substance XY will, if 
 its attraction for Y is stronger than that of X, form YOH, 
 leaving X~ free as an unsatisfied ion. This can either 
 break the union of H and O H in a molecule of water, or, 
 perhaps more probably, it unites with an H ion already 
 present in the solvent. This leaves a fresh OH ion to 
 carry on the action. 
 
 OH" + XY = YOH + X' 
 X" + H+ + OH" = HX + OH". 
 Similarly if an H ion is assumed free, 
 H+ + XY = HX + Y+ 
 Y+ + H+ + OH" = YOH + H+. 
 Of course the existence of a free ion would be only 
 momentary. The action of the H or OH ions must start 
 before they are freed from their electrostatic attraction, 
 and must depend on the superior attraction of Y or X for 
 them over the attraction exercised on them by the radicle 
 with which they are combined. 
 
 Now if H and OH as ions can produce hydrolysis there 
 ought to be evidence that water, which contains them and 
 them only, can act as a powerful hydrolyser provided the 
 concentration of the ions can be raised to any considerable 
 extent. In water at o° C. the concentration is very low, 
 0*35 X io~', but it rises rapidly with the temperature. 
 
CHEMICAL ACTION. HYDROLYSIS 8i 
 
 -7. 
 
 At i8° C. it is o-8o x lo"', and at 50° C. 2"48 x 10 
 That the hydrolysing action becomes evident in water 
 when the temperature is suiificiently raised is shown by its 
 action on glucose at 100° C, on salicin at 150° C, and on 
 red-hot sodium chloride, when the water is in the spheroidal 
 state, as already mentioned. It follows, therefore, that if 
 any method can take the place of raising the temperature 
 in increasing the concentration of the H and OH ions in 
 an aqueous solution, either generally or at the point of 
 action, this method must be indirectly a powerful hydro- 
 lyser. If then acids, bases, or salts can increase the 
 concentration of either ion generally, and enzymes can 
 increase it at the point of action, viz. the surfaces of their 
 particles, they themselves being either unchanged or else 
 reformed in the course of the reaction, these bodies will 
 indirectly be hydrolysers and will conform as regards 
 hydrolysis to the definition of catalysts. 
 
 Summary. 
 
 (i) Many different enzymes are known, but their 
 chemical action on their substrates in most cases is 
 hydrolytic. 
 
 (2) Hydrolysis is a kind of reversion of the process of 
 neutralisation of an acid by a base, or vice versa. Unless 
 both acid and base are weak the amount of hydrolysis is 
 small, and if both are strong the amount is insignificant. 
 
 (3) Hydrolysis belongs to the class of reversible reac- 
 tions. 
 
 (4) In some cases simple solution in water is sufficient 
 to produce hydrolysis, but in other cases the action is very 
 slow and may be hastened by catalysts. 
 
 (5) The action of enzymes on the three great classes of 
 food-stuffs, the fats, the carbohydrates, and the proteins, 
 is hydrolytic. 
 
 6 
 
82 ACTION OF ENZYMES 
 
 (6) The same is true of enzyme action on some simpler 
 bodies, such as urea, the amino-acids, and the purine 
 bodies. 
 
 (7) Processes accompanied by coagulation are, so far as 
 the coagulation is concerned, colloid reactions, and are not 
 enzymic. 
 
 (8) On the assumption that in certain decompositions 
 produced by enzymes there is formed an intermediate 
 compound combining water in an unstable form, these 
 processes also appear to be hydrolytic. 
 
 (9) Fermentation is a complex process involving a 
 series of reactions, but all these processes can be repre- 
 sented as hydrolytic and oxidative, through hydration and 
 its opposite, with accompanying rearrangement of the 
 H and OH radicles. 
 
 (10) An electrolyte is a substance which when dissolved 
 can conduct a current of electricity. It exists in the 
 solution partly undissociated, and partly as electrically 
 charged radicles. These charged radicles, called ions, are 
 the real conductors of the electric current. 
 
 (11) The facts on which the last statement is based are 
 given. They include the abnormal osmotic pressure of 
 electrolytes, their conductivity, and their obedience to 
 Ohm's law. 
 
 (12) The order of the strengths of the acids is the same 
 as the order of their electrical conductivities, and this is 
 determined by the degree of their dissociation. Hydrogen 
 ions are the common constituent of all acids, and it is to 
 these that their activity is due. Similarly the activity of 
 bases is due to their hydroxyl ions. 
 
 (13) In the purest water there is a certain small amount 
 of dissociation so that both H and OH ions are present. 
 Water can, therefore, act both as a weak base and a weak 
 acid. 
 
CHEMICAL ACTION. HYDROLYSIS 83 
 
 (14) The theory of Arrhenius attributes hydrolysis to 
 the existence of these ions in water, and the facts of 
 hydrolysis in the four possible cases, salt of a strong acid 
 and a strong base, of strong acid and weak base, of weak 
 acid and strong base, and of weak acid and weak base, can 
 be accounted for on this theory. 
 
 (15) Werner has pointed out that the theory is not 
 applicable to some complex salts, and on this account he 
 has formulated a theory which attributes hydrolysis to 
 previous hydration of the salt and subsequent division of 
 the complex at the junction of the H and OH radicles of 
 the water so taken up. 
 
 (16) Werner's theory assumes that molecular combina- 
 tion between solute and solvent occurs. The facts bearing 
 on this are : (i) The effect of electrolytes, etc., on the 
 solubility of gases in water ; (2) the effect of concentration 
 or dilution of a solution on the absorption of light when 
 the number of molecules in the path of the rays is kept 
 constant ; (3) the influence of temperature on the electro- 
 lytic resistance of dilute solutions of salts ; (4) the abnormal 
 depression of the freezing-point in certain salt solutions ; 
 (5) the rates of migration of different ions as depending 
 on the temperature ; (6) the behaviour of double salts in 
 solution. 
 
 (17) A resume oi Werner's theory of valency is given so 
 far as it bears on hydration and hydrolysis. He gives 
 evidence of the existence of complex salts which include 
 water combined in their molecules, also of the existence 
 of combinations of water with acids. 
 
 (18) The disposal of the acid radicle in the case of the 
 precipitation of a suspensoid colloid by a divalent positive 
 radicle like barium with combination of colloid and positive 
 radicle is explained on Werner's hypothesis. 
 
 (19) Since hydrolysis involves the union of H and OH 
 
84 ACTION OF ENZYMES 
 
 radicles with the parts into which the hydrolysed sub- 
 stance divides, it follows that the concentration of the 
 hydrogen ions in the solution is a matter of great import- 
 ance. 
 
 (20) A scheme is given showing that the presence of 
 either the H or the OH ion is sufficient to account for 
 hydrolysis. 
 
 (21) The effect of temperature on the hydrolytic power 
 of water can be traced to the change in the concentration 
 of the H and OH ions so produced. 
 
 (2a) If inorganic catalysts and enzymes can take the 
 place of a rise of temperature in this respect their action 
 as hydrolysers could be explained. 
 
 Chemical Action (continued). Oxidation and the 
 Oxidases. 
 
 The processes of oxidation and reduction are not merely 
 opposite, they are also complementary, for except an 
 oxidation takes place in the presence of free oxygen, 
 e. g. in air, the oxygen required for the oxidation must be 
 derived from some substance containing it, and this latter 
 substance, in virtue of giving up its oxygen, is reduced at 
 the same time as the oxidation takes place. Probably 
 even oxidations in air are not direct. 
 
 Oxidation of saturated substances, either in the labora- 
 tory or in the living organism, usually consists in the 
 replacement of hydrogen atoms by hydroxyl groups. An 
 example of this is furnished by the combustion of a hydro- 
 carbon. Ordinarily a hydrocarbon burned with an adequate 
 supply of oxygen yields nothing but carbon dioxide and 
 water without any indication of the formation of inter- 
 mediate compounds, but it has been shown by suitably 
 modifying the conditions that unstable hydroxylated 
 molecules are formed which subsequently undergo decom- 
 
OXIDATION AND THE OXIDASES 85 
 
 position into simpler products. For example, formaldehyde 
 and steam may be detected in the combustion of methane, 
 and the changes which occur are believed to be as follows : 
 CH4 - CH3OH - CH3(0H)j, which on loss of water 
 - CHaO (formaldehyde) - (Hj + CO) - (HaO + COj). 
 A similar change occurs in the oxidation of an aldehyde 
 to an acid : 
 
 The origin of the OH groups in such cases is to be traced 
 to the presence of water, whether in the form of liquid or 
 vapour, for thoroughly dried carbon monoxide does not 
 burn in thoroughly dried air, but when a trace of moisture 
 is present combination takes place at once. Pure metallic 
 sodium retains its bright colour in an atmosphere of dry 
 oxygen, but is instantly tarnished the moment that a trace 
 of moisture is admitted. On the other hand in perfectly 
 pure air-free water, metals like zinc and iron retain their 
 lustre undiminished, but if air is admitted they soon rust. 
 
 When ozone was discovered and its extremely powerful 
 oxidising properties became known, it was suggested that 
 all oxidations were due to its formation. That the forma- 
 tion of ozone sometimes occurs is shown by its occurrence 
 during the slow spontaneous oxidation of phosphorus in 
 air. But the oxidating substances produced in living cells 
 have an oxidation potential which is not so high as that 
 of ozone ; the latter liberates iodine from potassium iodide 
 with great rapidity, the former very slowly. The theory 
 that ozone is the active agent in all oxidations cannot be 
 maintained, but outside the organism it may play a minor 
 part. 
 
 Oxygen, however, in the molecular form, as in air, is a 
 very slow oxidiser, and it is necessary, therefore, to account 
 for the rapid oxidations which occur of substances which 
 
86 ACTION OF ENZYMES 
 
 when exposed to molecular oxygen remain practically 
 unoxidised. In some way the oxygen must be rendered 
 active. 
 
 Now it has been proved beyond doubt that if two 
 oxidisable substances are exposed to oxygen, one of which 
 is easily oxidisable and the other is not, during the oxi- 
 disation of the first the second also will become oxidised, 
 although by itself it would remain unaffected. If sodium 
 sulphite in solution is shaken with air it is rapidly oxidised ; 
 if sodium arsenite is similarly treated no effect is produced ; 
 but if both together are shaken with air both are oxidised. 
 Such a reaction is called a " coupled " reaction. The active 
 substance in this case oxygen, is called the actor, the 
 readily oxidised substance the inductor, and the third, or 
 difficultly oxidisable substance is known as the acceptor. 
 
 To account for the facts of oxidation there have been 
 three main theories. 
 
 (i) The ionisation theory of Van't Hoff assumes that very 
 small amounts of atomic (ionic) oxygen are constantly 
 being produced from molecular oxygen, and that it is with 
 these that the oxidisable substance enters into combina- 
 tion. That such gas ions exist is rendered probable by 
 the facts of the conduction of electricity in gases, and the 
 condensation of aqueous vapour in the steam jet as the 
 result of the oxidation of various substances in close 
 proximity, but only while the process of oxidation is 
 going on. Van't Hoff believed that oxygen dissociated 
 into positive and negative ions, and that the readily 
 oxidisable — or " autoxidisable " — substance took one kind 
 and the oxidisable substance the other. The facts, how- 
 ever, can be explained on theories of more general applica- 
 tion. 
 
 (2) Hoppe Seyler's theory. Hoppe Seyler believed that 
 oxygen is endered active by nascent hydrogen. Hydrogen 
 
OXIDATION AND THE OXIDASES 87 
 
 is liberated in many processes of anaerobic fermentation 
 in an active form, and he believed that it attacked 
 molecular oxygen, appropriating one atom and leaving 
 the other free to unite with an oxidisable substance, if 
 present, if not, then with water forming hydrogen peroxide, 
 HOOH. That nascent hydrogen is capable of effecting 
 oxidations as well as reductions is shown by the fact that 
 hydrogen absorbed by palladium can oxidise benzene to 
 phenol, and toluene to benzoic acid in the presence of 
 water and oxygen. The weak points of the theory are» 
 according to Kastle, that it does not take into account the 
 formation of other peroxides known to be formed besides 
 hydrogen peroxide, and that it accounts for the latter by 
 the oxidation of water. Kastle states that HOOH is 
 never produced by the action of oxygen on water, even 
 under the influence of light and heat, and in the presence 
 of dilute sulphuric acid ; but this is not strictly accurate, as 
 ultra-violet rays will decompose water into hydrogen per- 
 oxide and hydrogen. Nevertheless it appears to be true 
 that HOOH is not formed by the oxidation of water as a 
 rule, but is formed as a secondary product of the hydrolysis 
 of another peroxide, or sometimes by the oxidation of a 
 labile hydrogen atom or ion of some compound. 
 
 (3) The peroxide theory founded by Traube and elabo- 
 rated by Engler and others, depends on the fact that 
 oxidations cannot proceed in the total absence of water. 
 Traube believed («) that the oxygen molecule as a whole 
 combines with the oxidisable substance or with the 
 hydrogen of water to form a peroxide ; (6) that water 
 actively participates in these autoxidations and that 
 HOOH is formed as a primary product. For example, 
 the oxidation of zinc in air and dilute sulphuric acid 
 results not merely in its own oxidation but in the forma- 
 tion of a peroxide, HOOH, as well. 
 
88 ACTION OF ENZYMES 
 
 Zn + gg-g + Os, = Zn(OH)j, + HOOH. 
 
 He also believed (c) that the peroxide formed in this 
 coupled reaction acts as a carrier of oxygen to the 
 acceptor, and (d) that water is decomposed in oxidations 
 with the formation of atomic hydrogen, and that this 
 combines with molecular oxygen to form HOOH. 
 
 An objection to the theory is that it has not been 
 found possible to prove the presence of HOOH in all 
 oxidations; but then it may have only a momentary 
 existence. 
 
 In view of the objections raised the theory has been 
 somewhat modified, and may be stated thus : Molecular, 
 or possibly ionic oxygen, unites with an autoxidisable 
 substance producing a very unstable higher oxide, possibly 
 by molecular combination, which immediately breaks 
 down or is rearranged into a lower oxide and a peroxide, 
 and it is the peroxide which, either with or without the 
 intermediate formation of HOOH, acts as a carrier of 
 oxygen to the acceptor, the peroxide in giving up its extra 
 oxygen becoming the lower oxide. That this is more than a 
 mere theory has been proved by the facts of the oxidation of 
 indigo in the presence of benzaldehyde, C^HjCHO. The 
 ultimate products are benzoic acid and oxidised indigo, 
 but it has been shown that benzoyl hydrogen peroxide, 
 CgHjCO.O.OH, is formed as an intermediate compound. 
 Further, whatever may occur in the laboratory, the action 
 of a peroxide in the living organism is the only hypothesis 
 which will account for the fact than many of the oxidation 
 processes which take place in vivo can be imitated in vitro 
 only by hydrogen peroxide ; e. g. the oxidation of the 
 normal saturated fatty acids in the /3-position ; 
 
 CH3.CH2.CH2.COOH - CH3CO.CH2.COOH 
 Butyric acid. Acetoacetic acid. 
 
OXIDATION AND THE OXIDASES 89 
 
 and indole is oxidised to indoxyl in the body, but outside 
 the body by HOOH only. 
 
 Most peroxides are hydrolysed in water in two stages, 
 thus: 
 
 <l + OH = KoXiH = AO + HOOH. 
 
 Therefore a peroxide arising by autoxidation of an 
 oxidisable substance produced by a cell gives rise, as a 
 rule, to the formation of hydrogen peroxide. 
 
 There is evidence, however, that HOOH is not the 
 eroxide concerned. The fresh juice of Lathnea squamaria, 
 which contains an oxidase, was exposed to a current of air. 
 This ensures the formation of a peroxide, if none was 
 actually present in the fresh juice. If one be present it 
 can be separated and precipitated by barium hydroxide, 
 and as a matter of fact a precipitate is thus produced. 
 The precipitate is filtered off and decomposed by sulphuric 
 acid to set free the peroxide from its combination with 
 barium. The proof that a peroxide is present is the blue 
 coloration produced by adding the solution to a mixture 
 of potassium iodide and starch. This peroxide is not 
 HOOH, for if so it would give an orange-yellow colour 
 with titanic acid, and it does not give this reaction. 
 Nitrites can be proved to be absent ; these would equally 
 give the blue coloration with potassium iodide and starch. 
 It follows that a peroxide is present which is not HOOH, 
 and it is practically certain to be an acylhydroperoxide. 
 That such a substance could be activated by peroxidase is 
 proved by the action of the latter on ethyl hydrogen 
 peroxide. 
 
 Apart from this there is also no necessity to assume the 
 formation of hydrogen peroxide to account for the oxida- 
 tions which occur in the organism. It has been pointed 
 
go ACTION OF ENZYMES 
 
 out that these are replacements of H by OH, and it 
 follows that in the oxidising substance there is a comple- 
 mentary replacement of OH by H. First, as regards the 
 replacement of H by OH, the formation of alcohols, of 
 aldehydes, and of acids can be represented as successive 
 replacements of H by OH, thus : 
 
 /H /OH /OH /OH 
 
 CH3.C-H CHs.C-H CH3.C-OH CH3.C-OH 
 
 \H \h \h \0H 
 
 Ethane. Ethyl Ethyl Acetic acid, 
 
 alcohol. aldehyde. 
 
 The forms shown lose a molecule of water where more 
 
 than one OH radicle is attached to one carbon atom. 
 
 Second, as regards the complementary replacement of 
 
 OH by H, the example of a superoxide like HOOH or 
 
 CgHjCO.O.OH may be taken. In general terms the 
 
 formula is R.O.OH, in the first case R = H, in the second 
 
 R = CgHsCO. 
 
 CH,C<' 
 
 U 
 
 O + I HO^.O.R = CH3C/OH ^ jj Q^ 
 
 \o 
 
 Aldehyde. Acetic acid. 
 
 H.O.R. or ROH is in the first case water, in the second 
 CgHjCOOH, or benzoic acid. If this be a correct state- 
 ment of the case it is not the oxygen connecting the 
 radicle R with OH which leaves its combination with R, 
 but the OH itself. It is true that superoxides in general 
 readily give off oxygen on treatment with dilute acid, but 
 they do this not directly, but probably after first forming 
 hydrogen peroxide. BaOj + H^SO^ = BaSO^ + HOOH. 
 Since the oxidation can be perfectly well represented as 
 an exchange of H and OH between the reacting substances 
 it is unlikely that an intermediate stage involving the 
 giving off of oxygen is inserted. Further, of the two 
 radicles, O and OH, in R.O.OH, it is on the face of it 
 
OXIDATION AND THE OXIDASES 91 
 
 probable tbat the OH is the more weakly attached of the 
 two, for the attachment of OH, itself negative, is to a 
 negative radicle, whereas O is in part attached to a positive 
 one. Oxidation, therefore, by a superoxide like HOOH, 
 the type that occurs in the living organism, and therefore 
 the type which is a priori probable in any enzyme reaction, 
 can be resolved into an exchange of places by the H and 
 OH radicles. To account for all the cases, however, it is 
 necessary to include a subsequent rearrangement of the 
 atoms of the reacting molecules. This appears to occur 
 with indigo when oxidised by CgHjCO.O.OH. 
 
 .NH NH. 
 
 CgHA >C=C< XgH, + zCeHjCO.O.OH 
 
 Indigo. 
 
 N.OH N.OH 
 = C6H,< >C=C<( >CeH, 
 CO CO 
 
 Hypothetical intermediate compound. 
 
 H-zCaHjCOOH. Benzoic acid. 
 
 Hypothetical intermediate compound 
 
 = 2C6H,<^q)>C.0H. Isatin. 
 
 The rearrangement breaks the double bond. 
 
 The theory of oxidation as a replacement of H by OH 
 applies to the saturated compounds, but not to the 
 unsaturated. For example, sulphurous acid, HjSOgr 
 becomes sulphuric acid, HjjSO^, apparently by the simple 
 addition of oxygen : 
 
 o=s<gg + o = °>s<gg 
 
 and if SO.; mixed with oxygen is passed over platinised 
 asbestos at 400° C. SOj is formed. In the latter case it is 
 
92 ACTION OF ENZYMES 
 
 not clear that the gases are thoroughly freed from traces 
 of moisture, while in the former the reaction takes place 
 in aqueous solution. Further, the oxidation of sulphurous 
 acid by chlorine and its reduction of iodine to hydriodic 
 acid include the action of water. The reaction with 
 chlorine may be represented as follows : 
 
 0=S<'^S + aliOH + CI2 = OH-S/^S + 2HCI 
 \0H Qjj/ \0H 
 
 = o/KoH + "°^ + ^^^^• 
 It follows that the oxidation of an unsaturated compound 
 is due, not to the replacement of H by OH, but to the 
 addition of OH, and that the OH required can be obtained 
 from water, provided that the hydrogen radicle of water 
 has first been removed. 
 
 Such a reaction occurs with enzymes in the oxidation 
 of the polyphenols. Catechol, ortho-, and resorcinol, 
 meta-dihydroxybenzene, in alkaline solution are autoxi- 
 disable in air, but hydroquinone, or quinol, the para- 
 compound, is not ; it is easily oxidised by laccase to 
 quinone. Quinone is not a benzene derivative; it is a 
 ■cyclic diketone containing two double bonds, the para- 
 
 diketone of a dihydroxybenzene. 
 
 OH O 
 
 /\ /\ 
 
 Hydroquinone I | | |j Quinone 
 
 OH O 
 
 The reaction probably results in the addition of OH thus: 
 
 HOx^^OH 
 
 HC^OH 
 Since each carbon atom in benzene can unite with only 
 one monad atom the formation of this double hydroxide 
 
OXIDATION AND THE OXIDASES 93 
 
 involves the disappearance of the benzene structure, two 
 bonds which were formerly internal having become 
 external, and the four remaining bonds unite in two pairs 
 as shown. As usual, the combination of carbon with more 
 than one OH is unstable and HOH separates, leaving : 
 O 
 
 [I II or HO.CeH,,OH + HOOH = g^QH^gg 
 
 O = OCfiH^O + jHOH. 
 
 Quinone 
 
 The HOOH represents the peroxide supplied by laccase. 
 
 Since a superoxide is concerned, it follows that the 
 characters of any superoxide oxidation are to be found with 
 the whole group of superoxides, hence reactions which in 
 the organism may be carried out by organic peroxides can 
 be carried out in the laboratory by hydrogen peroxide, 
 and in experimenting with the oxidases it is legitimate to 
 use HOOH as the peroxide, whether this is the actual 
 peroxide used by the cells or not. 
 
 A difficulty may be felt here from the point of view of 
 catalytic or enzyme action. Since the formation of the 
 peroxide implies the simultaneous formation of the lower 
 oxide, and itself in giving up its oxygen or hydroxyl 
 becomes converted into the lower oxide, the previously 
 autoxidisable substance now exists in the form of a lower 
 oxide, i. e. it is no longer autoxidisable, its special quality 
 has disappeared. There is evidence that*this occurs in 
 oxidase action, for Kastle and Loewenhart and also Bach 
 have pointed out that the oxidases are not true ferments 
 or oxygen-catalysts, because they are unable to accom- 
 plish the oxidation of practically unlimited amounts of 
 oxidisable material. This is intelligible if a peroxide is 
 concerned in the process, since it cannot be reformed 
 from the same material. There is, however, evidence that 
 
94 ACTION OF ENZYMES 
 
 autoxidisable substances exist which can be reformed on 
 parting with their oxygen. Hserngglobin is the best 
 known case. Since its absorption of oxygen appears to 
 have relation both to its chemical constitution and to the 
 tension of the oxygen to which it is exposed, Bayliss 
 suggests that both chemical and adsorption combinations 
 are concerned, but from the point of view of enzyme 
 action haemoglobin does not enter into the question except 
 as the means of providing oxygen for oxidases, and this 
 seems not to occur through enzyme action, for the oxygen 
 might be equally well supplied to the enzyme by shaking 
 its solution with air. If the oxidases cannot oxidise 
 unlimited quantities of material, and if the peroxides, by 
 means of which they act, cannot be reformed from the 
 same material, it would appear that enzyme action in the 
 process of oxidation is excluded. There is evidence, 
 nevertheless, that it does occur, and this will now be 
 considered. 
 
 The oxidases, or oxidising enzymes, which are known 
 are very few, though there are indications of the existence 
 of others. They are : (i) Laccase, originally found in the 
 juice of the lac tree, an enzyme which, when the juice is 
 allowed to stand in air, gradually turns it black ; the 
 boiled juice does not show this change, nor does the 
 change occur in the absence of oxygen. (2) Tyrosinase, 
 which turns tyrosin black, forming the melanin pigments. 
 (3) Indophenol oxidase, which oxidises a mixture of 
 «-naphthol and paraphenylene diamine to indophenol, a 
 blue compound. (4) The purin oxidases, which convert 
 xanthin and hypoxanthin to uric acid, and in some cases 
 oxidise uric acid itself. (5) Catalase, which acts specifically 
 on HOOH, producing molecular, t. e. inactive, oxygen. It 
 is, properly speaking, no*^ an oxidase. Aldehydase is 
 sometimes included, but is rather to be classed with the 
 
OXIDATION AND THE OXIDASES 95 
 
 reducing enzymes. Of these, laccase, or enzymes acting 
 in the same way, is almost universally distributed in the 
 plant world, but is found in certain animal cells only. 
 Tyrosinase is widely distributed among plants, but not so 
 widely as laccase ; it is, however, much more abundant in 
 the animal kingdom, where it plays an essential part in 
 the formation of animal pigment. 
 
 Laccase may be taken as the type of oxidising enzymes, 
 as its method of action appears to be common to the 
 oxidising enzymes, though minor differences are to be 
 found, especially in the influence of acids and alkalies and 
 of hydrogen peroxide on each. 
 
 The reaction by which the presence of laccase or 
 laccase-like enzymes is recognised is the guaiacum blue 
 reaction. The gum-resin guaiacum contains guaiaconic 
 acid, and it ,is this which on oxidation gives the blue 
 colour. 
 
 The fundamental experiments are : 
 
 (i) To a scraping from the surface of a potato apply a 
 drop of alcoholic solution of guaiaconic acid: a blue 
 colour is produced. The same effect is produced by 
 scrapings from the root of the horse-radish and many 
 other plants. 
 
 (2) Hydrogen peroxide does not give this blue colour 
 with guaiaconic acid. 
 
 (3) A solution can be prepared from the root of the 
 horse-radish by a complicated method (principally by the 
 use of alcohol), which with guaiaconic acid does not give 
 the blue colour. 
 
 C4) A mixture of this preparation, hydrogen peroxide, 
 and guaiaconic acid gives the blue reaction. 
 
 (5) If in the first experiment, before guaiaconic acid is 
 added to the potato scrapings, the tube containing the 
 scrapings is exposed to a current of hydrogen or any 
 
96 ACTION OF ENZYMES 
 
 neutral gas till the oxygen is wholly displaced, a blue 
 colour is no longer obtained on addition of guaiaconic 
 acid. 
 
 From these experiments it is inferred that a peroxide 
 like hydrogen peroxide must be present, and that it is this 
 which oxidises the guaiaconic acid, but not until it is 
 activated. The activator, because it acts on peroxides, is 
 called peroxidase, and is the true enzyme of oxidation. 
 It has the general properties of an enzyme, for it is 
 destroyed by heat and it can act on practically unlimited 
 quantities of the peroxide provided that an oxidisable 
 substance is present and that this is removed when 
 oxidised. In contact with HOOH alone it is gradually 
 destroyed. In all these points it acts like a true enzyme. 
 
 It is now clear why oxidases did not appear to be true 
 enzymes. Oxidases like laccase, tyrosinase, etc., are 
 really compound bodies consisting of the true enzyme 
 peroxidase, a peroxide, and, it may be, an autoxidisable 
 substance. Their defects as enzymes are due to the 
 peroxide constituent, for this must be gradually used up in 
 the course of the reaction. 
 
 The oxidases in general appear to be less specific in 
 their action than the hydrolytic enzymes. Laccase oxi- 
 dises not only laccol but also guaiacum, guaiacol, hydro- 
 quinone, phenolphthalin, adrenalin, and a large number of 
 phenols and aromatic amino-compounds. There is a 
 certain specificity, however, as the only substances easily 
 oxidised by laccase are those of the benzene series con- 
 taining hydroxyl or amino groups in the ortho- or para- 
 positions. Tyrosinase acts most readily on the homo- 
 logues of phenol in which the side chains occupy the 
 para-position. 
 
 Like the hydrolytic enzymes the oxidases are sensitive 
 to the action of acids, alkalies, and salts. Laccase is 
 
OXIDATION AND THE OXIDASES 97 
 
 poisoned or paralysed by most acids, at any rate beyond a 
 very low concentration, but some, such as carbonic, boric, 
 and phosphoric, have no effect at any concentration. 
 Hydrocyanic acid and hydrogen sulphide, hydroxylamin, 
 phenyl hydrazin, and similar reducing bodies destroy the 
 action of all oxidases. Tyrosinase is inhibited by N/ioo 
 HCl and is considerably retarded by N/ioo NaOH. 
 Neutral salts in general retard its action. 
 
 There are also certain salts which, like the co- 
 enzymes of the hydrolytic enzymes, are either absolutely 
 necessary to the action of the oxidases or very greatly 
 stimulate their action. Bertrand found that laccase 
 always contains small amounts of manganese, and that 
 its oxidising power is proportional to the amount of 
 manganese present ; further, that laccase oxidations are 
 greatly accelerated by small amounts of manganese salts, 
 and that no other metal is capable of taking its place. 
 The tyrosinase found in the skins of animals acts upon 
 tyrosin only in the presence of small amounts of iron. 
 Slowtzoff claims to have prepared a laccase free from 
 manganese, but, on the other hand, Bertrand showed that 
 a specimen poor in manganese was comparatively inac- 
 tive, and that the effect of the addition of manganese to 
 this preparation was to increase its oxidising power 
 twenty-five times. Manganese salts by themselves can 
 bring about the oxidation of various oxidisable substances, 
 such as hydroquinone, in air, and the quantity of oxygen 
 absorbed varies with the nature of the manganese salt, 
 being greater with the salts of the organic acids. It 
 appears, therefore, that those manganese salts which are 
 most easily hydrolysable are the most efficient oxygen 
 carriers. It is clear, therefore, that the condition of the 
 manganese salt itself is important, and that the active 
 state is the hydroxide. Iron salts have a similar action. 
 
 7 
 
98 ACTION OF ENZYMES 
 
 Schinoxidase from the latex of Schinus molle gives the 
 same reactions as laccase, but its ash contains no manga- 
 nese and is rich instead in iron. Again, ferrous salts in 
 extremely small amounts strongly accelerate the action of 
 HOOH on oxidisable substances. Ferrous salts, too, as 
 also those of copper and manganese, accelerate the oxi- 
 dising powers of oil of turpentine, benzaldehyde, etc., no 
 doubt by action on the peroxides produced. This is in 
 line with the dependence of the oxygen-carrying power of 
 haemoglobin on its iron content, for the guaiacum test for 
 blood, which is an indication of oxidising power, is given 
 even by the iron-containing derivative hasmatin, but not 
 by the iron-free hsematoporphyrin. In the molluscs and 
 Crustacea hsemocyanin, a copper-containing substance, 
 acts like haemoglobin as an oxygen carrier. Zinc is found 
 in the blood of certain marine forms, and salts of zinc in 
 extraordinary dilutions have a great effect on the growth 
 of moulds. 
 
 Peroxidase is the most widely distributed of enzymes. 
 It can certainly be said of any living tissue or organ that 
 it is dead when it fails to show the reactions of the peroxi- 
 dases. It is also extraordinarily resistant to decay. All 
 seeds which retain their germinating power contain per- 
 oxidases, and in corn they have been recognised in 
 samples over two hundred years old. Peroxidase outlasts 
 the power to germinate by a hundred years. It therefore 
 appears that peroxidase activity is one of the most 
 characteristic and persistent properties of living material. 
 
 Catalase is a remarkable enzyme. Loew has arrived at 
 the conclusion that there does not exist a cell which does 
 not contain some catalase. Its action is exclusively on 
 hydrogen peroxide, which it decomposes with the forma- 
 tion of molecular, i. e. inactive, oxygen. In this respect 
 it is unique, for all other substances which decompose 
 
REDUCTION AND THE REDUCASES 99 
 
 hydrogen peroxide activate the oxygen, e, g. even finely- 
 divided platinum. The simplest explanation of this 
 action is that it always reacts with two molecules of 
 HOOH and never with one. 2HOOH = 2HOH + Oj. 
 As it does not activate oxygen it is not an oxidase 
 proper, though its enzyme action is undoubted. It is 
 believed to exert a protective action against hydrogen 
 peroxide formed as a by-product, and possibly acts also 
 as a regulator of peroxidase action, for in a mixture of 
 HOOH with peroxidase and catalase active oxygen and 
 molecular oxygen are formed in proportion to the relative 
 amounts of the two enzymes. 
 
 Reduction and the Reducases. 
 
 There appears to be some doubt as to the existence of 
 these enzymes. Euler says that most apparent cases of 
 reducase action are really stoichiometric reductions by a 
 readily oxidisable substance, and that catalytic action is 
 not concerned. The following experiments by Schardinger 
 seem, however, to be conclusive : 
 
 (i) Fresh milk has no action on methylene blue. 
 
 (2) Fresh milk reduces methylene blue to the leuco or 
 colourless base if an aldehyde such as acetic or formic is 
 present. 
 
 (3) Boiled milk has no action in the presence of an 
 aldehyde on methylene blue. 
 
 (4) The action of bacteria in the milk can be excluded. 
 Reductions in general are due to the action of nascent 
 
 or active hydrogen, and the source of this is of import- 
 ance. 
 
 Analogies with reducase action have been found in the 
 action of the platinum group of metals which give the key 
 to the source. If a hypophosphite is dissolved in water 
 
100 ACTION OF ENZYMES 
 
 no change takes place, but if finely divided palladium is 
 also present it is oxidised. 
 
 Hv /H Hv nu 
 
 >P< becomes >P< 
 
 O^ \0H O/ \0H 
 
 The oxygen which enters the compound in the form of 
 OH can come only from the water of the solution. Thus : 
 H3PO3 + 2HOH = H3PO3 + Hg + HOH. The hydrogen 
 represented as being evolved is absorbed by the palladium 
 and is later set free. If an aldehyde is taken in place of 
 the hypophosphite the presence of metals of the platinum 
 group has no effect, but if an acceptor for the nascent 
 hydrogen is also present the aldehyde is oxidised to the 
 corresponding carboxylic acid. Acceptors are methylene 
 blue, nitrates, indigo, etc., all easily reducible substances, 
 but none of them is reduced at any perceptible rate by 
 formaldehyde alone without platinum. Since methylene 
 blue contains no oxygen, the oxygen used in the oxida- 
 tion of the aldehyde must come from the water of the 
 solution. 
 
 R.CHO + HOH + meth. blue = R.COOH + H. meth. 
 
 blue., i. e. reduced. 
 The enzj'me which appears to be contained in fresh milk 
 takes the place of finely divided platinum in the above 
 reaction. 
 
 To explain the reducing action of milk, etc.. Bach has 
 suggested the existence of a perhydride of oxygen in water, 
 H4O, on the ground that oxygen may be quadrivalent, and 
 on analogy with the action of peroxidase on peroxides he 
 has suggested the name, perhydridase, for the milk enzyme. 
 Similar reducing enzymes have been described in the 
 tissues, whose powerful reducing action while fresh is well 
 known, and a reducing enzyme has been extracted from 
 the liver which reduces methylene blue in the presence of 
 an aldehyde. 
 
REDUCTION AND THE REDUCASES loi 
 
 From the point of view of the oxidation of the aldehyde 
 the enzyme has been described as an aldehydase. It 
 appears to be independent of the presence of atmospheric 
 oxygen, for liberal supplies of fresh oxygen diminish the 
 rate of oxidation and may even suppress it altogether. 
 The process resembles anaerobic fermentation where the 
 oxygen required is furnished not by the air but by oxygen- 
 containing compounds, in this case, water. 
 
 Bach's assumption of a perhydride in water seems to 
 me totally unnecessary. It has already been pointed out 
 that any oxidation involves a reduction, in general of a 
 peroxide, but in the case of anaerobic fermentation the 
 reduction must be of some substance containing oxygen 
 and not a peroxide. Now, as an oxidation involves a 
 reduction, so a reduction must involve an oxidation, and 
 while the action of a reducing enzyme is reducing on the 
 substance regarded— in the case under consideration, 
 methylene blue — it is equally an oxidising enzyme from 
 the point of view of the substance oxidised, viz. aldehyde. 
 Treating it then as an oxidising enzyme, it derives its 
 oxygen from the water of the solution, and the oxidation 
 conforms to the usual type of oxidations in being a replace- 
 ment in the aldehyde of H by OH; 
 
 CH,C 
 
 H 
 
 O + 
 
 OH 
 
 H = CHoC/S" + H + H. 
 
 The nascent hydrogen so formed reduces the reducible 
 substance, methylene blue. Plainly the action of the 
 enzyme is to activate water. Hence the assumption 
 of the presence of H4O is unnecessary, and the fact that 
 it has never been isolated renders its existence very 
 problematical. 
 
 The oxidation of salicylic aldehyde by the juice of the 
 potato in the presence of potassium chlorate is the work 
 of an aldehydase, salicylic aldehyde being a substance 
 
102 ACTION OF ENZYMES 
 
 difficult to oxidise. Formic aldehyde and benzyl alcohol 
 are more easily oxidised, and for them the presence of a 
 nitrate is sufficient. Tha^ plant and animal tissues have 
 the power of reducing nitrates to nitrites has long been 
 known, and in the light of these facts appears to be a 
 special case of the power of certain enzymes to transfer 
 oxygen (probably as OH) from a substance rich in oxygen 
 to one that is poorer. 
 
 Summary. 
 
 (i) The processes of oxidation and reduction are not 
 merely opposite, they are also complementary, one implying 
 the other. 
 
 (2) Oxidation of saturated substances, either in the 
 laboratory or in the living organism, usually consists in 
 the replacement of hydrogen atoms by hydroxyl groups. 
 
 (3) The source of the hydroxyl groups can in some 
 cases be traced to the hydroxyl group of the molecule of 
 water. 
 
 (4) There is no evidence that ozone takes any part in 
 biological oxidations. 
 
 (5) Owing to the inactivity of molecular oxygen as an 
 oxidiser, its oxygen must be activated before it can be 
 utilised. 
 
 (6) In a coupled reaction the oxidation of an easily 
 oxidisable substance brings about the simultaneous 
 oxidation of a difficultly oxidisable substance associated 
 with it. 
 
 (7) Three main theories have been formulated to 
 account for the known facts of oxidation : (i) Van't 
 Hoffs ionisation theory, (2) Hoppe Seyler's nascent 
 hydrogen theory, (3) Traube's peroxide theory. 
 
 (8) There is evidence that oxidations take place at 
 
REDUCTION AND THE REDUCASES 103 
 
 times which can be explained in terms of any one of 
 these theories, but the third or peroxide theory explains 
 all the known facts and is open to fewest objections. 
 
 (9) The peroxide theory postulates the combination of 
 molecular oxygen with an autoxidisable substance to 
 form a higher oxide, which is immediately transformed 
 into a lower oxide and a peroxide, and the peroxide so 
 formed acts as the oxygen carrier to the substance to be 
 oxidised. 
 
 (10) That a peroxide is concerned in biological oxida- 
 tions is proved by the cases in which hydrogen peroxide 
 is the only known substance which can imitate in the 
 laboratory certain biological oxidations. 
 
 (11) It has been thought that hydrogen peroxide is the 
 peroxide concerned, but it can be proved by direct experi- 
 ment in certain cases that it is not the peroxide formed, 
 and if an organic peroxide is formed there is no necessity 
 to assume that hydrogen peroxide is formed from it before 
 oxidation can take place. 
 
 (12) Even on the peroxide theory of oxidation it is 
 probable that the process of oxidation in saturated com- 
 pounds consists in the replacement of H by OH, the latter 
 being directly derived from the peroxide. In unsaturated 
 compounds oxidation consists in the addition of OH 
 derived from the same sources. 
 
 (13) To account fully for the results of oxidation in 
 some cases a subsequent rearrangement of the atoms of 
 the molecule must be assumed. 
 
 (14) As the peroxide does not appear to be reformed 
 from the same material, one of the essential qualities of 
 catalytic action appears to be lacking, viz. unlimited 
 action ; this appears to be the case with the oxidases. 
 There is evidence, nevertheless, that an enzyme is con- 
 cerned. 
 
104 ACTION OF ENZYMES 
 
 (15) The best known oxidases are enumerated with their 
 main actions. 
 
 (16) The cardinal experiments proving that an oxidase is 
 compounded of a peroxide and a peroxidase are described. 
 Peroxidase has all the properties of a true enzyme. 
 
 (17) The specificity of the oxidases is not so great as 
 that of the hydrolytic enzymes. 
 
 (18) The oxidases are specially sensitive to the action 
 of certain electrolytes. 
 
 (19) Analogies with the co-enzymes of the hydrolytic 
 enzymes are found in the action of the hydroxides of 
 manganese, iron, copper, and possibly zinc. 
 
 (20) Peroxidase is the most widely distributed of the 
 enzymes. Its activity appears to be one of the most 
 characteristic and persistent properties of living material. 
 
 (21) Catalase is an important enzyme with a unique 
 action on hydrogen peroxide, but as it does not activate 
 oxygen it is not an oxidase. 
 
 (22) While some doubt has been thrown on the existence 
 of reducases certain experiments given seem to prove their 
 existence. 
 
 (23) Analogies with the action of the reducases are to 
 be found in the action of the platinum group of catalysts. 
 
 (24) Bach has suggested the existence of a perhydride 
 of oxygen, H^O, in water, and an enzyme acting on it 
 which he names perhydridase, to account for the forma- 
 tion of nascent hydrogen, as it is this which carries out 
 the reduction. 
 
 (25) Aldehydase as an oxidising enzyme derives its 
 oxygen from water. 
 
 {26) The origin of the nascent hydrogen required for 
 reduction can be traced to the activation of water without 
 the necessity of assuming the existence of such a substance 
 as H4O. 
 
METHOD OF ENZYME ACTION 105 
 
 (27) The reduction of nitrates by plant and animal 
 tissues appears to be a case of the transfer of oxygen from 
 a substance rich in oxygen to one that is poorer. 
 
 The Method of Enzyme Action. A Hypothesis. 
 
 If the conclusions derived from the study of hydrolysis 
 and oxidation in the preceding sections are admitted, it 
 follows that hydrolysis and its converse, synthesis, are due 
 to the addition or removal of the H and OH groups, and 
 also that in saturated compounds oxidation is a replace- 
 ment of H by OH and reduction a replacement of OH 
 by H, while in unsaturated compounds oxidation is an 
 addition of OH and reduction an addition of H. The 
 influence of free oxygen is an indirect one as an acceptor 
 for the hydrogen set free. In other words, the whole of 
 ■enzyme action has been reduced to the action of hydrogen 
 and hydroxyl radicles. 
 
 That this should be so is really to be expected if one 
 thinks of hfe and living tissue as a development in the 
 presence of the two universal substances, water and air. 
 The processes of hydrolysis and dehydration, oxidation, 
 and reduction can take place without the intervention of 
 catalysts, and it may be that the earliest forms of life 
 depended on the naturally occurring reactions. As, how- 
 ever, life developed and increased in complexity an 
 increase in the speed of the reactions would become 
 necessary, and at the same time a limitation of their 
 sphere of action, for a reaction which at one place would 
 be beneficial at another might be injurious. Two powers 
 are, therefore, required : 
 
 (i) Control of the rate of reaction. 
 
 (2) Control of the sphere of reaction. 
 The control of the rate of reaction resolves itself into a 
 
io6 ACTION OF ENZYMES 
 
 means of controlling the amount and the movements of 
 the H and OH radicles wherever found. The presence of a 
 substance which had an attraction for these radicles and 
 yet did not hold them so firmly as to be unable to give them 
 up if a substance presented itself with greater attractions 
 in the given circumstances, would solve the problem of 
 increasing the rate of reaction, since now the reaction 
 would be due not to the naturally occurring, sparsely distri- 
 buted radicles, but to masses of them. A general increase, 
 however, of these radicles would act on all parts of the vital 
 structure equally ; this danger must be avoided by control 
 of the sphere of reaction. This can be attained by limiting 
 the masses of radicles to definite points to which the sub- 
 stances to be acted on are attracted, and not other bodies 
 present in the solution. The limitation to definite points 
 is secured by the colloid nature of the enzymes, as the 
 action takes place not generally in the solution but only 
 on the surfaces of their particles. The further limitation 
 that only certain substances of those present are to be 
 acted on is ensured by attracting only these substances to 
 the active points. The attraction may be either physical 
 or chemical, but as the physical in the shape of adsorption 
 appears to precede the chemical action in all cases, we may 
 assume that the enzyme forms an adsorption compound 
 with the substrate always. By these means complete con- 
 trol of the reaction is obtained, its speed is increased, and 
 its action limited to the desired point and the desired 
 substances. 
 
 Any enzyme, therefore, possesses two qualities. 
 
 (i) A power common to it and all other enzymes 
 of attracting one or both of the H and OH radicles. 
 
 (2) A specific power of adsorbing a particular sub- 
 strate or substrates. 
 
 These powers, the specific and the general or unspecific,, 
 
METHOD OF ENZYME ACTION 107 
 
 are to be found in all enzymes, but the degree of the 
 power varies ; one enzyme may be weaker than another 
 in its attraction for H or OH, and the specificity of a 
 particular enzyme may on the one hand be limited to a 
 particular substrate and on the other may extend to a 
 large group of substances. 
 
 While the specific attractions of enzymes indicate 
 extreme diversity of composition, the possession of a power 
 common to all enzymes indicates the action of a limited 
 class of substances. These two apparently contradictory 
 inferences are only reconcilable by assuming that the 
 majority of enzymes are compounded of two substances, 
 one exercising the specific and the other the general func- 
 tion. A small class of enzymes may exist single in 
 composition because that member of the lirnited class 
 which can exercise the general function happens also to 
 attract a particular substrate. In general, however, the 
 double action must indicate a double composition. 
 
 While it seems almost impossible to arrive at the com- 
 position of the specifically acting member of the alliance 
 it ought not to be impossible to discover the class of sub- 
 stances which can exercise the general function, as this 
 class is likely to be limited, and members of it should be 
 discoverable by a study of those enzymes about whose 
 composition most is known. 
 
 In spite of the complication of the entire process which 
 takes place in oxidation, when the actual enzyme action 
 takes place the process appears to be among the simplest 
 of these actions, as it has been reduced to peroxide- 
 peroxidase-substrate, and there are other indications that 
 in oxidation enzyme action is to be found in its simplest 
 form. In the first place the extreme specificity of some 
 of the hydrolytic enzymes is not met with among the 
 oxidases as a rule, laccase oxidising not merely laccol but 
 
io8 ACTION OF ENZYMES 
 
 many aromatic hydroxy-derivatives as well ; secondly, 
 laccase at any rate is not so sensitive to heat as are the 
 hydrolytic enzymes, for its solutions can be boiled for 
 three minutes without destroying their activity. While 
 this enzyme is especially stable to heat, other oxidases are 
 destroyed only at 8o-go° C, though the maximal tempera- 
 ture for the hydrolytic enzymes varies from 60° to 75° C. 
 Lastly, the extraordinarily wide distribution of peroxidase 
 or the peroxidases, for they are practically universal in 
 living cells, indicates a simple constitution, since other- 
 wise it is difficult to understand how it or similar 
 -substances could be found in cells of such diverse nature. 
 That the oxidising enzymes are the simplest also 
 appears from the fact that most is known of their action 
 and of the bodies which influence them. It has already 
 been mentioned that the salts of manganese and iron 
 seem to be intimately connected with oxidation, and in a 
 lesser degree copper is also active, and possibly zinc, and, 
 further, that the active preparation is the hydroxide. 
 Manganese and iron, even apart from their relation to the 
 oxidising enzymes, can greatly influence the process of 
 oxidation, e.g. manganese salts will bring about the oxida- 
 tion of hydroquinone in air. If a trace of ferrous sulphate 
 {less than one part per million) be added to old tincture 
 of guaiacum or to a fresh tincture of guaiacum containing 
 a trace of hydrogen peroxide, a very intense blue coloration 
 is obtained. On heating the solution containing ferrous 
 sulphate and hydrogen peroxide it loses its power to blue 
 guaiacum, and traces of mineral acids also prevent this 
 action just as they prevent the action of peroxidases, the 
 <iosage being the same in both cases. Again, Wolff 
 claims to have reproduced all the actions of the oxidases 
 with colloidal ferrocyanide of iron. It acts as peroxidase 
 towards phenols : it is filterable without loss of activity ; 
 
METHOD OF ENZYME ACTION 109 
 
 its activity is weakened after one minute's boiling, and 
 traces of mineral acids reduce its activity ; it is also 
 sensitive to excess of hydrogen peroxide. Sjolleman has 
 made a colloidal solution of manganese oxide which gives 
 the characteristic reactions of the oxidases and decom- 
 poses hydrogen peroxide like potassium permanganate. 
 
 The resemblance of manganese and iron hydroxide 
 action to the oxidases is increased when in the presence 
 of albumin and similar colloidal substances. In dilute 
 solution of fresh egg albumin made faintly alkaline a small 
 amount of manganese chloride will give the guaiacum 
 reaction, and heat destroys the oxidising power of this 
 like other similar compounds. Dony Renault obtained a 
 very active preparation by precipitating a solution con- 
 taining 10 grams of gum arable, i gram of manganese 
 formate, and 0*4 gram of crystallised sodium carbonate in 
 50 c.c. of water with alcohol ; this was filtered off, washed, 
 reprecipitated, and redissolved. In the end it resembled 
 natural laccase very closely. In all the cases mentioned 
 the solutions were slightly alkaline, and the OH ions, if 
 not absolutely necessary, certainly greatly stimulate the 
 process. 
 
 The hydroxides of manganese and iron are substances 
 insoluble in water ; they can be obtained in the colloidal 
 form, but being suspensoid colloids they are easily pre- 
 cipitated. Suspensoid colloids can have conferred on 
 them the properties of emulsoid colloids in this respect if 
 a certain amount of emulsoid colloid is added to the 
 suspensoid, and it is believed that adsorption between the 
 two takes place. Hence the action of albumin, gum 
 arable, etc., may be a stabilising action on the manganese. 
 If such a compound is formed it ought to show the other 
 properties of the emulsoid from which it is derived, such 
 as sensitiveness to heat and to alcohol, and since the 
 
no ACTION OF ENZYMES 
 
 number of emulsoids from which to choose is practically 
 unlimited, the specificity of the combination would also be 
 due to the emulsoid. Bayliss concludes, therefore, that a 
 peroxidase is in all probability a peculiarly active form of 
 the colloidal hydroxide of manganese, iron, or copper, 
 preserved in this active state by the presence of an emulsoid 
 colloid, and that it is to this stable colloid that the enzyme 
 owes its precipitation by heat and alcohol, and possibly 
 any degree of speciiicity that it may possess. He states 
 that a view essentially the same as this was suggested by 
 Perrin. 
 
 If manganese and iron hydroxides are the active mole- 
 cules they must possess some property relatively to oxygen 
 which is peculiar to them, and not shared by the hydroxides 
 of other metals, or by very few of them. That there is 
 such a property is shown by the fact that there are at 
 least two simple hydroxides of manganese, and the same 
 is true of iron. When manganese chloride, MnCl^, is 
 treated with an alkaline hydroxide in the absence of air a 
 colourless flocculent precipitate of manganous hydroxide, 
 Mn(OH)2, is formed. Exposed to air the white precipitate 
 rapidly absorbs oxygen and passes into manganic hydroxide, 
 Mn(0H)3. Again, white ferrous hydroxide is precipitated 
 when an alkaline hydroxide or ammonia is added to.a solu- 
 tion of a ferrous salt, provided air be entirely absent. Ex- 
 posed to air the white precipitate rapidly absorbs oxygen 
 and passes into ferric hydroxide, Fe(OH)3. Therefore for 
 both manganese and iron there are two hydroxides pos- 
 sible, the higher differing from the lower by the possession 
 of an extra OH, and also by being the more stable form. 
 That, however, Fe(OH)2 can exist as well as Fe(OH)g 
 while there is no indication of the existence of FeOH, 
 proves that the third OH of the hydroxide Fe(OH)g is not 
 so firmly bound to Fe as the other two. This agrees with 
 
METHOD OF ENZYME ACTION m 
 
 the fact that reducing substances can reduce ferric salts 
 to ferrous. It follows, therefore, that Fe(OH)j is such a 
 substance as appeared to be demanded in an enzyme, viz. 
 one that had an attraction for either H or OH, in this 
 case OH, yet did not hold it so firmly as to be unable to 
 give it up in the presence of a substance with greater 
 attractions in the given circumstances. But if a substance 
 exist which can take up OH and give it out again as 
 required, then that substance would at the end of the 
 reaction appear unchanged, and in this respect would 
 conform to the definition of a catalyst. 
 
 In an oxidase reaction, therefore, Fe(0H)3 or 
 Mn(0H)3 ought to be able to take the place of peroxidase, 
 and that this is possible is proved by the facts already 
 given, which show the immense stimulating effect of 
 ferrous and manganous salts. The mechanism of the 
 reaction may be conceived to be as follows. Three sub- 
 stances are present — a peroxide, R — O — OH, manganous 
 hydroxide, Mn(0H)3, and a substance to be oxidised, or 
 Fe(OH)j[ might be used instead. Assuming that the sub- 
 stance to be oxidised is a saturated compound, its oxidation 
 will consist in the replacement of H by OH, and it must, 
 therefore, contain replaceable H. It may be represented 
 as H.Sub. So long as it is in this form the OH of the 
 peroxide, though, as has been pointed out, but weakly 
 attached, cannot detach the H from H.Sub, therefore 
 R — O — OH -1- H.Sub produce no reaction. But what is 
 impossible for weakly attached OH is quite possible for 
 the strongly negative radicle R — O — if it be brought into 
 existence by the detachment of OH. This is effected by 
 the attraction of Mn(OH)2 for OH, which is stronger 
 than the weak attraction of R — O — for it. Hence the 
 reaction is : 
 
 R— O— OH + Mn(0H)8 = RO" + Mn(OH)s 
 
112 ACTION OF ENZYMES 
 
 The strongly negative radicle R0~ has a great attraction 
 for positive H and detaches it from H.Sub. 
 
 R0~ + H.Sub = ROH + Sub±. 
 It follows from this that RO is much more strongly nega- 
 tive than Sub ; in fact, in most oxidations in vivo Sub 
 must be practically amphoteric in its reactions, being 
 negative to strongly positive radicles like H and positive 
 •to strongly negative radicles like OH. It is, therefore, 
 represented as Sub±. In virtue of its positive character 
 to OH and the fact that it cannot exist uncombined it 
 must have a stronger attraction for OH than has Mn(0H)2, 
 which is able to exist as such. Therefore the last stage 
 of the reaction is : 
 
 Sub+ + Mn(0H)3^ Sub.OH + Mn(OH)„ 
 and as a result the substrate is oxidised and the catalyst 
 reformed. 
 
 This appears to be satisfactory for saturated substances, 
 but not for unsaturated where simple addition of OH 
 takes place, and no H is present to unite with the residue 
 of the peroxide. In such cases the strongly negative RO 
 appears to be able to derive the necessary hydrogen from 
 water. 
 
 R.O.OH + Mn(0H)3 = R0~ + Mn(0H)3 
 
 R0~ + HOH = ROH + OH~ 
 
 aSub + Mn(OH)3 + OH~ = aSub.OH + Mn(OH)j. 
 In this case an OH radicle is freed in addition to the 
 OH which is attached to Mn(OH)2, and consequently two 
 molecules of the unsaturated substance can be oxidised, 
 one deriving its OH from Mn(OH)3 and the other from 
 the OH radicle freed in the reaction. Probably the action 
 of a catalyst would be rarely required, however, with an 
 unsaturated substance which as a strongly reducing body 
 can take its OH direct from the peroxide. 
 
METHOD OF ENZYME ACTION 115 
 
 R.O.OH + Sub = RO" + Sub.OH 
 R0~ + HOH = ROH + 0H~ 
 OH" + Sub = Sub.OH. 
 
 Since water must be dissociated in this reaction two 
 molecules of the substrate are oxidised. 
 
 The whole explanation is based on the assumption of 
 intermediate compounds with the catalyst, which later 
 break up with reformation of the catalyst. That such 
 intermediate compounds are formed in all cases is not 
 certain, but that they are found in some appears to be 
 clear from the action of MnOg in the decomposition of 
 potassium chlorate described in the section on catalysis, 
 and it has been proved by Erode in one case, that of the 
 acceleration of the reaction between hydriodic acid and 
 hydrogen peroxide by molybdic acid. A series of permo- 
 lybdic acids is formed by the action of hydrogen peroxide 
 on the catalyst ; these are formed with great rapidity, and 
 when formed they react with great rapidity also on 
 hydriodic acid with separation of iodine and return of the 
 catalyst to its original form of molybdic acid. 
 
 As regards enzymes, the existence of intermediate 
 adsorption compounds appears to be beyond doubt, but 
 whether the enzyme enters into the chemical reaction is 
 unknown. A further examination of the reactions given, 
 however, seems to throw some light on the point. 
 
 There seem to be two weak points in the equations : 
 (i) The absence of the action of water in the case of 
 saturated substances ; (2) the splitting of undissociated 
 molecules of water by RO . As regards the first, if water 
 enters into the reaction in the case of an unsaturated 
 substance it is hardly likely to be inactive in the case of 
 a saturated. Taking in the action of water the first set of 
 equations becomes : 
 
 8 
 
114 ACTION OF ENZYMES 
 
 R.O.OH + Mn(OH)2 = R0~ + Mn(0H)8. 
 
 RO + HOH = ROH + OH". 
 
 H.Sub + OH" = Sub.OH + H+. 
 
 H+ + Mn(0H)8 = Mn(OH)2 + HOH. 
 It has already been pointed out that the radicle Sub 
 must have but small attraction for H, and if the above be 
 the correct form of the equations it must have less for it 
 than for OH. This is likely, as it is a substance capable 
 of being oxidised, and the oxidised form is usually more 
 stable than the unoxidised. The hydrogen radicle set 
 free reforms the catalyst by uniting with its feebly com- 
 bined OH. 
 
 With regard to the second point — the dissociation of 
 HOH — it is true that RO is strongly negative, but it is 
 hardly likely to be so strongly negative as OH, and there 
 does not seem any good reason why it should attract H 
 away from it. It is also true that H and OH ions occur 
 in water, but they are so small in amount that it is diffi- 
 cult to believe that they have any appreciable effect on the 
 reaction. 
 
 But while it is unlikely that RO" could detach H from 
 OH in a free molecule of water it might be able to do this 
 provided the attachment of OH to H were weakened in 
 any way. Now, Mn (OH)^ has a considerable attraction 
 for OH, and though this is not nearly sufficient to break 
 its connection with H (unless an acceptor for the sepa- 
 rated H is present like dissolved oxygen) it might weaken 
 the connection if its attraction for OH were sufficient to 
 attach a molecule of water to itself, thus : 
 
 Mn(OH)2--OH'H. 
 In such a compound the attraction of OH for H would 
 not be so great as in the free water molecule, and the 
 radicle R0~ might be able to break the connection, par- 
 ticularly as this does not involve the appearance of a free 
 
METHOD OF El^ZYME ACTION 115 
 
 OH radicle. The final form of the equations then would be : 
 
 R.O.OH + Mn(OH)i,--OH'H = ROH + Mn(OH), 
 + 0H~ 
 
 H.Sub + OH" = Sub.OH + H+. 
 
 H+ + Mn(OH)3 = Mn(OH)g--OH'H. 
 The reformation of the catalyst in this conception 
 certainly means the extraction of OH from its combination 
 with Mn(OH) 3 but not its complete severance, and the 
 real catalyst is not Mn(0H)2 but the hydrated form 
 Mn(0H)2 --OH'H. Such a body would not be a 
 chemical compound proper, it would be a molecular com- 
 pound, yet one depending on chemical affinity. 
 
 In the case of an unsaturated compound the only 
 difference would be that the OH'H molecule is not re- 
 formed by the course of the reaction, but is derived from 
 a fresh molecule of water. 
 
 At the commencement of this section the presence of a 
 substance capable of massing the H and OH radicles by its 
 attraction for them, yet not holding them so firmly as to 
 be unable to give them up, was postulated. If Mn(OH)j 
 can really form such a compound as Mn(OH)a — OH'H, 
 then that compound has the qualities required. 
 
 An objection may be raised to the equations given on 
 the ground that R.O.OH would react with Mn(OH)g - - 
 OH'H as follows: 
 
 R.O.OH + Mn(0H)2--0H'H = ROH -|- Mn(OH)„ 
 Mn(OH)^ = HOH + MnOCOH)^ (manganous acid), 
 and that there would then be no oxidation of the sub- 
 strate and no reformation of the catalyst. It does actually 
 happen, if a solution containing a peroxide and a peroxi- 
 dase is allowed to stand without any substrate, that the 
 solution loses its enzymic activity, and the reason is now 
 clear. Mn(OH)^ is a very unstable compound and 
 rapidly loses water, hence there is no great tendency to 
 
ii6 ACTION OF ENZYMES 
 
 its formation, and a substrate, if present, takes up OH in 
 preference. 
 
 Granting that such a compound as Mn(0H)2 — OH'H 
 can exist, a compound formed on the opposite model 
 attracting H and not OH ought to exist, X - - H'OH. It 
 would not be a peroxidase, for the semi-detached radicle 
 now is OH, not H. If this OH were taken up by a 
 substance containing H, which had an attraction for OH 
 and the H were replacedj nascent hydrogen would be set 
 free and this could perform reductions. This appears to 
 occur with the platinum group of metals, and especially 
 with palladium. 
 
 R.CHO + Pd--H'OH = R.COOH + Pd.H + H+. 
 
 H+ + oxidised indigo = reduced indigo + 0H~. 
 
 Pd.H + 0H~ = Pd - - H'OH. 
 Hence reducing enzymes may be of the type X - - H'OH. 
 It has been recognised that in these reactions another sub- 
 stance is oxidised, and that the necessary oxygen is 
 derived from water. Both these facts are satisfied by the 
 above equations. 
 
 If the existence of Mn(0H)3 — OH'H be granted as a 
 chemical molecular compound, then in a number of such 
 compounds the strength of the attraction for OH, or, in 
 the case of X — H'OH, for H must vary, some attracting 
 the radicle but weakly and others strongly. It the attrac- 
 tion be very strong the second water radicle must be very 
 readily formed, and the catalyst be a powerful one. 
 Hence palladium is a more powerful catalyst for certain 
 reactions than platinum, for while platinum black can 
 occlude one hundred times its volume of hydrogen, palla- 
 dium black can occlude six times as much. It is unlikely 
 that any chemical-atomic compound is formed with the 
 hydrogen, and if not, such cases are of great interest, as 
 they indicate that if a physical attraction for one com- 
 
METHOD OF ENZYME ACTION ii7 
 
 ponent of a chemical compound is powerful enough, that 
 compound can be broken up provided an acceptor for its 
 other component is present. 
 
 With regard to the specific element of the oxidases 
 there is little information to be had except for one striking 
 fact. There seems to be a difference between the laccase 
 derived from the Rhus genus of plants and that from the 
 Medicago genus. The former always contain manganese, 
 the latter, though their activity is greatly intensified by 
 manganese compounds, do not contain it, nor do they 
 contain iron, etc., which might take its place. Further, 
 they are much less sensitive to heat, for while the former 
 are destroyed by boiling for a short time the latter are 
 unaffected. On chemical examination the latter are 
 found to be composed of a mixture of calcium salts of 
 organic mono- and polybasic hydroxyacids, among which 
 are glycollic, CH2OH.COOH, malic, COOH.CHOH. 
 CHjj.COOH, mesoxalic, COOH.C(OH)2.COOH, and 
 citric acids. When a mixture of the pure salts is made, 
 its catalytic power is found to be practically that of 
 Medicago oxidase. From a consideration of the chemical 
 formulae it is clear that, apart from their combination 
 with calcium, their point of agreement is the possession 
 of alcoholic hydroxyl. It may be that there is a tendency 
 to further oxidation in the group containing this, and that 
 this tendency leads to the formation of molecular com- 
 pounds with water. However this may be, the catalyst 
 so formed is to a certain extent specific in that it acts on 
 the group of substances attacked by laccase, and it might 
 be possible by examination of the physical and chemical 
 qualities of the salts of these hydroxyacids to say wherein 
 the attraction resides. 
 
 Medicago laccase, therefore, does not conform to the 
 type of double composition which the facts seem to 
 
ii8 ACTION OF ENZYMES 
 
 require in the majority of enzymes. It is representative 
 of that small class, where the substance exercising the 
 general function also possess a specific attraction for the 
 substrate. 
 
 It is now possible, so far as the oxidases are concerned, 
 granting the existence of the compound with water, to 
 answer the question whether enzymes take part in the 
 chemical reaction which they accelerate. The answer is 
 that they do not directly. Their function is confined to 
 enabling the hydrogen radicle obtained from molecules of 
 water to carry out the reaction. A similar position is 
 occupied by the reducing enzymes, the only difference 
 being that these activate the hydroxyl radicle of water. 
 In neither case does the enzyme take part directly in the 
 reaction ; its function is indirect and consists in activating 
 one or other of the water-radicles. 
 
 The composition of the hydrolysing enzymes is not so 
 well known as that of the oxidases. There is nothing to 
 compare with the constancy of manganese or iron in the 
 latter. It is true that they are colloidal, and that they 
 have a double action, specific in relation to their sub- 
 strates, but general in their action on those substrates. 
 This, however, does not prove that they are necessarily 
 double in composition, or even if they are, that the 
 unspecific component is a comparatively simple com- 
 pound. Still, analogy with the oxidases is in favour of a 
 similar composition, and the fact that simple bases and 
 acids can carry out hydrolyses in proportion to their 
 ionisability is greatly in favour of the simplicity of the 
 unspecific component, for ionisation is not met with to 
 the same extent in the more complex organic bases and 
 acids. 
 
 The existence of co-enzymes is another indication of 
 
METHOD OF ENZYME ACTION 119 
 
 the simplicity of the unspecifically acting component of a 
 hydrolytic enzyme. In many cases none are known, in 
 others they are known to exist but their composition is 
 unknown, in still others they are familiar chemical sub- 
 stances. If an unspecific component exists, it might be 
 thought that it should be capable of detection in all cases, 
 but this leaves out of account the very variable character 
 of the firmness of attachment in adsorption compounds. 
 Fixation often occurs, e. g. trypsin to charcoal, so that the 
 compound cannot be broken by mere washing with water, 
 and yet it can hardly be described as chemical, since 
 another substance more strongly adsorbed can release it. 
 We may expect to find some co-enzymes very loosely 
 attached, others more firmly, while again we may suspect 
 the existence of others which we cannot detach. As 
 examples, it may be mentioned that pancreatic lipase can 
 be separated from its co-enzyme by mere filtration of the 
 glycerol extract. In this case the co-enzyme may be a 
 bile-salt. Sodium chloride, the co-enzyme of liver 
 amylase, is more firmly attached, for it can be removed 
 only by dialysis. The co-enzyme of yeast juice requires 
 for its separation high pressure on a gelatinised filter- 
 Where none can be found by these methods, the ash may 
 give evidence of the presence of a more or less simple 
 co-enzyme, but not necessarily, for if it be an organic 
 compound or an ammonium salt it would not leave traces 
 in the ash. It may be asserted that co-enzymes are only 
 adjuvants of enzymes, but in face of the fact that the 
 enzyme action in many cases cannot be carried on without 
 them, they are more likely to be essential parts of the 
 enzymes. 
 
 That mineral acids and bases can catalyse hydrolyses is 
 well known, but the method of action is not known. It 
 has been pointed out previously that the existence of 
 
120 ACTION OF ENZYMES 
 
 either a free OH or a free H ion would account for the 
 hydrolysis. Now, since a base, e.g. KOH, ionises into 
 K and OH, and an acid, e. g. HCl, into H and CI, it follows 
 that the ion required is in either case present, so that the 
 action may be on the following lines, XY being the sub- 
 stance to be hydrolysed : 
 
 K+ + OH" + XY = X" + YOH + K+ 
 
 K+ + H+ + OH~ = KOH + H+ 
 
 H+ + X" = HX 
 For an acid : 
 
 H+ + cr + XY = HX + Y+ + cr 
 
 Cr + H+ + OH" = HCl + OH" 
 
 Y+ + OH" = YOH. 
 There are, however, difficulties in accepting these equa- 
 tions as a true picture of the reactions. The difficulty 
 does not lie in the decomposition of XY, for in such a 
 substance as ethyl acetate, which is a typical substance 
 hydrolysed by hydrochloric acid, the acetic residue 
 represents a weak acid only, not to be compared with 
 hydrochloric. The difficulty is in explaining why a double 
 decomposition of the ordinary type does not take place, 
 HCl + XY = HX + YCl. In part the explanation may 
 be the small attraction of Y, i. e. ethyl, for CI, since the 
 formation of ethyl chloride only takes place in a mixture 
 of hydrochloric acid and ethyl alcohol when both alcohol 
 and acid are as nearly pure as possible. There remains 
 the difficulty of explaining why the H radicle should leave 
 HCl to attach itself to the acetic radicle when acetic is a 
 so much weaker acid than hydrochloric. The objection 
 appears to be fatal to any attempt to represent the facts 
 on these lines. If, however, it were got over, the further 
 reaction with water is explained by the small attraction of 
 Y for CI and the presence of the naturally occurring ions 
 of water. When these are removed by combination with 
 
METHOD OF ENZYME ACTION I2i 
 
 Y and CI fresh ions are formed to restore the equilibrium 
 Avhich exists between the undissociated water molecules 
 and the water ions. These criticisms and explanations 
 apply also to hydrolysis by KOH. Another explanation 
 must, therefore, be sought for. 
 
 The process would be represented on Werner's scheme 
 as follows : HCl is not the true active acid but an anhydro- 
 acid which has a strong attraction for the OH of HOH 
 through its H component ; hence it hydrates, OH entering 
 the first or non-dissociable zone, and H the second or dis- 
 sociable zone, thus : 
 
 HCl + HOH = (CIH.OH)H 
 so forming the true or aquo-acid. This dissociates : 
 
 (CIH.OH)H = (CIH.OH)" + H+. 
 With a substance XY a double decomposition occurs : 
 (CIH.OH)" + H+ + XY = (CIH.OH)Y + HX. 
 The fate of (CIH.OH)Y depends on the strength of the 
 attraction of CIH for OH. The whole ionises into 
 (CIH.OH)" + Y+. It may go further by hydrolysis : 
 
 (CIH.OH)- + Y+ = CIH + OH +Y+. 
 Being a hydrolysis this is a reversible equation, Y appear- 
 ing on both sides of the equation is without effect. 
 Therefore by the law of mass action the product of the 
 concentrations of CIH and OH divided by the concentra- 
 tion of (CIH.OH) is a constant. If OH increases 
 CIH.OH must increase, i. e. in alkaline solution there will 
 be practically no hydrolysis. But if OH diminishes CIH 
 must increase to keep the product constant. Now the 
 solution is not alkaline but acid owing to the presence of 
 HX, therefore hydrolysis occurs : 
 
 (CIH.OH)Y = CIH -1- YOH. 
 The anhydro-acid is reformed and hydrolysis of XY has 
 laken place. 
 Similarly with bases, KOH is the anhydro-base which 
 
isa ACTION OF ENZYMES 
 
 has a strong attraction for the H of HOH through the 
 OH component, and there is formed the aquo- or true base 
 (KHO.H)OH. Double decomposition occurs with XY, 
 forming (KHO.H)X + YOH, the latter ionising as Y+ and 
 0H~. As the solution is therefore alkaline, hydrolysis of 
 (KHO.H)X takes place with the formation of HX and the 
 reformation of KOH, 
 
 It is possible to use the notation already explained 
 when describing the action of the oxidases, the apos- 
 trophe ' being used as before to indicate weakened bonds. 
 KOH + HOH = KHO - - H'OH 
 KHO — H'OH + XY = KHO — H'X + YOH 
 KHO - - H'X = KOH + HX. 
 With the necessary changes the same equations apply to 
 the action of HCl. 
 
 There is not, however, the same reason for the molecular 
 union with water as there was, in the case of Mn(OH)3, and 
 if it occurs it must be put down to some such cause as the 
 auxiliary valency which Werner postulates. The argu- 
 ments for the union of water with acids, bases, and salts 
 have been given earlier and are very cogent. Werner 
 states that HC1(0H3)3 has actually been proved to exist. 
 
 An objection may be raised on the ground that in acid 
 hydrolysis (CIH.OH)Y (or CIH - - OH'Y, according to 
 the notation employed) is supposed to be formed, and in 
 the case of the hydrolysis of methyl acetate by HCl this 
 would be methyl chloride. Now Tafel has pointed out 
 (i) that methyl chloride is not formed with any rapidity 
 under the conditions of the reaction, and (2) it cannot 
 replace HCl as a catalyst. The answer to the first objec- 
 tion is that the hydrated methyl chloride supposed to be 
 formed appears to have only a momentary existence, as it 
 must be unstable in the presence of excess of H ions ; alsa 
 that it is a hydrated methyl chloride, and it is doubtful 
 
METHOD OF ENZYME ACTION 123 
 
 that methyl chloride added in the anhydrous state would 
 hydrate so readily in solution as HCl. The answer to the 
 second point is that if a catalyst acts by forming inter- 
 mediate compounds, methyl chloride cannot act as a 
 catalyst unless such an unlikely body as methyl chloride 
 of methyl were formed. 
 
 It follows from the views propounded in this discussion 
 of the action of acids and bases that the H and OH radicles 
 required are derived from the water of the solvent, just as 
 occurs with the oxidases. 
 
 While acids and alkalies are efficient hydrolysers they 
 are not to be compared with the enzymes as regards 
 power. Boiling with hydrochloric acid for hours is needed 
 to produce the same change in proteins that trypsin can 
 produce in an hour or two at body temperature, and the 
 great power of lactase on milk sugar in comparison with 
 a mineral acid has already been mentioned. On this 
 account it might be thought that enzyme action was 
 essentially different from the H and OH action of acids 
 and bases, were a simpler explanation not available. The 
 enzymes concentrate the radicles at the point of action 
 and also the substrate, viz. the surfaces of their particles, 
 whereas in acid or alkali hydrolysis both substrate and 
 ions are evenly dispersed through the solution ; hence 
 much fewer collisions between ions and substrate molecules 
 take place with the latter in a given time. 
 
 The hydrolytic action of salts, which such facts as the 
 necessity of sodium chloride for liver amylase and bile- 
 salts for pancreatic lipase oblige us to assume, is more 
 difficult to account for. Of the four possible classes of 
 salts, (a) those of a strong acid and a strong base, (6) of 
 strong acid and weak base, (c) of weak acid and strong 
 base, and (rf) of weak acid and weak base, there is no 
 difficulty in seeing how H ions can be formed in (6) and 
 
124 ACTION OF ENZYMES 
 
 OH ions in (c), for these salts hydrolyse to some extent in 
 aqueous solution, liberating a strong acid from class {b) and 
 a strong base from class (c). These give rise to H or 
 OH ions in the manner described in the preceding section. 
 As regards class {d) hydrolysis, it is true, is much more 
 marked than in the other two classes, but there is little 
 ionisation and, presumably, little attraction for water, 
 •hence few H or OH ions are formed. Class (a), however, 
 is of special interest because practically no hydrolysis 
 takes place in aqueous solution, yet the facts compel us 
 to assume that H or OH radicles arise if the hypothesis 
 of H or OH action in all cases is correct. Such facts are 
 the activation of liver amylase by sodium chloride, and the 
 activation of trypsin by calcium chloride. 
 
 While these salts of a strong acid and a strong base are 
 not hydrolysable by simple solution in water, there is 
 evidence that in certain circumstances they do hydrolyse. 
 Emich's experiment with red-hot sodium chloride and 
 water in the spheroidal state shows the influence of 
 temperature ; but more important in the present connec- 
 tion is the acidity of the supernatant fluid when a 
 suspensoid colloid is precipitated by barium chloride. It 
 was shown in a previous section that this could be 
 accounted for satisfactorily only on the hypothesis that 
 •hydrolysis of the barium chloride had occurred. Barium 
 as an alkaline earth forms a strong base, and, therefore, 
 barium chloride falls into the class of salts under con- 
 sideration. Now the compound formed by the colloid and 
 barium is clearly an adsorption compound ; if, therefore, 
 adsorptive attraction be strong enough it can induce a 
 chemical action between two other substances which 
 would not react without it — in this case barium chloride and 
 Avater. The action seems to be similar to that of palladium 
 in oxidising a hypophosphite in solution ; the hypo- 
 
METHOD OF ENZYME ACTION 125 
 
 phosphite under its influence reacts with water. The 
 action has been traced already to the combination of 
 palladium with the hydrogen of water, and this combina- 
 tion, considering how it is affected by physical agents, is 
 not likely to be a chemical one, though it may be due to 
 solid solution not adsorption. 
 
 It is true that monovalent radicles like sodium will not 
 precipitate an emulsoid colloid except in comparatively 
 high concentration, but nevertheless when present in 
 lesser concentration the tendency must exist, i. t. the 
 hydrolytic action of the colloid on the salt must be in its 
 first stage. Since there is evidence that salts in solution 
 unite with water, the combination with water is not the 
 first stage of the hydrolysis, for the combination takes 
 place whether the colloid is present or not. The first 
 stage then must be a weakening of the bond between the 
 atoms composing the salt : NaCl becomes Na'Cl. The 
 combination with water only can be represented in 
 Werner's form as CNaHO.H)Cl, or in the notation used in 
 
 this section as : 
 
 NaCl 
 I I 
 
 I I 
 OH'H 
 
 If the bond between the Na and CI is weakened by the 
 colloid the attraction of Na for OH, on the one hand, and 
 of 01 for H on the other, will be strengthened, and this 
 will be accompanied by a corresponding weakening of the 
 bond between OH and H, thus : 
 
 Na'Cl 
 
 II II 
 
 II II 
 OH',H 
 
 In presence of a substrate, XY, with which either 
 NaOH or HCl can react, I imagine there would be a 
 
126 ACTION OF ENZYMES 
 
 momentary snapping of the bond between Na and CI, with 
 the result that two reactions would take place : 
 
 Na OH + XY = NaX + YOH 
 and 
 
 HCl + XY = YCl + HX. 
 Of the four bodies so formed two would react as follows : 
 
 NaX+YCl = NaCl + XY, 
 and there would be left NaCl reformed, XY, substrate, 
 still attracted to the enzyme, while HX and YOH, the 
 products, would diffuse away, the reaction recommencing 
 as before. 
 
 This is the simplest form of the reaction. It is probable 
 that at the moment of formation of NaOH and HCl 
 other molecules of water enter the reaction, preventing the 
 formation of NaX and YCl and conforming the reaction 
 to the equations given previously for hydrolysis by an 
 acid and an alkali. 
 
 So far the explanations have dealt with hydrolysis ; it 
 remains to show how synthesis may occur. Synthetic 
 reactions occur when water is in defect, while yet the 
 attraction of the catalyst for either H or OH remains as 
 before. Unable to obtain the required radicle from water 
 it finds it in one of the bodies to be united, thus : 
 
 HCl + YOH = CIH - ■> OH'Y. 
 Double decomposition then occurs with HX owing to the 
 attraction of OH for H above its attraction for Y : 
 
 CIH -- OH'Y + HX = CIH -- OH'H + XY. 
 An equilibrium exists between the concentration of the 
 substances on the enzyme and that of those in the solu- 
 tion which this increase of water on the enzyme has 
 disturbed, therefore the water attached to the catalyst 
 must be detached and diffuse away. 
 
 CIH - - OH'H = HCl + HOH. 
 
METHOD OF ENZYME ACTION 127 
 
 Equilibrium will be reached when the concentration of 
 water in the solution has so increased that there is no 
 tendency for water formed by the above reaction to diffuse 
 away ; the catalyst is then hydrated and cannot act on 
 HX and YOH. 
 
 Similarly for an alkali catalyost : 
 
 KOH + HX = KHO - - H'X 
 
 KHO--H'X + YOH = KHO--H'OH + XY 
 
 KHO--H'OH = KOH + HOH. 
 
 For a salt of the NaCl type, where owing to its colloid 
 connection NaCl has become Na'Cl, the reactions may be 
 thus represented : 
 
 Na'Cl + YOH + HX =Y'OH — Na'Cl — H'X 
 
 Y'OH -- Na'Cl- - H'X=NaOH + X~ and HCL+Y+ 
 =NaX + YCl + HOH 
 NaX + YCl =NaCl + XY. 
 
 Hence for synthesis as well as hydrolysis the efficient 
 agents are the H and OH radicles. 
 
 So far the unspecific element of a hydrolytic enzyme 
 has been dealt with. As regards the specific element 
 little seems to be known. A hint may possibly be 
 found in the fact mentioned already : " An addition of 
 0-05 grm. of asparagine to 100 c.c. of a starch solution 
 containing amylase increases the velocity of the reaction 
 sevenfold." Asparagine is the monamide of aspartic acid 
 and is perhaps the most common amide in plants. Its 
 chemical solution is HOOC.CH2.CH(NH3).CONHj, and 
 it exists in optically isomeric forms. It is a weak acid, 
 and as such may have some slight effect in increasing the 
 hydrogen ion concentration, but it ought to be far sur- 
 passed in this particular by stronger acids. It follows, 
 therefore, that its extraordinary influence cannot be due 
 to this, and that it is more probably due to its action on 
 
ia8 ACTION OF ENZYMES 
 
 the specific component of the enzyme. If this specific 
 component were asparagine, or were in part composed of 
 asparagine, then the addition of asparagine would be 
 equivalent to adding fresh enzyme, for the unspecific 
 component is probably present in the solution ready to 
 combine with it. A study of the physical and chemical 
 qualities of asparagine and its action on starch — whether, 
 for example, it forms adsorption compounds with starch, 
 with peculiar facility — ought to throw light on the con- 
 stitution of amylase. 
 
 Summary. 
 
 (i) The whole of the chemical action of enzymes may 
 be reduced to the action of hydrogen and hydroxyl. 
 
 (2) This is to be expected, as the two substances 
 everywhere present when life first developed were water 
 and air. 
 
 (3) The use of enzymes is to control the rate and the 
 sphere of naturally occurring reactions. The former is 
 secured by increasing the number of the active H and OH 
 radicles ; the latter by limiting them to definite points 
 to which the substrate of the reaction is also attracted. 
 
 (4) Any enzyme possesses a general or unspecific power 
 of attracting one or other of the H and OH radicles, and 
 a specific power of adsorbing a particular substrate. 
 
 (5) This double power probably indicates a double 
 constitution. 
 
 (6) The enzyme action which takes place in oxidation 
 appears to be one of the simplest forms. 
 
 (7) The hydroxides of manganese and iron are intimately 
 connected with peroxidase action. They can by them- 
 selves influence the process of oxidation, and their 
 resemblance to the enzymes is greatly increased in the 
 presence of colloids. 
 
METHOD OF ENZYME ACTION 129 
 
 (8) A peroxidase is probably a hydroxide of manganese, 
 iron, etc., preserved in the colloidal form by an emulsoid 
 colloid. It is to the latter component that the reactions 
 of the enzyme to heat and alcohol and its specificity are due. 
 
 (9) The hydroxides of manganese and iron are found in 
 two forms, differing from each other in stability and in 
 the possession or not of an extra OH radicle. 
 
 (10) Equations are given showing the reactions that a 
 hydroxide of manganese in the presence of a peroxide and 
 a substrate would be likely to initiate, in both saturated 
 and unsaturated substances. 
 
 (11) The action depends on the formation of inter- 
 mediate compounds ; the existence of these is proved in 
 some cases of catalytic action. 
 
 (12) On close examination the equations given are 
 unsatisfactory unless the entrance of water into the 
 reaction is assumed. It is shown also that the water so 
 entering must be in a form in which the bond between its 
 H and OH radicles is weakened. 
 
 (13) It follows that the hydroxide of manganese as 
 such is not capable of carrying out the functions of 
 peroxidase ; it must first be hydrated. The act of 
 hydration loosens the bonds connecting H and OH in the 
 water combined. 
 
 (14) Since substances such as the lower hydroxides of 
 manganese and iron exist which have the power of 
 attracting OH, it is likely that substances can be found 
 which have a similar power of attracting H. Such sub- 
 stances would act as reducing catalysts. 
 
 (15) The action of palladium and platinum black is 
 explained on this hypothesis. 
 
 (i5)The specific element in the oxidases is unknown, 
 but some of the laccases give evidence bearing on its 
 constitution. 
 
 9 
 
130 ACTION OF ENZYMES 
 
 (17) Enzymes do not take part directly in the chemical 
 action which occurs ; their part is the activation of one 
 or other of the water-radicles, and it is this latter which 
 carries out the reaction. 
 
 (18) That the hydrolytic enzymes have a similar double 
 composition may be conjectured from analogy with the 
 oxidases, and also from the existence of co-enzymes. The 
 latter appear in many cases to be essential to the action 
 of the enzyme. 
 
 (19) The catalytic action of the mineral acids and bases 
 in hydrolysis is discussed, and it is shown that the facts 
 are difficult to explain except on the hypothesis of 
 hydration of the acid or base, resulting in the weakening 
 of the bond between the H and OH radicles in the com- 
 bined water. 
 
 (ao) Objections raised in the particular case of the 
 hydrolysis of methyl acetate in the presence of hydro- 
 chloric acid, based on the improbability of methyl chloride 
 being an intermediate compound, are considered and 
 possible answers given. 
 
 (21) The more powerful action of enzymes, as compared 
 with acids and bases, is traced to concentration of the H 
 and OH radicles. 
 
 (22) The hydrolytic action of salts in connection with 
 enzymes is discussed. It is shown that of the four classes 
 of salts, two form acids and bases by hydrolysis, and 
 another is probably not concerned with enzyme action. 
 
 (23) The large class of salts formed from a strong base 
 and a strong acid raises the greatest difficulty, but it is 
 shown that in special circumstances they can hydrolyse 
 into acids and bases, and evidence is given for the proba- 
 bility that union with a colloid leads to the first step 
 towards this hydrolysis, viz. a weakening of the bond 
 which holds their component radicles together. 
 
DEDUCTIONS 131 
 
 (24) Union between the salt and water takes place 
 apart from the presence of a colloid, and this union 
 weakens the bond between the H and OH radicles of the 
 combined water. When the union with the colloid has 
 -weakened the bond between the components of the salt, 
 -the bond connecting the H and OH of the combined 
 water is still further weakened. 
 
 (25) In presence of a substrate which can be acted on, 
 both acid and alkali may be formed simultaneously from 
 the salt, and these, reacting with the substrate and with 
 each other, cause hydrolysis of the substrate and reforma- 
 tion of the salt. 
 
 (26) It is shown that synthetic reactions can be 
 accounted for on the same basis of union of the catalyst 
 with H and OH, but in synthetic reactions this union 
 takes place with the H and OH radicles of the two sub- 
 strates, not with those of water. 
 
 (27) The specific elements of the hydrolytic enzymes 
 are unknown, but facts bearing on the constitution of this 
 component of amylase are mentioned. 
 
 Deductions. 
 
 The hypothesis of enzyme action to which this inquiry 
 has led is based on (i) the possibility of combination 
 between molecules, and (2) the hypothesis that this 
 molecular combination loosens the internal bonds in one 
 or both of the combining molecules. As regards the 
 possibility of combination between molecules the existence 
 of the double salts appears to put this beyond doubt, and 
 for the existence of combinations of solute and solvent in 
 the case of water arguments have already been given. 
 To these may be added the fact that when crystals of 
 almost any salt are dissolved in water a lowering of the 
 
132 ACTION OF ENZYMES 
 
 temperature takes place owing to the development of 
 osmotic pressure. The development of this pressure 
 implies that work has been done, and for this work energy 
 must be obtained from somewhere. It is obtained by 
 extracting heat from surrounding objects. If, on the 
 other hand, the anhydrou's salts are dissolved, there is often 
 a rise of temperature which must be due to combination 
 with the solvent setting free an amount of heat which is 
 not merely sufficient to supply all the energy required for 
 the work done in developing osmotic pressure, but which 
 is able also to supply heat to surrounding objects. With 
 regard to the second point — the loosening of internal 
 bonds — this may furnish an explanation of chemical 
 action between non-electrolytes where ions do not appear 
 to be formed, for the loosening of the internal bonds must 
 render the substances affected more reactive, in fact it 
 may be an essential condition of reaction. One is tempted 
 to suggest that molecular combination precedes chemical 
 action in all cases, and is the analogue in the molecular 
 world of the adsorption which precedes chemical action in ■ 
 the case of colloids. 
 
 Natura non facit saltum is an aphorism which is applic- 
 able. There ought to be all stages between that where 
 two bodies can remain in intimate juxtaposition without 
 any kind of reaction and the stage where the two react 
 with explosive violence, and one of these stages may be 
 that of molecular union with loosened atomic bonds. 
 Such a conception implies that a valency on an atom is 
 not an individual unitary force pointed in a definite 
 direction, but a certain quantity of affinity which is^ 
 divisible and may be altered in position. This view is 
 held by Werner. It is usual to speak of residual affinity 
 as accounting for molecular compounds, but there does 
 not seem any necessity to assume the existence of this in 
 
DEDUCTIONS 133 
 
 order to explain these compounds. The proof of the 
 existence of unsatisfied affinities in certain cases depends 
 on quite other considerations. If the affinity of an atom 
 is concentrated on one part of its surface (if surface can 
 be said to exist on an atom) directly opposite to a similarly 
 affected surface on another atom with which it is com- 
 bined, it is conceivable that the entrance of a compound 
 on the scene containing an atom with which the first 
 could combine if both were free, may cause a certain 
 amount of the affinity on both to leave its previous position 
 and be concentrated on those sides of the atoms which 
 face. If the change in distribution of the affinity is suffi 
 •cient, the combination on both sides will break up and 
 double decomposition occur ; but if not, a certain attrac- 
 tion will still be exercised, resulting in molecular combina- 
 tion. As the affinity of the atom is to some extent 
 redistributed, there is less attracting that atom with which 
 it is already combined, and this implies a loosening of this 
 bond. The loosening of the bond implies that a certain 
 amount of affinity on the other combined atom is unsatis- 
 fied, and this is indicated by an increase in its reactivity. 
 In the most stable compounds, no doubt, the amount of 
 affinity which is subtracted from that uniting the partners 
 is insufficient to weaken the union materially. The 
 affinity is eo great that there is, so to say, plenty to spare. 
 Any hypothesis of enzyme action must account for the 
 properties of enzymes. Most of those described in the 
 section dealing with these properties have already been 
 accounted for, e. g. specificity, synthetic or reverse action, 
 combination with substrates and products, catalytic 
 action, and co-enzymes. Of the remainder, the influence 
 of electrolytes which are not eo-enzymes may be 
 mentioned. The very great influence of acids and alkalies 
 can now be easily accounted for by their production in 
 
134 ACTION OF ENZYMES 
 
 the solution of H or OH ions. Since the whole enzymic 
 process, so far as it is chemical, depends on the H and 
 OH radicles, the addition of hydrogen or hydroxyl ions 
 derived from an outside source must have a profound in- 
 fluence. If the action is due to the hydrogen radicles, the 
 addition of a small amount of hydrogen ions may assist 
 the process, but if they are added in excess the production 
 of the radicles by the enzyme may be interfered with and 
 even suppressed. Since the rapidity and power of the 
 enzymic reaction depends on the local concentration of 
 these radicles, a suppression of this concentration by acid 
 will reduce the process to one of ordinary acid hydrolysis, 
 which is much slower than enzyme hydrolysis. The same 
 remarks apply to the addition of alkali to a solution con- 
 taining an enzyme acting by the production of OH 
 radicles. If, on the other hand, hydrogen ions are added 
 to an enzyme acting by OH radicles, or hydroxyl ions to 
 one acting by H radicles, then a neutralisation of the 
 enzymic radicle will take place and the enzyme action 
 disappear. 
 
 The action of salts is more complicated. If they do 
 not combine with the unspeciiic component of the enzyme 
 and yet are adsorbed by its colloid they may aid the 
 reaction, but if they combine they may destroy the 
 enzyme action altogether. They may also combine with 
 the substrate, as indeed may acids and alkalies, and the 
 combination may be- more readily adsorbed by the 
 enzyme, or adsorption may be prevented altogether. 
 However they act, the fact that they must practically 
 always have some action on one or other of the com- 
 ponents of the enzyme reaction is amply sufficient cause 
 for their well-known great influence on enzymes. 
 
 The great influence of heat may be accounted for in 
 several ways. Chemical reactions are accelerated by 
 
DEDUCTIONS 135 
 
 increasing the temperature ; hence, in moderation a rise 
 of temperature may increase the speed of an enzymic 
 reaction, but an immoderate increase destroys the enzyme. 
 It has been pointed out that colloids are sensitive to heatj 
 in many of them irreversible coagulation occurring if the 
 temperature rises too high. This appears to be due to 
 aggregation of their particles and probably also to a 
 change in the composition of these particles. As enzymes 
 have a colloid component a rise of temperature beyond a 
 certain point must affect this component and therefore the 
 whole enzyme. ~ Coagulation would remove the unspeci- 
 fically acting component in a state of adsorption. There 
 is, however, probably another cause at work. If mole- 
 cular combination precedes chemical action, it may be that 
 the adsorption of the general component to the specific is 
 due to the possibility of chemical combination taking 
 place between them in other circumstances. The rise of 
 temperature may render this chemical combination 
 possible, and then the enzyme ceases to be an enzyme. 
 A condition of this kind is seen in the combination of the 
 blue acid of Congo-red with a colloidal hydroxide of 
 aluminium, iron^ etc. The adsorption compound is blue, 
 the colour of the free acid, but if it be heated to 100° C, 
 combination takes place and the red colour of the salt 
 appears. 
 
 The existence of anti-enzymes is disputed, but they 
 may exist if the colloid component of the enzyme can act 
 as an antigen, i. e. if it be a protein, as it may be in some 
 cases. An antibody appears to unite with its antigen, for 
 the neutralisation of antigen by antibody is quantitative. 
 The combination so formed may cease to attract the 
 generally acting component or the substrate, and the 
 enzyme as enzyme cease to exist. On the other hand, 
 merely altering the hydrogen ion concentration must have 
 
136 ACTION OF ENZYMES 
 
 an enormous effect on any enzyme, so that it must be 
 difi&cult to decide which cause is at work. 
 
 Zymogens may be accounted for in certain cases where 
 an acid or alkaU can act as activator by a chemical 
 decomposition setting free the enzyme from an inactive 
 combination, but it is also possible that the zymogen is the 
 colloidal component of the enzyme without its generally 
 acting component, and that the action of the activator is 
 to supply this. 
 
 The ultimate proof of an hypothesis is the demonstra- 
 tion of the truth of deductions made from it. If the 
 hypothesis I am urging is a correct explanation of enzyme 
 action it ought to be possible to manufacture enzymes. 
 Clearly defined principles from which the constitution of 
 the enzyme requiired by a particular substrate could be 
 theoretically deduced are still wanting, but it is possible 
 to devise an empirical method for arriving at the same 
 result. It is necessary first of all to discover a colloid 
 with special attractions for the substrate under considera- 
 tion, and tests for the presence of this might be based on 
 the following points. It is clear that a sufficient aggre- 
 gation of molecules of substrate about a colloid particle 
 will increase its size, so that a tendency to turbidity if not 
 precipitation may occur. The disturbing effect of electro- 
 lytes in altering the electric charge must be eliminated. 
 This increase in the size of the colloid particles might be 
 seen in the ultramicroscope, or may be macroscopically 
 visible as an increased turbidity ; or again it might be 
 proved by precipitation of the combination by an electro- 
 lyte which was previously unable to effect this. An 
 example of the last is furnished by mixing dialysed 
 amylase and starch, which produces no precipitation ; if 
 to the mixture sodium chloride is added precipitation 
 
DEDUCTIONS 137 
 
 occurs, though sodium chloride was unable td precipitate 
 either component separately. 
 
 The unspecific component of the required enzyme may 
 be found by adding various salts, acids, or alkalies to the 
 solution containing the specific colloid. While it is not 
 necessary that the salt, acid, or alkali to be found should 
 be inseparable from the colloid, it certainly should be 
 difficult of removal, this indicating the formation of an 
 adsorption compound. The number of salts, acids, and 
 alkalies utilisable is not innumerable. Apart from the 
 oxidation processes, the electrolytes of living tissues are 
 practically summed up in four acids and five bases and 
 their combinations. These are : hydrochloric, sulphuric, 
 phosphoric, and carbonic acids, and the bases of sodium, 
 potassium, calcium, magnesium, and ammonium. Some 
 idea of the strengths in which these substances should be 
 used may be obtained firom the analysis of the ash of the 
 purest known enzymes. As all unite with water it is 
 probable that more than one would serve as the non- 
 specific component, though probably one would be more 
 efficient than the others. It might be objected that the 
 efficient hydrolytic agent to be sought for would be an 
 organic compound, but the facts learnt from the co-enzymes 
 leave little doubt that, as a rule, the common electrolytes 
 are the active agents. By proceeding on somewhat 
 similar lines Dony Renault and others have been suc- 
 cessful in making artificial oxidases ; artificial hydrolytic 
 enzymes ought also to be possible. 
 
 The composition and method of action of enzymes may 
 seem a limited subject to expend energy on, but its 
 importance is really considerable. The insight an accurate 
 knowledge of the subject (which is not yet attainable) 
 would give to a physician into the metabolism of special 
 
138 ACTION OF ENZYMES 
 
 tissues might lead to true views as to the cause of any 
 fault in this metabolism, and might enable him to rectify 
 it by comparatively simple methods such as the injection 
 of a simple electrolyte or non-electrolyte into the general 
 circulation or into the part affected. The cytologist 
 would find a knowledge of the enzymes of a cell and their 
 action of still greater interest, as the major part of cell 
 chemistry is based on them. He would have at his 
 disposal the means of influencing the functions of a par- 
 ticular cell by supplying it by, for example, Barber's 
 method of puncture, with a slight excess beyond normal 
 of a particular electrolyte, for the enzyme formed in 
 cbnnection with that particular electrolyte would be iii- 
 creased in quantity and its action in the cell increased 
 to an equivalent degree. It would be more difficult ta 
 influence the specific component of the enzyme, as this,, 
 being of a complex nature, is composed of large molecules 
 or aggregations of molecules. Nevertheless even this 
 component might be influenced if fractions of it were 
 supplied, e. g. in the case of the proteins, the amino- 
 acids or the products of their decomposition. These 
 on reception into the cell might be built up into new 
 colloid particles, which in their turn might form particular 
 enzymes. Finally, some of the most characteristic pro- 
 perties of living tissue might be conferred on synthetic 
 colloids, for the combination of these with the appropriate 
 non-specific component might mean the formation of 
 substances which would no longer be, so to say, the 
 passive subjects of reactions, but would be active agents 
 in attacking other substances. So one step more would 
 be made on the road to the great goal of the biologist, the- 
 synthesis of life itself. 
 
INDEX. 
 
 Acceptor, 86 
 Acetaldehyde, 66 
 Acids, strengths of, 69 
 Acids and alkalie5,.action on enzymes, 
 47, 119-123, 133 
 
 effect on colloids, 1 7 
 
 Acid salts, 51 
 
 Activators of enzymes, 4B, 52 
 
 Actor, 86 
 
 Adsorbed material, condition of, 27 
 
 Adsorption, 22 
 
 — causes of, 27 
 
 — and chemical attraction, 29 
 
 — as a cause of hydrolysis, 78, 124 
 
 — compounds, 22, 23 
 definiteness, 24 
 
 — distinguished from solution, 23 
 
 — effect of heat on, 26 
 
 — and electrolytes, 28 
 
 — and enzymes, 41 , 49, 50 
 
 — and fixation, 25, 129 
 
 — formula, 25 
 
 — rapidity of process of, 26 
 
 — reversibility of, 25 
 
 — and surface tension, 28 
 Aldehydase, loi 
 Amino-acids, hydrolysis of, 63 
 Amphoterous electrolytes, 18 
 Amylase, 9, 41, 46, 49, 50,51, 54. I27. 
 
 128, 136 
 Anaerobic fermentation, oxidation in, 
 
 lOI 
 
 Anti-emulsion, 54 
 Anti-enzymes, 54, 135 
 
 Antiseptics, effect on enzymes, 8, 44 
 
 Antitryptic serum, 55 
 
 Armstrong's view of synthetic action, 
 
 39 
 Asparagine and enzyme action, 54r 
 
 127 
 Atomic bonds, loosening of, 132-133 ' 
 Autolysis, 9 
 Autolytic enzymes, 47 
 Auxiliary ^valencies, 75 
 
 BasES, strengths of, 69 
 Bile-salts as co-enzymes, 53, 119 
 Brownian movement, 13 
 
 Calcium salts, action on enzymes, 49, 
 52,56 
 
 Cane-sugar, inversion of, 62 
 
 Carbohydrates, hydrolysis of, 62 
 
 Catalase, 50, 51, 94, 98 
 
 Catalysis, i 
 
 — of oxygen production from potas- 
 sium chlorate, 2 
 
 Catalytic action, resemblances ta 
 enzyme action, 42 
 
 differences from enzyme action, 
 
 44 
 
 Charcoal as adsorbent, 22 
 
 Chymosin, 64 
 
 Co-enzymes, 52, 118 
 
 Cold, no effect on enzymes, 46 
 
 Collodion membranes and enzymes, 
 
 S« 
 Colloidal complexes, 20 
 
140 
 
 INDEX 
 
 Colloids, 9 /" 
 
 — and chemical constitution, 20 
 
 — chemical reactions in, 19 
 
 — and crystalloids, 10 
 
 — effect of electrolytes on, 14, 16 
 
 — action of neutral salts on, 18 
 
 — osmotic pressure of, 13 
 
 — precipitation or coagulation of, 16 
 
 — and surface tension, 1 1 
 
 — viscosity of, 14 
 
 ■Colloid solutions, causes of perma- 
 nency of, 12 
 Combination of enzyme and substrate, 
 
 39 
 
 — solute and solvent, 72, 131 
 Combustion, intermediate compounds 
 
 in, 84 
 Condensation of carbon dioxide on 
 
 charcoal, 4 
 Consecutive reactions, 3 
 Continuous phase, 10 
 Copper and oxidation, 98 
 Coupled reactions, 86 
 
 Dialysis, 10, 49, 52 
 
 Diastase, 47 
 
 Diffraction of light by colloid particles, 
 
 II 
 Disperse phase, 10 
 Double salts in solution, 74 
 
 EcTO- and endo-enzymes, 8 
 Ecto-enzymes, preparation of, 8 
 Electric charge on colloid particles, 12 
 Electrolytes, «ffects of, on colloids, 14 
 
 16 
 on enzyme action, 44, 47, 123- 
 
 126, 133 
 
 — effect of passage of current through 
 solutions of, 68 
 
 Electrolytic dissociation, 69 
 Emich's experiment, 71, 124 
 Emulsion, 35, 37, 40, 41, 47 
 Emulsoid colloids, 14, i'5 
 Endo-enzymes, preparation of, 9 
 
 Enterokinase, 56 
 
 Enzyme action, effect of concentra- 
 tion, 43 
 
 — methods of investigation, 35 
 
 — the method of, 105 
 
 ■ — power of, compared with inorganic 
 catalytic action, 34, 1 23 
 
 — specificity of, 35 
 Enzymes as catalysts, 52 
 
 — classification of, by action, 61 
 
 — definition of, 8 
 
 r- effect of (heterogeneity of system, 
 
 42 
 
 — function of, on chemical reactions, 
 118 
 
 — general considerations on, 7 
 
 — irreversible changes in, 51 
 
 — action of, in low concentrations, 40 
 
 — manufacture of, 136 
 
 — properties of, 34, 133 
 
 — stability of, influenced by acids and 
 alkalies, 48 
 
 — two qualities of, 106 
 
 — universal occurrence in organism, 7 
 Equilibrium position under enzyme 
 
 action, 44 
 
 — in solution, 5 
 Erepsin, 38,51 
 Esterases, 62 
 
 Ethyl acetate, hydrolysis of, 5, 62, 120 
 External phase, 10 
 
 False equilibrium, 43 
 Fats, hydrolysis of, 62 
 Fibrin ferment, 64 • 
 Fermentation, 65 
 
 Gases, solubility of, in solutions, 72 
 Gelatinisation, 15, 16 
 Gels, IS, 16 
 Gibbs theorem, 27 
 Glucose, hydrolysis of, 65 
 Glucosides, action on isomeric, 35 
 Glyceric aldehyde, 65 
 Guaiacum blue reaction, 95 
 
INDEX 
 
 141 
 
 H.S.MOCYANIN, 98 
 
 Ha-'tioglobin, 94 
 
 HEemolysis and lipase, 54 
 
 Hardy's rule, 17 
 
 Heat coagulation, 46 
 
 He^t, influence on enzymes, 45, 134 
 
 Hofmeister series, 18 
 
 Hoppe Seyler's theory of oxidation, 
 
 86 
 Hydration oiE acids, 77 
 
 — of salts, 76 
 
 Hydrochloric acid as catalyst, 5 
 and hydrolysis of ethyl acetate, 
 
 62 ' 
 
 and pepsin, 56 
 
 Hydrocyanic acid and catalase, 50, 97 
 Hydrogen peroxide and catalase, 50 
 
 catalysis of decomposition of, 2 
 
 in oxidation, 87 
 
 oxidation by, compared with 
 
 biological oxidations, 88 
 Hydrolysis, 61 - 
 
 — through adsorption,_78, 124 
 
 — effect of concentration of hydro- 
 gen ions on, 79 
 
 — .of fats, 62 
 
 — general equation, 63 
 
 — of protein, 63 
 
 — Arrhenius' theory of, 70, 72 
 
 — by I water alone, 42 
 
 — Werner's theory of, 71 
 Hydrolytic enzymes, 1 18 
 
 — — specific elements of, 127 
 Hydroquinone, 35 
 
 Hydroxides of iron and manganese, 
 
 no 
 Hydroxynitriles, decomposition of, 64 
 
 Imbibition, 17 
 
 — effect of acids and alkalies on, 17 
 Indigo, oxidation of, 88, 91 
 Indophenol oxidase, 94 
 
 Inductor, 86 
 
 Intermediate compounds in oxidation 
 processes, 113 
 
 Intermediate compounds in catalysis, 
 
 3 
 
 Internal phase, 10 
 Inversion of cane-sugar, 62 
 Invertase, 34, 40, 47, 51, 52 
 Ions, 67 
 
 — combination of, with solvent, 74 
 
 — of gas, 86 
 
 Iron hydroxides, no 
 
 — and oxidation, 98, 108-111 
 
 — salts, effect on pepsin, 52 
 
 — and tyrosinase, 97 
 Irreversible changes in enzymes, 51 
 
 Laccase, 45, 92, 94, 95, 96, 97, 107, 
 
 108, 117 
 Lactase, 34, 37, 47, 123 
 Lactic acid, 66 
 LiSsegang's phenomenon, 20 
 Light absorption by solutions, 73 
 
 — effect on enzymes, 46 
 
 Lipase, 36, 41, 47, 48, 49, 52, 53, 54, 
 
 62, 119 
 Lyophile emulsions, 14 
 
 Maltase, 35, 37 
 
 Manganese dioxide as catalyst, 2, 3 
 
 — hydroxides, no 
 
 — in laccase, 97 
 
 — and oxidation, 97, 108-1 16 
 " Masked " salts, 27 
 
 Methyl chloride as intermediate pro- 
 duct, 4, 122 
 Methylene blue, reduction of, 99 
 Microscopic vision, limitation of, 1 1 
 Minute amount of catalyst, 2 
 Molecular compounds, 76, 131, 133 
 Molybdic acid and oxidation, 2, 113 
 
 Narcotics, 51 
 
 Neutral salt action on colloids, 18 
 Nitriles, hydrolysis of 65 
 Nuclease, 47 
 
 Optical isomers, action on, 36 
 
T42 
 
 INDEX 
 
 Optimum temperature, 46 
 Organised and unorganised ferments, 8 
 Ortho-esters, relation to hydration; 65 
 Osmotic pressure, 13, 132 
 
 and electrolytic dissociation, 74 
 
 of solutions of electrolytes, 68 
 
 Ostwald's generalisation of Gibbs' 
 
 theorem, 27 
 Oxidase action not truly enzymic, 93 
 Oxidases, 45, 94, 96, 108 
 
 — specific elements of, 117 
 Oxidation, 84 
 
 — in anaerobic fermentation, 101 
 
 — by enzymes, 107 
 
 -^ Hoppe Seylers' thepry of, 86 
 
 — and hydroxyl, 90 
 
 — of indigo, 88, 91 
 
 — and iron, 98, 1 08-1 1 1 
 
 — and manganese, 97, 108-116 
 
 — of the polyphenols, 92 
 
 — of saturated compounds, 84 
 
 — Traube's theory of, 87, 88 
 
 — of unsaturated compounds, 91 
 
 — Van't HotE's theory of, 86 
 Ozone, 85 
 
 Palladium as catalyst, 100, 1 16, 125 
 
 Papain, 55 
 
 Pepsin, 47, 52 
 
 Pepsinogen, 48, $6 
 
 Perchloride of mercury and enzymes, 
 
 SI 
 Perhydridase, 100 
 Perhydride of oxygen, 100, 101 
 Peroxidase, 47, 96, 98, 108, no 
 Peroxides in oxidation, 87, 89 
 Phases, homogeneous'and heterogene- 
 ous, 9 
 Phosphates and enzymes, 50, S3, S4 
 Platinum as catalyst, 1, 2, 99 
 Poisoning of enzymes, 51 
 Polymerisation of complex molecules, 
 
 30 
 Polyphenols, oxidation of, 93 
 
 Potassium chlorate, decomposition 
 of, 2 
 
 — permanganate, action on lipase, $z 
 Products, effects on enzymes, 40, 45 
 Proenzymes, 47 
 
 Proteins as colloids, 18 
 
 — hydrolysis of, 63 
 
 — lonisation of, 18 
 Ptyalin, 49, 54 
 Purin oxidases, 94 
 Pyrocatechol, 35 
 Pyruvic acid, 66 
 
 — aldehyde, 66 
 
 Radium rays and enzymes, 46 
 
 Reducases, 99, 116 
 
 Reducing bodies and oxidases, 97 
 
 Reduction, 84, 99 
 
 Rennet, 64 
 
 Resorcinol, 35 
 
 Reverse action, 38, 136 
 
 Reversible equations, 5 
 
 Retarding catalysts, i 
 
 Rontgen rays and enzymes, 46 
 
 Salts, action on enzymes, 49, 123- 
 
 126, 134 
 Silicic acid, 15 
 Sodium chloride and enzymes, 50, 54, 
 
 119, 136 
 Sols, 15 
 Specific adsorption, 30 
 
 — surface, li 
 
 Specificity among catalysts, 6 
 
 — of enzyme action, 35 
 
 — of non-enzymic actions, 35 
 Stalagmometer, 28 
 Substrate, 36 
 
 — effect of concentration of, 43 
 Sulphur dioxide, reaction catalysed. 
 Surface film, compression in, 27 
 solubility in, 27 
 
 — tension, 27 
 Suspensoid colloids, 14 
 Synthesis of maltose 38 
 
INDEX 
 
 143 
 
 Synthetic action, 38, 126 
 
 Temperature, effect on electrolytic 
 resistance of dilute solutions, 74 
 
 on enzymes, 45 
 
 on hydrolysis by water, 81 
 
 Toluene not injurious to enzymes, 44 
 Traube's theory of oxidation, 87, 88 
 
 Trypsin, 4S, SI, SS. 123 
 Trypsinogen, 49, 56 
 Tyndall- Faraday phenomenon, 10 
 Tyrosinase, 94, 96, 97 
 
 Ultrahicroscopk, 10 
 Ultraviolet rays and enzymes, 36 
 Urea, hydrolysis of, 63 
 Urease, 51. 
 
 Valency, definition of a, 133 
 — of ions, effect on colloids, 16 
 
 Valency, Werner's theory of, 75 
 Van't Hoff's theory of oxidation, 86 
 Velocity of action of enzymes and 
 catalysts, 43, 44 
 
 factors of, 43, 48 
 
 Viscosity of colloids, 14 
 
 Water and combustion, 85 
 
 — electrolytic dissociation of, 69 
 
 — as a hydrolyser, 70, 80 
 
 — molecular combination of, 76 
 
 — necessary for synthetic action, 38 
 
 — and oxidation, 85, 1 13 
 Werner's theory of valency, 75, 132 
 
 Yeast juice, 8, 53, 119 
 
 Zinc and oxidation, 98 
 Zymase,SO, 51,53, 54 
 Zymogens, 47, 49, 52, 55, 136 
 
 ADLARD AND SON AND WEST NEWMAN, LTD., IMPR., LONDON AND DORKING.