Principles of General Physiology

If this shifting of the equilibrium position is due to the supply of energy from some component of the system, it seems difficult to suppose that it can be from the enzyme itself : if so, the equilibrium should not be the same with different amounts of enzyme, whereas \\e have seen that, in experiments in which it was certain that equilibrium \v;is really attained, the concentration of enzyme played no part in the equilibrium. It may be, nevertheless, that the amount of energy required is so small that it was supplied by the smallest coin-eiitiati.ni used.

A subsidiary point is worth mention here. We have seen that lipase is increased in its act i\ it y by bile-salts, and the question arises, does this increased activity affect both tincomponents of the reversibler eaction? It has been shown by Hamsik (1910) that it does ; the position of equilibrium is unaffected. The fact serves to confirm the view taken of the mode of action of the co-enzyme in this case, namely, that it increases the active surface of the en/vine.

Bourquelot et Bridel (1914) made the interesting observation that, if maltase and eimilsin together act on glucose and alcohol, so that a mixture of the a- and /3-ghtooadee is formed, the same composition of the sj'stem in equilibrium is attained, whatever the relative amount or the order in which the enzymes are added. There must, therefore, be a conversion of the one glucoside into the other, presumably after previous hydrolysis. The manner in which catalysts act appears to be of more than one kiml. s.> that no satisfactory general statement can he made.

Formation of Intermediate Compounds.— In one case, that of the acceleration of the reaction between hydriodic acid and hydrogen peroxide by molylidic arid, this stage of intermediate combination has been satisfactorily shown by Brode (1901) to be passed through. A series of permolybdic acids is formed by tinaction of hydrogen peroxide on the catalyst ; these are formed with great rapidity and, when formed, they react also with great rapidity on hydriodic acid, with separation of iodine and return of the catalyst to its original form of molybdic acid. Both these reactions together occur at a greater ratethan the original uncatalysed reaction between hydriodic acid and hydrogen peroxide, so that the criterion of Ostwald (1899, p. 517) as to the conditions to be satisfied for such an explanation to be admissible are'present.

It must l>e admitted, however, that this case of catalysis with the formation of an intermediate comjMmnd of a chemical nature appears to be an exceptional one. In some cases, indeed, as in thfe catalysis of methyl acetate by hydrochloric acid, it is found that the reaction by way of methyl chloride takes loiujrr than the spontaneous actual one, so that this obvious intermediate compound is excluded. Adsorption. — It was suggested above that the increased concentration produced by adsorption might perhaps be sufficient to account for the greater rate of reaction. But it scarcely seems possible to explain the existence of such

a variety of enzymes on this hypothesis alone, although one must not be too hasty in making such a statement until more is known as to the nature of adsorption in its manifold aspects. With respect to the numerous theories of catalysis that have been suggested, the reader may consult the work of Mellor (1904, chapter x.). Before proceeding to a discussion of what we know as to the mode of action of enzymes, a brief description of their physical and chemical properties, as far as they are known, is requisite.

Physical Properties of Enzymes. — They are all in the colloidal state in solution. They do not diffuse through thick parchment paper, but, as samples of this paper vary in the dimensions of their pores, it may be found that an enzyme in a highly dispersed condition may diffuse slowly through some papers. This was the case with the amylase of Fraenkel and Hamburg (1906). As colloids, they have an electrical charge, varying with the electrolytes present with them. This charge appears to play some part in the mechanism of their action, as will be seen presently.

There is indirect evidence that many, if not all, are optically active. The Chemical Nature of Enzymes. — It is obvious that great practical difficulty exists in the investigation of this subject, owing to the minute amounts of these intensely active substances which we have at our disposal. It was thought at one time that they had the composition of proteins, but, as preparations were made of greater purity, it was found that the protein reactions disappeared more and more, although the preparation gained in activity. Moreover, according to Beijerinck, they are incapable of serving as nitrogen food for bacteria or yeast. It is probable that, like inorganic catalysts, they are of very varied chemical nature, but what this is cannot as yet be stated definitely in respect of any one of them.

It is possible that they are not single chemical individuals, but complex systems, as was suggested by Bertrand some years ago. In an address to the French Association for the Advancement of Science in 1909, the theory is stated as follows : — One of the constituents of the system is capable, on its own account, of producing the reaction in question to a slight degree, but requires the presence of another substance, inactive in itself, before its activity becomes appreciable. The former is, according to the case, some such substance as acid, alkali, calcium or manganese salt, etc. The latter is a more complex substance, often similar to egg-white, colloidal in character. This view is similar to that stated by von Wittich (1872, p. 469) as regards pepsin, which is held merely to intensify the action of hydrochloric acid. It is not quite clear, however, whether von Wittich intended to make the general statement that all enzyme actions are of this nature, although it seems implied. This view receives support also from the facts connected with the "artificial laccase " prepared by Dony-Henault (1908, p. 151), in which the active agent is colloidal manganese hydroxide, but protected from aggregation by the presence of a " stable " colloid, gum arabic.

At this point I feel bound to make a slight protest against Bunge's gibe at physiologists (1907, p. 241), in which he says that "the less a physiologist knows about chemistry, the greater is he inclined to work at the most difficult chemical subjects — the proteins and ferments." If the chemistry to which reference is made here is pure statical, structural, organic chemistry, as would appear, it is a remarkable fact that such a mode of attack has taught us practically nothing about the nature of enzymes, and has only led to the multiplication of names, on which Bunge himself justifiably throws contempt as "a drag and a brake to science." It is only since the question has been attacked from the kinetical standpoint of physical and colloidal chemistry that we are beginning to see light. It is, of course, far from my intention to undervalue the work of organic chemistry as one of the helps to the comprehension of our difficult problems, as must be apparent from the previous pages of the present book, and would be of self-evident absurdity ; but, in view of opinions sometimes expressed, it is necessary to point out that there are other bodies of doctrine of equal importance in the study of physiology.

Enzymes Act at their Surfaces. — The clearest direct proof of this fact is that emulsin, lipase, urease, and trypsin exert their activity in alcoholic media of such a strength that the enzyme is completely insoluble, and can be filtered off. In such cases, where the enzyme is not uniformly distributed, rate of diffusion must play a part in the first stage of the particular heterogeneous reaction, as, in fact, is found by experiment, since shaking such systems accelerates the rate of change. When the enzyme is in colloidal solution, although it forms a separate phase, it is comparatively uniformly distributed, so that the diffusion distances are very small and we can, with caution, apply the formulae of velocity of reactions developed for homogeneous systems. Adsorption of substrate on the surface of the enzyme phase is the next stage, as we saw in describing heterogeneous reactions in general. This probably takes place with great rapidity as soon as the components are sufficiently near together. Chemical reaction follows ; but, under conditions in which it takes place slowly, cold for example, it is possible to separate the actual adsorption compound of enzyme and substrate. The " compound " of starch and amylase has been prepared by Starkenstein (1910) and by Philoohe (1908, p. 393), that of fibrin and pepsin by von Wittich (1872, p. 444), those of trypsin with starch, caseinogen, and charcoal, and of amylase with caseinogen by myself (1911, 1). It will be noted that it is not necessary that the substrate should be one on which the enzyme acts in order that adsorption may take place.

A further point of interest is that electrolytes behave in this process in the same way as that in which they behave in what we have called above "electrical adsorption" (page 58), as shown by myself in the case of trypsin (1911, 1). If the enzyme and the substrate are both negatively charged, a certain obstacle to adsorption exists, since, if it took place, it would increase the electrical energy of the surface. If a bivalent ion, say Ca' ', is present, the charge on the surface is reversed and adsorption facilitated. In this way the favourable action of electrolytes in many cases can be explained.

Whether the formation of an intermediate compound of a chemical nature between the enzyme and adsorbed substrate takes place as the next stage is, as yet, uncertain. It has not been shown to occur, so that the precise nature of what happens after adsorption still remains in the dark. The work of Wohler, Pliiddemann, and Wohler (1908) is of some importance in this connection. Their investigations concern the catalytic action of various oxides and of platinum on the oxidation of SO0 in the manufacture of sulphuric acid. They show that any sulphites or oxides of the ordinary kind are inadmissible as intermediate chemical compounds between catalyst and substrate. If such a compound is to be assumed, it must be an endothermic one, such as a peroxide. They regard their experiments as more favourable to the theory of acceleration by increased concentration due to adsorption, but do not consider them as definitely deciding the question.

The possibility of increased chemical potential brought about by molecular forces in the act of concentration on the surface, as pointed out by Hardy, must not be forgotten. A difficulty should be mentioned here. We suppose that the natural state of equilibrium is brought about rapidly by increased concentration on the surfaces of enzymes. But, as Prof. Hopkins reminds me, supposing that the different 'components taking part in the reaction are not equally adsorbed, the position of equilibrium would not be the same on the enzyme as in the body of the solution. It will be clear, however, that the conditions controlling adsorption are so complex that no statements can be made with regard to the case of enzymes until actual experiments have been made with pure preparations.

The following illustration may assist the reader in understanding the facts of heterogeneous reactions. I must apologise for its apparently trivial nature. Imagine a number of snails in the neighbourhood of a strawberry. As soon as a snail, in the course of its wanderings, becomes sensible of the presence of the food, it proceeds towards it. This is the preliminary diffusion, and would perhaps be more like the real kinetic process if we suppose that the snail was insensible of the existence of the strawberry until it accidentally came into contact with it. The next stage, that of adsorption, follows rapidly as the animal attaches itself to the fruit. If nothing more happens, there is no chemical reaction. The final, chemical stage is the devouring of the food and its subsequent hj'drolysis. It is obvious that the rate of this final stage is proportional to the number of snails "adsorbed." It will also be noted that it is not in linear ratio to the number at work. The more there are, the more they interfere with one another, and, when the strawberry is completely covered, the advent of more snails will not further increase the rate of disappearance, since the newcomers cannot get at the fruit. The strawberry here corresponds to the enzyme ; we may imagine that, instead of the fruit, we

have a powerful chemical substance which induces the disintegration of the snails, representing the substrate, which are adsorbed on its surface. The exponential ratio of the concentration of the enzyme to its activity receives a satisfactory explanation on this adsorption theory, as will be plain from the above illustration. On the other hand, it seems that we must either attribute some special properties to the enzyme surface itself, which may be of the nature of configuration, chemical or physical, or else we must suppose the formation of an intermediate chemical compound between enzyme and substrate, to be afterwards broken up into enzyme and products. The case of the relation between the a- and /3-glucosides to maltase and emulsin will serve to show what is meant here. The a-glucosides are scarcely acted on at all by emulsin, perhaps not at all, but rapidly by maltase, and vice versa with regard to the /3-glucosides. Now it does not seem possible that any ordinary surface could distinguish to such a degree between the properties of two substances so nearly alike as the a- and /3-glucosides of methyl are. At the same time, apart from their optical isomerism, they have certain other differences, solubility for example. As was remarked before, until we know more as to the possibilities of adsorption, it would be rash to be dogmatic on the question.

As to the configuration of the surface, it is quite conceivable that a particular pattern, so to speak, may allow closer approximation of reacting molecules than another pattern does. As a very rough illustration, a surface beset with projecting spikes would not allow so close an approximation of a flat surface as would another flat surface. We must be careful, however, not to be misled by too statical a conception of the phenomena. Moreover, there may be true chemical combination with the actual chemical substance of the surface of a colloidal aggregate, without the phenomena losing their characteristic adsorption nature. See also Bayer and W. W. Starling (1915).

When enzymes arise in the course of the growth of cells, it is plain that they must pass through preliminary stages and it seems that what are called "zymogens" constitute a stage of this kind. Sometimes we find the enzymes secreted to the exterior in the inactive form ; the trypsinogen of the pancreatic juice is such a case ; it requires the action of another enzyme, enterokinase, to convert into active trypsin. Details of the phenomenon may be found in my monograph (1913, 2, p. 132) and to some extent in the following chapter of this book.

We must note the difference between a zymogen and an enzyme which is inactive on account of the want of its co-enzyme. The conversion of a zymogen into an enzyme cannot be reversed by any process at present known to us, whereas the co-enzyme can be added or removed at will. There appears to be some evidence that enzymes may make their appearance in response to the presence of an appropriate stimulus, or rather substrate. Thus Duclaux (1899) stated that Penicillium glaucum, grown on different media, produced enzymes which hydrolysed these media, enzymes which were absent in other cases.

A significant point with regard to the nature of enzymes is to be found in their occurrence in situations where they have never had the opportunity, in the course of evolution, of meeting with their special substrates, lactase in the almond, for example. If this lactase is a selective enzyme acting only on lactose it must have been produced, accidentally, as it were, as a by-product of metabolism. Otherwise it must be regarded merely as an incidental property of emulsin.

The appearance of enzymes in the blood in response to injection of proteins or carbohydrates, Abderhalden's "protective enzymes," requires further investigation. They are not specific, that is, a particular sugar may set free the enzyme which hydrolyses it or another in addit on. They are probably set free from some situation in the organism. The production of an enzyme, not found somewhere in the organism, has not been shown to occur. We are quite justified in speaking of the relation between the u- and (3 glucosides and maltose and emulsin as a "specific" one, although the difference may be merely quantitative, as we shall see presently. There are, however, many degrees of specificity ; emulsin acts on a great variety of glucosides, trypsin on all proteins, while invertase is said to have no action on any substance but ( amsugar. This last fact places a difficulty in the way of accepting Bertrand's hypothesis, at least in its simplest form. Since, if invertase merely activates acid, it should be capable of hydrolysing maltose and lactose as well as saccharose.

But it seems to me that the practice of some investigators in assuming a separate enzyme for every substrate acted upon is not warranted by the facts. When we say that there is a salicinase in what is usually called emulsin, if we mean anything more than that emulsin hydrolyses salicin, we are going beyond what is justified by the experimental evidence. It is true that, under some conditions, extracts containing emulsin may act more powerfully on salicin than upon some other glucoside, while other extracts may act better on the latter ; but it has not been shown that this is due to anything other than different conditions. It is to be remembered that even acid will hydrolyse some glucosides much more readily than others. Until a separate enzyme is prepared which acts on no other substrate but salicin, under any conditions, the name salicinase should not be used. At the present time it would be more profitable to devote attention to the various ways in which the rate of action of an enzyme on various substrates can be modified by change of conditions.

The multiplication of names may even be mischievous in leading to the belief that new knowledge has been obtained when a phenomenon is described by a name derived from a classical tongue instead of in English. There is risk, for example, that when we say that the injection of a foreign protein causes the production of a "precipitin" for the protein, we may imagine that this "precipitin" has been shown to be a definite chemical individual, instead of a mere description of the fact that a precipitate is formed. The "side-chain theory" of Ehrlich, great as has l>een its use in suggesting problems for investigation, is, at present, overburdened with multitudes of names, which consist, for the most part, merely of descriptions of the phenomena, although they suggest actual substances. There are many signs that one or two simple explanations, on the basis of colloidal chemistry, will be found to put an end to most of these names.

One is tempted, indeed, to make a well-known quotation from Moliere (1673). The reader will remember that in the ballet of " Le Malade Imaginaire," which ballet is a satire on medical examinations, one of the medical students sings (tome v. p. 308 of Hachette's edition) : — " Mihi a docto doctore Domandatur causam et rationem quare Opium facit dormire. A quoi respondeo, Quia est in eo Virtus dormitiva, Cujus est natura Sensus assoupiie." Which I may venture to translate thus : —

"The learned doctor asks me The cause and reason why Opium sends to sleep. To him I make reply, Because there is in it A virtue dormitive, The nature of which is The senses to allay." For this profound answer the candidate receives his diploma with acclamation, together with his licence to "kill and to cure." Incidentally, I would call attention to the fact that this play was the last written by its author, although it is regarded by many as his best work. This fact may be commended to

the attention of those who wish to prevent men at any particular age from taking part in the work c f the world. Galileo was over seventy years of age when he wrote one of his best works, and thought out, amongst other things, the application of the pendulum to the regulation of clocks. In one of Leeuwenhoek's letters we find the words: "A certain gentleman, who was with me some months ago, intreated me to go on in making observations, adding that the fruit which ripen'd in autumn was the most lasting. This is now the autumn of my life, I being arrived at the age of 88J years " (H. G. Plimmer, 1913, p. 135). Many other instances might be given, from the sphere of "action" as well as that of scientific discovery.

To return to our theme. As already remarked there are various facts which are calculated to give us pause before accepting, as an article of faith, the doctrine of the perfect specificity of enzymes. They will be found in my monograph (1913, 2, pp. 150-156) by those interested. There are one or two points of general interest which may be mentioned here. Dakiii (1904) found that, when lipase was used for hydrolysis of the optically inactive mixture of the two mandelic ethyl esters, one of the isomers was hydrolysed more rapidly than the other, although finally both were completely decomposed. This was brought into relation with the probable optical activity of the enzymes, so that the " compounds " of this with the two forms of the ester would not be symmetrical, and would therefore decompose at an unequal rate. Similar facts are described by Fajans (1910) with regard to the decomposition of the two camphor-carboxylic acids by optically active bases, acting as catalysts, and, as regards synthesis, by Rosen thaler (1909) in the case of emulsin forming benzaldehyde-cyanhydrol. We have seen that there are many cases known where living organisms consume preferably the one isomer, but when this has disappeared, the opposite one is also attacked. Dox and Neidig (1912) show how extracts of Aspergillus hydrolyse both a- and /3-methyl glucosides, but at an unequal rate. There are other cases known where extracts of tissues, which were originally supposed to contain only one kind of enzyme, say maltase, have been found to act, slowly, on the opposite isomer. It seems to be simpler to regard these as due to a slow action of the same enzyme on both isomers than as due to the presence in traces of another enzyme, especially when this other enzyme is one which, under natural conditions, would never have had any opportunity of action. Fajans (1910) has shown in detail how much more satisfactorily the various experimental data can be explained on this hypothesis.

The results of Erlenmeyer, mentioned above (page 285), show the possibility of an optically active enzyme acting more rapidly on the appropriate component of a racemic mixture if an indifferent optically active substance is present. Such a process of conversion appears to be independent of chemical combination in the usual sense. There are a large number of reactions which proceed by themselves very slowly, or sometimes, apparently, not at all, but which can be enormously accelerated by the presence of small amounts of various foreign substances.

The characteristic property of such accelerating agents, known as " catalysts," is that they do not form part of the system in its final equilibrium, and either appear at the end in their original form or, in some cases, are partially destroyed or removed from the sphere of action in the form of constituents of some subsidiary reaction. When the system is one that reaches a definite equilibrium under the conditions of the experiment, the position of this equilibrium is unaffected by the presence or the amount of the catalyst, which merely hastens the time taken for the process, and this in proportion to its concentration.

Two important things are shown by this fact, namely, that the catalyst does not supply or remove energy from the system, and that it accelerates both the hydrolytic and synthetic components of a reversible reaction. In living organisms there are a large number of substances which behave like catalysts, and are known as " enzymes." They are extremely active, and explain the occurrence in the organism of reactions which require, in the laboratory, powerful reagents anil high temperatures. Lactose is hydrolysed by both hydrochloric acid and by an enzyme, lactase ; but weight for weight, the latter is, at least, five thousand times as powerful as the acid.