Principles of General Physiology

Light causes changes of permeability in the cell membrane and, in plants, has been shown to have the effect of retarding growth. Certain animals adapt the colour and pattern of their skin markings to suit the background against which they are seen. In the frog, the receptors for the reflex are situated in the toes and the eyes play no part. In the fish, the eyes are th'e receptors. The effects of radium and of X-rays on living tissues are similar to those of intense ultra-violet light. X-rays are generally regarded as being light waves of extremely short wave length.

IN the preceding chapter we have seen how, by the aid of light energy, the chemically stable system of CO2 + H2O is converted into one of higher potential energy, carbohydrate and oxygen. In reconversion to its original state, the energy of this system is utilised by living organisms for various purposes. But, although the system possesses considerable potential energy, it is, chemically, a stable one. This is, indeed, necessary, in order that its energy should not be given off spontaneously at all times, but only when required. Molecular oxygen is unable i to oxidise carbohydrate, except at an extremely slow rate ; although there are ( certain substances, such as simple aldehydes and those compounds called "unsaturated," which are "autoxidisable," that is, capable of oxidation by molecular oxygen. In this process, moreover, by a mechanism which will require discussion later, other substances, not themselves oxidisable by molecular oxygen, undergo simultaneous oxidation ; we have a " coupled reaction."

This mechanism alone, however, will not satisfy the requirements of the case. ( We have, accordingly, a catalytic mechanism in addition, which has the effect \ itself of " activating " oxygen. It may be remarked here that the processes of oxidation and reduction are not merely of use for the purposes of obtaining energy by complete combustion. Intermediate stages result in the formation of substances required for use in chemical reactions of importance for other purposes. The monograph by Dakin (1912) will serve to show the numerous cases of interest in this respect.

In the discussion of the question it must not be forgotten that the oxidation of one substance is always accompanied by the reduction of another. As Hardy points out (note appended to Drury's paper, 1914, p. 175), the place where oxidation takes place in a cell may also be a reduction place, if a different zero of oxidation potential be taken. A convenient one is that of atmospheric oxygen. A region of such a chemical potential would be a reduction place for compounds whose oxygen potential is higher than that of atmospheric oxygen, but an oxidation place for substances in which it is less than that of atmospheric oxygen. The absence of agreement as to the ZCFO may lead to confusion.

The first question to be investigated is the nature of the state into which oxygen is put, so as to be able to oxidise substances upon which it has no action in its ordinary molecular state. It is sometimes stated that it is in the " atomic " state, but this suggestion does not really help much, since we do not actually know what the difference between the atomic and molecular state is. Again, the active state is sometimes spoken of as the " nascent " condition. It is a matter of experience that chemical elements or groupings are more ready to enter into combination at the moment of their liberation from previous combination. It appears that, in the process, chemical energy is made use of before it . has become degraded into heat, hence more free energy is available.

The most probable view seems to be that it is in the process of changing its I valency, or electric charge, that oxygen is in the active state. At all events, I electrical phenomena are associated with the activation of oxygen, as shown by the following facts. Oxidation and reduction do not always mean the addition or removal of oxygen or hydrogen. The change from ferric to ferrous salts is a reduction, but consists in the conversion of trivalent iron to bivalent iron.

Haber (1898) has shown that the reducing action of hydrogen, developed on an electrode, depends on the electrical potential there. In this way, a reduction process can be carried to a particular point and no further. Thus, nitre-benzene can be reduced to azoxy -benzene and no further, if the cathode potential is low. Dony-Henault (1900) also showed that alcohol can be quantitatively oxidised as far as aldehyde, with a proper anode potential. The phenomena connected with the autoxidation of phosphorus and its effect in condensing a steam jet were referred to above (page 31). We may note that the effect consists in the production of " gas ions " and is not shown by the products of the reaction, but only by some process taking place in the actual course of the reaction itself.

Ostwald (1890, p. 76) suggested that reduction means a diminution of charge, that is, a loss of (negative) electrons. Oxygen, as we know, may be bi- or quadrivalent. In the peroxides it is probably the latter, and, when split off in a particular way, it has unusually powerful oxidising properties, a fact which is of great importance in physiological oxidations. Its activity appears to depend on its readiness to give up its extra charges. The theory of the process which takes place in the spontaneous oxidation of phosphorus, benzaldehyde, or other such substances, was suggested by Bach (1897), and by Engler and Wild (1897), independently, and was adopted by Ostwald (1900, 1). It has been observed as an experimental fact that, in such reactions, there are formed simultaneously two oxides in equivalent proportion, a lower oxide and a peroxide. Now, in the production of the former, energy is given out, whereas the latter has a higher oxidation potential than the oxygen gas, and requires energy to form it. This energy is derived from that afforded by the production of the simple oxide.

We saw, in speaking of "coupled reactions," that these reactions, in which energy afforded by one reaction is used to enable another to take place, must be capable of being expressed as parts of one and the same complete reaction ; we see, then, why there is always a quantitative, equivalent relation between the simple oxide and the higher oxide. Schonbein (see the monograph by Engler and Weissberg, 1904, p. 9) was the first to point out that half the oxygen is used to oxidise the substance itself, while the other half is "activated." The peroxide obtained in the oxidation of phosphorus is ozone and its formation should be described thus. In the process there is first formed a peroxide of phosphorus, which then splits up into ozone, on the one hand, and a lower oxide of phosphorus on the other hand. Thus : —

Whether this intermediate oxide is to be detected or not depends on the rate of its decomposition. The way in which the above reaction is described is that of Ostwald. It certainly is in agreement with experimental facts and explains why the two products of an autoxidation are always in equivalent proportion, since they were at one time combined in one substance. Ostwald also points out that it is a general rule that the most unstable product of a reaction makes its appearance first and is then decomposed.

It is interesting to call to mind also what Larmor has pointed out (1908). If we consider the great distance between the molecules of a gas as compared with their own dimensions, it is easy to see that an impact between molecules takes place only at a comparatively rare frequency. Suppose that the molecules are of two different kinds, capable of reacting together ; the impacts will, in a certain proportion of cases, be between different kinds of molecules, and some of them will give rise to combination. If there is also a third kind of molecule present, the compound molecule fonned by the first two will occasionally meet with this third kind, and combination of three kinds of molecules result. But the chances are almost infinity to one against three different kinds of molecules meeting simultaneously in such a way as to combine together. We are, no doubt, justified in extending these considerations to substances in solution. It appear^. therefore, to be extremely improbable that a reaction between three different molecules ever occurs in one stage, or, indeed, a reaction between two of one kind and one of another kind, or between three of one kind. All reactions should, if possible, be represented as taking place in stages between two molecules only at a time. Nernst (1913, pp. 475 and 595) calls attention to the same fact with reference to the improbability that reactions of a higher order than bimolecular should be anything but very rare. In any case, their velocity must be very small. Reactions which appear to be trimolecular are often found, on investigation, to take place in two bimolecular stages (see also van't HofTs Lectures, 1901, Heft 1, p. 196).

The peroxide, ozone, which is produced in such autoxidations as that of phosphorus, has a powerful oxidation potential, so that, for example, it liberates iodine from potassium iodide with great rapidity. Now, the peroxides which we find produced in living cells have an oxidation potential which is not so higli as this ; they consist either of hydrogen peroxide, or have a similar constitution. Their appearance in the photo-chemical reactions of the green leaf has been met with in Chapter XIX. In fact, in the presence of water, the organic peroxides of the latter type readily form hydrogen peroxide.

Peroxides of this type only liberate iodine from potassium iodide very slowly and their power of oxidising such substances as sugar is practically nil. We have, however, already seen an example of a typical catalytic process, with formation of an intermediate compound, in the increased action of hydrogen peroxide on hydriodic acid when minute amounts of molybdic acid are added (Erode). We note that the molybdic acid is found at the end unchanged, so that, to all appearances, it may have been merely an onlooker in the reaction. This is 1>\ no means the case, as we saw.

There are other substances, such as ferrous iron in the well-known Fenton's reaction (1894), which act as catalysts on hydrogen peroxide with the separation of what we may, for convenience, continue to call "active" oxygen. Moreover, from various animal and plant tissues, enzymes have Ixvn prepared which have the same effect. These have been called by Bach (1903) " peroxidases." The nature and properties of the numerous substances concerned with physiological oxidations and reductions have led to much work and caused much difficulty in the interpretation of the complex phenomena observed. A consistent and intelligible theory was first proposed by Bach and Chodat (1904), to whom we owe the greater part of the accurate investigation of the subject (see the article l>v Bach, 1913). In the following pages I describe the phenomena on the Imsis of this theory, although further research may make necessaiy some modification in it, and it cannot be regarded as altogether complete as yet.

The intervention of cell structures will come up for discussion in a later paragraph. There is an enzyme, to which we have already referred, called catafase, which has the property of decomposing hydrogen peroxide without activating the oxygen given off. The result of its action is molecular oxygen merely, given off as gas. Although catalase is of very common occurrence in plants and animals, the part it plays is, at present, somewhat uncertain. In any case, it does not directly concern us here, since it does not bring about oxidation, although, according to Bach (1913, p. 182), it plays an important part in protecting sensitive parts of the cell mechanism from the easily diffusible hydrogen peroxide, formed in the oxidative processes. Bach states, also, that in a mixture of hydrogen peroxide with both catalase and peroxidase, part of the peroxide is decomposed with production of active oxygen,

the other part into molecular oxygen, according to the relative amount of the two enzymes present. Catalase might thus act as a regulator of the oxidation process. Peroxides are characterised by the presence of two atoms of oxygen, directly I united together. If we take oxygen as quadrivalent, such a- substance as hydrogen ' peroxide contains two atoms of oxygen united by three valencies, and when one atom is split off in the active form, this atom possesses four free valencies to be I satisfied by combination with an oxidisable substance. Or we may put it thus, I in the formation of the peroxide, the valency of the oxygen is changed from two ^ to four. A substance gives none of the reactions of a peroxide, however many oxygen atoms it may contain, unless some are directly connected together. Persulphuric acid, HSO4 - O4SH, thus behaves as a peroxide, similarly other peracids and their salts. Since the atoms of molecular oxygen are united together, it seems that, when it combines with an autoxidisable substance, the primary product must be a peroxide, as is assumed in the equation given above (page 581). At the same time, ordinary oxygen has no peroxide properties, -although by the addition of another atom to make ozone, we obtain a powerful peroxide. In a peroxide, therefore, the two atoms directly united must, apparently, be themselves united to some other atom or group, which may be oxygen itself. The meaning of this is not clear. These groups rnay, indeed, be either " electronegative," as in persulphuric acid, or "electro-positive," as in sodium peroxide, NaO - ONa.

Most peroxides are hydrolysed by water in two stages, thus : — 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. Catalytic Activation of Peroxides. — Indigo blue is very slowly oxidised in air, presumably with the usual production of a peroxide. Oil of turpentine is oxidised in a similar manner at a considerable rate. In the presence of the latter, the oxygen of its peroxide is transferred to the indigo, which thus undergoes a rapid oxidation.

Now Bach holds (1913, p. 148) that the oil of turpentine should be called a catalyst in this reaction, since it does not appear as a constituent of the oxidised indigo, although it is not itself in its original form at the end of the reaction. I think, however, that it tends to confusion to speak of a catalytic process, where energy for a reaction is afforded by the agent called catalyst, as in this case, and that it is better to call it a coupled reaction. At the same time, it must be confessed that it is difficult to draw a very marked line of demarcation between this kind of reaction and that of the acceleration of the action of hydrogen peroxide on hydriodic acid by molybdic acid. The only essential difference is that in the latter, true catalytic action, the catalyst is recovered as in the beginning. This must be considered to be the real criterion, for even when the catalyst is not recovered intact, the change it has undergone is independent of the main reaction, merely incidental, whereas the oxidation of the oil of turpentine is an essential part of the reaction.

The cases where colloidal platinum and related metals act as catalysts are regarded by Bach (1913, p. 149) as precisely similar to that of indigo and oil of turpentine, since a peroxide like that of oil of turpentine is. supposed to be formed, but, in this case, becoming metal again. It seems to me, however, that these phenomena of heterogeneous catalysis are not, as yet, satisfactorily explained by the hypothetical assumption of various oxides of the metal, for whose actual existence there is not sufficient evidence. There is the fundamental difference, also, that the energy needed to raise the oxidation potential of the system is not afforded by chemical degradation of the platinum, as it is in the case of oil of turpentine and similar cases of autoxidation. The bearing of this question on the nature of Bach's " oxygenase " will be seen presently.

The process of autoxidation results, then, in the production of a peroxide and in the simultaneous oxidation of certain other substances present in the system, which are not, by themselves, accessible to molecular oxygen. In the presence of water we generally find that the peroxides form hydrogen peroxide, which has not a high oxidation potential. But it was known already to Schonbein (1860) that ferrous salts, in extremely small amount, strongly accelerate the action of hydrogen peroxide on oxidisable substances. Ferrous salts, in fact, as also those of copper and of manganese, accelerate the oxidising power of oil of turpentine, benzaldehyde, etc., no doubt by action on the peroxides produced. It is supposed by some that these metallic salts combine with the peroxides to form unstable "complexes," which split off the peroxide oxygen more readily than the original peroxides, like the permolybdic acids of Brode's experiments. In any case, the metal reappears in its original form and is thus a true catalyst.

When we come to apply the above phenomena to the process of oxidation in living cells, we find that the problem is by no means easy. The oxidation systems met with are often of great complexity and the enzymes unstable. The result has been that, although we are in possession of a large number of facts, their relation to a general theory is not a simple matter to make out. The reader will find an excellent account of the subject in the monograph by Kastle (1910) ; we must confine ourselves here to those facts which seem to give most guidance in the formation of a general theory. When we refer to certain preparations as coming from this or that plant or animal organ, it is not to be supposed that similar substances are not of general occurrence. It happens that, for various reasons, particular enzymes and so on are more readily isolated from their admixture with other substances in some cases than in others.

The gum-resin, guaiacum, happens to be a convenient test for the presence of active oxygen, since one of its constituents, guaiaconic acid, is oxidised to a blue substance by active oxygen, but not by ordinary oxygen, nor even by such peroxides as that of hydrogen. It is best used in the form of a solution of guaiaconic acid in dilute alcohol. Suppose that we take a scraping from the surface of a potato, some fresh blood fibrin, or various other products of living cells, and apply a drop of guaiaconic acid solution. A blue colour is produced, showing the presence of active oxygen. But this simple experiment does not lead us far in the analysis of the mechanism.

Now, Bach and Chodat (1903) showed that from the root of the horse-radish a solution could be prepared which did not give the blue reaction spoken of, neither did it give off oxygen gas when hydrogen peroxide was added. But if to this solution we add hydrogen peroxide, guaiacum is oxidised with the production of the blue colour. The solution must therefore contain something which activates hydrogen peroxide. This constituent is destroyed by heat, precipitated by alcohol, and shows the general properties of an enzyme, and was therefore called " peroxida.se."

We are next naturally led to look for evidence of the presence of hydrogen peroxide, or similar peroxide, together with peroxidase, in those cases in which guaiacum is blued without the necessity of adding hydrogen peroxide. An experiment by Bach (1914, p. 225) is of interest here. Fresh potato juice oxidises tyrosine rapidly. If acted on by alcohol, a precipitate is formed which has scarcely any action on tyrosine, unless hydrogen peroxide is added. Hence hydrogen peroxide can take the place of a similar substance naturally present.

Now, when we take a solution of peroxidase and add guaiacum and hydrogen peroxide, we naturally obtain the blue colour whether free oxygen is present or not, since the hydrogen peroxide supplies what is required. But, suppose we take potato scrapings, put them into a tube through which we lead hydrogen or coal gas, until the oxygen is displaced, and then, by means of a tap funnel, previously fitted, we drop guaiaconic acid on to the potato, we see that no oxidation takes place until air is allowed to enter the tube. If we consider this result for a moment, we shall see that its meaning must be this : Peroxidase is present as usual, but the absence of active oxygen, unless air is present, shows that no peroxide is available for the peroxidase to act upon. The peroxide is therefore

formed when air is admitted by taking up of oxygen in the process of autoxidation of a spontaneously oxidising substance. This system of autoxidisable substance, peroxide and peroxidase, is that which was at one time thought to be itself an enzyme and called an "r^drtftf"— The actual enzyme concerned is peroxidase, as was shown by Bach ancTChodat (1904) ; the other constituent, which forms a peroxide in presence of oxygen, is called by them, " oxygenaseJi- Oxidase is, in fact, separated into two constituents, each inactive until mixed.

It may be noticed that the termination "ase" implies enzyme nature, but we have seen reason for regarding the production of peroxides in autoxidation as not being of the nature of catalysis. It does not seem to me, moreover, that the easier destruction by heat of the oxygenase than the peroxidase proves its enzyme nature. In this respect, I am in agreement with Moore and Whitley (1909), but it is, after all, only a question of the meaning of the word "catalyst." Otherwise the scheme of Bach satisfies the facts excellently.

We may summarise the matter thus : — the substrate, which is to be subjected to oxidation, is usually one that, by itself, takes up oxygen by autoxidation so slowly as to be imperceptible. This is accelerated by the presence of a genuine autoxidisable substance (Bach's " oxjgenase "), which acts in a way similar to that of oil of turpentine and such readily oxidised substances. This action is produced by the formation of peroxides. These peroxides are rendered still more active by the enzyme, peroxidase. which accelerates the transfer of oxygen to the substrate by splitting it off from the peroxide. The value of the enzymic last part of the process may be this. We saw that the oxidation of an autoxidisable substance may effect the oxidation of other substances present during the reaction itself, so that it necessitates the presence of both free oxygen and an easily oxidised substance. The peroxide formed in this reaction, on the other hand, may very well persist for an appreciable time and be thus available for the bringing about of an oxidation when acted on by peroxidase coming into play as required.

We have seen above that salts of iron, manganese, or copper act on peroxides in the same way as the enzyme, peroxidase, and Bertrand thought that his " laccase " owed its activity to the presence of manganese. It was found later that iron__ could take the place of manganese, and Bach (1910) states that he "has prepared " oxidases " free frombolh these metals; whether any other metal, acting similarly, was present is not stated. The hypothesis that peroxidases are particularly active forms of one of these metals is, at present, the one most in agreement with experimental facts.

The metals found to be active as "peroxidases" are those capable of existing in two states of different valency. By the addition of one of these metallic salts to a system of peroxide and peroxidase, a mixed system can be produced, with increase of activity. Further, Dony-Henault (1908) has prepared what he calls an "artificial . laccase " in the following way. A solution is taken containing, in 50 c.c. of water, 1 g. manganese formate, 0'4 g. sodium bicarbonate, and 10 g. gum arabic. This is precipitated by alcohol. The precipitate is redissolved in water and again thrown down by alcohol. This substance is of interest for two reasons: — 1. As an obvious adsorption compound, but yet precipitated unchanged by alcohol, no doubt because the precipitation is practically total. 2. As an enzyme made artificially. It is precipitated by alcohol. But the more important property is that it acts catalytically. It does not seem to be destroyed by heat, but a similar substance made from albumin and manganese by Trillat (1904) is destroyed by boiling, while it has been stated that natural laccase is not so destroyed. The property clearly depends on the nature of the emulsoid colloid in association with the metal.