Principles of General Physiology

The natural " oxidase-^-systems are more or less " specific " : each appears to act on a special group of substrates, or even on one only. There is reason to suppose that this depends on the particular organic peroxide constituent of the complex system, rather than 011 the peroxidase. This " specificity " is not unknown in inorganic catalysts, thus Wolff (1908) found that colloidal ferrous ferrocyanide acts as a peroxidase towards phenols, but does not accelerate the action of hydrogen

peroxide on hydriodic acid. The direct action of the substrate on the enzyme, chemically destructive or by alteration of its physical properties, has always to !><• taken into account in these specific relations. It may be asked, what is the function of the gum in our artificial laccase? Before we can answer this question, we must examine into the state of the metallic salt in solution, whether it be iron, copper, or manganese. Salts of all these metals, especially in -the dilute solutions in question, are hydrolysed in water. In other words, the solutions used consist of hydroxides ' >t the metals in the colloidal state. This seems to be their active state, although it is not easy to say why the colloidal hydroxide should be more active than the ion. The experiments of Moore and Webster (1913) on the formation of formaldehyde by ultra-violet light, in the presence of colloidal ferric hydroxide, may be called to mind. Bertrand also (1897) tested the oxidising effect of a series of manganese salts on hydroquinone and found those to be the most powerful which were the most hydrolysed in solution.

If, then, the colloidal state is of so much importance, it seems clear that the activity must be in direct relationship to the extent of the surface. Hence the use of gum, albumin, and so on. The way these "stable" colloids act in protecting a suspensoid colloid, such as ferric hydroxide, from precipitation by electrolytes, thus ensuring a high degree of dispersion, has been explained above (page 97). A peroxidase is, then, 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, such as gum or albumin. It is to this stable colloid that the enzyme owes its precipitation by heat or by alcohol, and, possibly, any degree of specificity that it possesses. A view essentially the same as this was suggested by Pen-in (1905, p. 103).

Although it has long been known that fresh animal and plant tissues have the power of reducing nitrates to nitrites, and it was held by some that the process is a catalytic one, the existence of reducing enzymes, analogous to the oxidising ones, has not been generally accepted. Schardinger (1902), however, made the observation that fresh milk rapidly reduces methylene blue, indigo, and so on, if an aldehyde, such as formic or acetic aldehyde, be present ; whereas it has no such action in the absence of the aldehyde. The property is abolished by boiling, and has been used as a test to distinguish fresh from sterilised milk. It was clearly proved by Trommsdorff (1909) that this reaction is due to an enzyme and not to bacteria. If microbes are present, the milk, after some hours, acquires the property of reducing methylene blue without the addition of an aldehyde.

Now it is clear that we must have a formation of nascent or active hydrogen, and that it must come from the water in the system. If a reaction is going on which takes up oxygen from water, hydrogen will Ije set free. It is probable, then, that we have to do with a reaction of the kind called by Bach (1913, p. 150) " hydrolytic oxidative-reducing reactions." A reaction of this kind has been already described (page 266) in the oxidation of a-amino-acids to aldehydes, as discovered by Strecker. To understand the mechanism, however, a simpler system is better and we may take that of the decomposition of water by hypophosphites in the presence of metallic palladium, as invested by Bach (1909).

To begin with, it must be admitted that the mechanism of such reactions is by no means clear as yet and I confess to a certain amount of misgiving as to the chemical nature of the intermediate compounds supposed to be formed. The system is a heterogeneous one, palladium and similar metals being insoluble, and the phenomena of adsorption or surface condensation, always present in such systems, should be kept in mind. In the following description when oxides of platinum, etc., are spoken of, it is very doubtful whether they really have t In- definite chemical formula? assigned to them, since they have not been isolat«d. Similarly, it may be remembered that the permolybdic acids formed in Brode's typical case (page 324) are said to be a series of this kind : —

The formulae rather suggest adsorption compounds. We may also call to mind the controversy between Faraday and de la Rive (see page 306 above). To return to the hypophosphite system, hypophosphites do not undergo oxidation in water alone at any measurable rate. But in the presence of finely divided palladium, this takes place. Bach puts it thus : the water is decomposed and its HO is used for oxidation of the hypophosphite, the hydrogen is taken up temporarily by palladium, and then set free. Thus : — Hx /H OHH Hx /OH

Palladium acts as a true catalyst ; minute amounts decompose indefinite amounts of hypophosphite. If an easily reducible substance is present, the nascent hydrogen reduces it. If we take an aldehyde in place of hypophosphite, we find that the presence of metals of the platinum group does not accelerate to any great degree the decomposition of water. A further addition is required in the form of an easily reducible substance as " acceptor " for the nascent hydrogen as it is formed ; such substances are methylene blue, indigo, nitrates, and so on. None of these are reduced at any perceptible rate by formaldehyde alone without platinum. In the reaction, the aldehyde is oxidised to the corresponding carboxylic acid. The case of methylene blue is instructive because this dye contains no oxygen, so that the additional atom of oxygen required to convert formaldehyde into formic acid must come from the water by some means.

The explanation of this fact suggested by Bach (1911, 1) is, shortly, as follows. Water may be looked upon as an unsaturated compound, H2O = , since oxygen is quadrivalent, at all events potentially. Since H' and OH' ions are also present in water, as we have seen, it seems probable that " unstable complexes " may be formed thus : — The first may be called " hydrogen suboxide " or " oxygen perhydride," analogous to the metallic salts M4O, such as Ag4O. The second is the hydrate of hydrogen peroxide. We have seen reason in Chapters VII. and VIII. to hold that ions are associated with water molecules.

The acceleration by platinum of the oxidation of aldehydes to form acids can be explained on the view of Engler and Wohler (1901) that colloidal platinum combines with molecular oxygen to form a peroxide, Pt02, which, in its turn, reacts with water to form the hydrate — This substance may also be supposed to be formed by reaction of platinum with the H20(OH')2 present in the water. It acts as a powerful oxidising agent on formaldehyde. Since H20(OH').2 is used up, the equilibrium is disturbed, more is formed, and so the catalytic process continues.

If we admit the presence in water of H40, it is not unlikely that platinum metals should form strongly reducing hydrides by combination with this. The two complexes of H' and OH' ions with water are only present in very small concentration, so that unless one of them is used up, say the hydride in reducing methylene blue, the oxidation of the aldehyde can only take place to a very small extent. I have already remarked that this view assumes the existence of chemical compounds of rather doubtful nature, and Bach himself states (1913, p. 154) that the question requires further investigation to make the mechanism clear. The view given certainly explains the occurrence of active hydrogen, which is otherwise difficult to account for.

We naturally turn next to look for the evidence of an enzyme, in milk and tissue cells, which plays a part similar to that of the platinum metals. Since a peroxidase causes the activation of peroxide1 oxygen, we may call, with Bach (1913, p. 161), an enzyme which causes the activation of perhydride hydrogen a " perhydridase." The enzyme shown by Schardinger and Trommsdorff to be responsible for the reduction by fresh milk of methylene blue, if an aldehyde be also present, has been referred to above. Now, reducing action of fresh tissues has been described by

various observers and ascribed to "reductases" or " reducases." In analogy with the oxidase system, as Schardinger's reaction appears then to be a hydrolytic-oxidative-reduction process, in which the aldehyde is oxidised by the oxygen of water, while the hydrogen thus set free reduces the methylene blue to its leuco-base, by the intermediation of the enzyme. Liver tissue, ground up with water, has been known for a long time to be capable of reducing methylene blue at a rapid rate. Bach (1911-1912) investigated this reaction from the above point of view. Is there an enzyme present like that in milk and an oxidisable substance acting like aldehyde ?

If liver is rubbed up with five times its weight of 2 per cent, sodium fluoride and filtered through linen, the emulsion, diluted five times, has very little action by itself on methylene blue. But, if a small quantity of acetic aldehyde be added, the reduction is rapid. If previously boiled, the emulsion has lost this property. Filtration through paper deprives it of the enzyme, which remains on the paper. But a solution can be made by extracting the liver with 1 per cent, sodium bicarbonate. This is filtered through linen, the filtrate neutralised exactly with acetic acid and again filtered through paper. Alcohol produces a precipitate in the filtrate, and, from this precipitate, the enzyme can be extracted by half per cent, sodium bicarbonate. The solution reduces methylene blue in the presence of aldehyde. The enzyme is very unstable. A similar preparation can be made from lung, spleen, kidney, or thymus.

This enzyme also reduces nitrates to nitrites in the presence of an aldehyde, just as fresh milk does, so that it is not of a specific nature. The nature of the substance which takes the place of aldehyde in the cell system is not yet known. It appears to be insoluble, since it is left with the cell debris on the linen in Bach's process, as given above. An oxidase (or phenolase) produces its oxidation by the aid of free oxygen, while the reducase acts by means of the combined oxygen in water, indirectly. Thus:—

oxidase = peroxidase + oxidisable substance (oxygenase) + oxygen reducase = Schardinger's enzyme + oxidisable substance (oxygenase) + water. In the first case, we may say that free oxygen is reduced, but the reduction process stops here and is covered by the oxidation process ; in the second case, hydrogen is set free by the decomposition of the oxidising agent, water, and further reduction processes are set in progress. Cannizzaro's Reaction. — This reaction consists in the simultaneous oxidation and reduction of aldehydes, by which the hydrogen of water converts one molecule to the alcohol and the oxygen converts the other one to the acid : —

Parnas (1910, 2) showed that liver tissue greatly accelerates this reaction, a result which is obviously an aspect of the action of Bach's perhydridase. The enzyme in extracts of organs which oxidise salicylic aldehyde appears to be similar and the reaction to be a case of acceleration o'f Cannizzaro's reaction. Plant Reducases. — There is a perhydridase in potato juice and other plant extracts (Bach, 1913, 2). This requires for its action the presence of one of the lower aldehydes and then reduces nitrates to nitrites, but apparently methylene blue is not attacked.

The increased consumption of oxygen by acetone yeast in the presence of methylene blue, as described by Meyerhof (1912, 3), seems to be a related phenomenon. The experiments of Palladin, Hubbenet, and Korsakov (1911) on the higher plants also bear upon the question. Methylene blue causes greater oxidation in etiolated plants and the presence of oxygen is necessary for the effect in the bean plant, although not in the pea ; a fact which appears to be connected

with the anaerobic production of alcohol in the latter. In the presence of oxygen, the dye does not suffer reduction. Haemoglobin has the power of oxidising guaiaconic acid. This has been shown by Buckmaster (1907) to be due to the iron contained in it; the iron-free derivatives do not give the reaction. We have seen reason for regarding iron as a peroxidase, but since a peroxidase requires a peroxide to act upon, it appears that, in the blood reaction, there must be some substance present which is autoxidisable.

It is interesting to note that lecithin is oxidised in air by ferrous-ammonium sulphate (Thunberg, 1911). It would thus form a peroxide, like benzaldehyde does. Certain other cell constituents, nuclein, albumin, glucose, oleic acid, are not oxidisable by iron alone. The production of pigments by the action of an oxidising enzyme on tyrosine has been referred to above (page 359). This enzyme appears to be of frequent occurrence, not only in fungi, where it was first found, but also in animal tissues, amongst others in insect larvae. According to Bach (1914), the system called tyrosinase is a complex one. The effect of one of its constituents is to reduce the tyrosine. The products are then easily oxidised by the oxidase also present. The behaviour is similar to that of the respiratory pigments of plants, described by Palladin (1909), which are alternately oxidised and reduced by the agency of enzymes.

So far as we have arrived, the chemical process of oxidation in the cell seems to be as follows. Some autoxidisable substance in the cell takes up molecular oxygen, with the formation of peroxides and activation of half of the oxygen. The other half of the oxygen serves for complete oxidation of part of the autoxidisable substance. These peroxides are acted upon by peroxidase, with further increase of active oxygen, which is able to bring about oxidation of substances not autoxidisable and otherwise difficult of oxidation. But when we come to apply the facts learnt by study of extracts or of disintegrated cells to the interpretation of phenomena taking place in the living cell, we find that there is something else to be taken account of. This we may call " structure," meaning thereby not merely the coarse structure seen under the microscope, which is probably less important than the ultra-microscopic structure, of colloidal nature, to which attention was called previously (page 19).

The suggestion made by Warburg (1914), as to the purpose of the energy set free by oxidation in cells which do no external work, has been referred to in an earlier chapter of this book (page 32). We have already met with cases in which the importance of structure forces itself upon the attention. The non-disappearance of lactic acid in muscle, after rubbing with sand (Fletcher and Hopkins, 1907), the inability of Harden and Maclean (1911) to obtain press juices from tissues which could continue to consume oxygen, the great effect on oxygen consumption of alkalies which do not enter the cell itself (Warburg, 1910), and other similar actions on the surface, may be mentioned.

It should be pointed out that it is impossible to draw a hard and fast line between the phenomena to be considered here and those of tissue respiration, to be dealt with in the following chapter, but I will attempt not to repeat statements more than is necessary. The observations of Warburg and Meyerhof (1912) serve to illustrate the problem before us. The red blood corpuscles of birds contain nuclei and, in their normal condition, consume oxygen in considerable amount. If a press juice is

made by Buchner's method, no oxygen consumption is to be detected. Similarly, mechanical disintegration puts an end to the process. Now, although yeast juice, as is well known, is still able to cause alcoholic fermentation, Warburg and Meyerhof have shown that the activity of yeast cells in this respect is greatly diminished by rubbing with sand. Similar observations on other cells were made by Battelli and Stern, Palladin and others, to which reference will be found in the article by Warburg (1914).

Now cells can be killed by such treatment as dehydration with acetone, etc., without obvious destruction of structure ; in fact, ordinary microscopic structure is intact. Warburg points out (1914, p. 317) that acetone and ether make a good fixing method for cells even of the delicacy of the eggs of the sea urchin in division. The important point in the process, as used for the investigations with which we are now concerned, seems to be the rapid drying. The chemical composition is practically unchanged, even the lipoids remain in the cells. The effect of this treatment on yeast cells is greatly to diminish their fermentative power (Buchner and Hahn, 1903, pp. 87 and 269). On bacteria (staphylococci), Warburg and Meyerhof found that its effect on oxygen consumption and carbon dioxide production was not to abolish them completely, although they were greatly diminished. Under favourable conditions, this respiratory process might remain constant for some hours, with a respiratory quotient of 0'65 to 0'9. The injury to the oxidation process was, in fact, less than to the fermentative power of yeast by similar treatment. Of course, in such experiments, it is essential to know that all the cells were " killed," that is, incapable of growth. To do this, after drying with acetone, they were heated to 100°, and shown to be sterile after the experiment was concluded. If treated with acetone alone, without heating, the oxygen consumption falls only to one-third of the normal, although cultures showed that nearly all the cells were killed. In absolute amount, the oxygen consumption of such completely sterile cells does not fall much below that of some other normal surviving cells, those of the liver consuming 2 '7 c.c. of oxygen per gram per hour, while the sterile staphylococci consumed 1-5 c.c.

If we compare these results with those on the eggs of the sea urchin, we find some instructive facts. The unfertilised eggs, rubbed with sand, show at first a nearly normal oxygen consumption ; this slowly decreases, so that in the third hour it is only one-quarter to one-third of the normal. Fertilised eggs, already divided, have, along with their more developed organisation, a greater consumption of oxygen than unfertilised ones. Moreover, on rubbing with sand, the decrease is greater, so that in thejlrst hour it only amounts to one-quarter to one-third of the normal. Acetone-dried unfertilised eggs also have a measurable oxygen consumption, although it is less than when they are simply rubbed with sand.

In order to obtain some further idea as to what is to be understood by cell structure, Warburg (1914, p. 315) calls attention to the fact that in the muscle cell a much larger proportion of the chemical energy appears as free energy, useful for doing work, than if the cell is disintegrated ; in the latter case the chemical energy obtained from oxidation processes is all degraded to heat. By cell structure, then, we mean those elements with which, or by whose aid, the work of the cell is carried on. They are arrangements by which the chemical energy of the oxidation processes is caught, as it were, before it has fallen to the state of heat. If we look upon the cell constituents as chemical compounds merely, without the assistance of some mechanism, nothing but heat could be obtained on oxidation. The same thing applies to a petrol motor with its fuel. If smashed up and mixed together, nothing but heat would be obtained by burning the mass.

If we divide up a cell nucleus into a thousand particles and consider them distributed throughout the cell, the nuclear structure is destroyed, as shown by the fact that the ordered movement shown in karyokinesis is no longer possible. As remarked above, thecell membrane is to be regarded as a very important element of the cell structure or mechanism. We see then that the oxidations effected by the aid of the enzyme mechanisms, treated of in the earlier parts of the present chapter, take place in the cell in the

presence of a mechanism which makes use of their energy in the actual progress of the reaction. Further than this it is scarcely possible to go in the present state of knowledge. A few further facts, nevertheless, are of interest. Permeability. — Although acetone produces no visible change in the eggs of the sea urchin, it renders the cell membrane permeable to electrolytes. A living egg placed in distilled water rapidly bursts, after swelling up. Acetone eggs, after soaking in sea water, undergo no change of volume in distilled water.

Effect of Nucleus. — Although blood corpuscles containing nuclei consume more oxygen than non-nucleated ones, the fact does not necessarily imply that it is the nucleus alone that is responsible. There is more protoplasmic material in the former kind of cells. Certain evidence, also, shows that fragments of protoplasm, free from nuclei, consume oxygen. The nucleus, in fact, counts as a part of the cell structure. If red blood corpuscles of birds be frozen and thawed, cytolysis occurs, the membrane is ruptured and certain cell constituents escape. It has been shown by Warburg (1914, p. 322) that some of the structural parts are not disintegrated, but, being insoluble, can be centrifuged off. By this process, we obtain an upper layer of structureless qell substance and a lower one of structural elements. When separated, and their oxygen consumption tested, it is found that this is scarcely to be detected in the upper structureless layer, but in the lower layer it is practically identical with that of the mixture before it is centrifuged.