Oxidative Enzymes

OXIDATIVE ENZYMES, a group of enzymes that bring about oxidation processes occurring in plant and animal cells both during their life and for some time after the destruction of protoplasm. From a biological point of view, the most important O. process of the living cell is respiration, during which organic substances are oxidized by atmospheric O₂ to final products of decomposition (CO₂ and water). As early as Lavoisier pointed out that this process is analogous to combustion. But whereas combustion can only occur with very strong heating of organic substances, respiration occurs at relatively low temperatures. Therefore, it should be classified as a group of phenomena very common in nature, usually called "spontaneous oxidation" or "slow combustion." These processes occur at a noticeable rate only in the presence of special accelerators-catalysts. As early as Schonbein pointed out the necessity of the presence in the living cell of such catalysts-O. enzymes. He believed that without these enzymes "animals would suffocate in an ocean of pure O₂, as in empty space." For the first time, an O. enzyme was isolated by the Japanese scientist Yoshida (H. Yoshida) from the sap of the lacquer tree, and therefore it was named laccase. Bertrand (G. Bertrand), who studied laccase in detail, showed that similar enzymes also exist in other plant objects. He combined all these enzymes that oxidize in the presence of atmospheric O₂ under the common name "oxidases." Bach together with Chodat (R. Chodat) subjected these enzymes to detailed research. On the basis of the data he obtained, he built a theory according to which the action of oxidases is based on the activation of atmospheric O₂. In this case, intermediate peroxide compounds of organic substances and hydrogen peroxide are formed. The latter in the presence of a special O. enzyme "peroxidase" can be used by the living cell for further oxidation of organic substances. However, neither oxidase nor peroxidase can oxidize carbohydrates, proteins, or fats. Bach and Chodat, through a series of careful experiments, proved that the action of these enzymes extends only to the following substances: phenols, aromatic amines, and hydroiodic acid. None of these compounds is a material oxidized in the process of respiration. Consequently, the presence of oxidases in the cell cannot yet account for the process of respiration. The opinion was even expressed (P. Portier) that oxidases are not at all "respiratory enzymes" and that their role is limited only to a protective effect, to the formation of substances that prevent the penetration of microorganisms into injured tissue. But this point of view proved to be incorrect, and at the present time the outstanding role played by oxidases in vital oxidation has been fully established. Respiration is a complex process consisting of a series of separate, interconnected and coordinated chemical reactions. Oxidation caused by oxidases is only one link in this long chain of phenomena. Therefore, it is impossible to reproduce the entire phenomenon with the help of oxidases alone. Another extremely important link in the respiratory process are the oxidation-reduction reactions occurring within the living cell. As early as Traube (M. Traube) showed that oxidation can occur not only due to free atmospheric O₂, but also due to bound O₂, in particular the O₂ of water. The simplest example of such hydrolytic oxidation can be the oxidation of alkali metals, which proceeds due to the hydroxyls of water with the release of the corresponding amount of H in the form of gas: 2Na+2H₂O = 2NaOH + H₂. But such a reaction can only occur when the oxidizing body has a sufficient reserve of energy to decompose water itself. Usually, oxidation due to the elements of water occurs by coupled reactions, in which of two bodies, unable separately to decompose water, one binds the O₂ of water, the other H. For example, metallic zinc in the absence of water is not oxidized by atmospheric O₂ at all and does not decompose water itself. But if zinc is shaken with water and O₂, the metal combines with the hydroxyl of water, giving zinc hydroxide, and the H of water with an O₂ molecule forms hydrogen peroxide. Another example of the same kind of oxidation-reduction reactions proceeding due to the elements of water can be the Cannizzaro reaction (see Cannizzaro reaction), which consists in the simultaneous reduction and oxidation of aldehydes occurring in the presence of alkali. + 1 + I = RCHO OH OH RCOOH+H₂O. In the case mentioned, one molecule of aldehyde is oxidized by the hydroxyl of water into the corresponding acid, while another is reduced by hydrogen into alcohol. In vitro this reaction can only proceed in an alkaline solution, but in the living cell it occurs in a neutral medium due to the presence here of special oxidation-reduction enzymes (oxidoreductases). For the first time, the action of these enzymes was definitely discovered by Schardinger (F. Schardinger) in milk. Schardinger showed that a mixture of formic aldehyde and methylene blue in an aqueous solution is decolorized by fresh milk in a few minutes. Boiled milk remains inactive. Later it was established that we have here an oxidation-reduction process proceeding due to the elements of water. In this case, the aldehyde is oxidized into the corresponding acid, and methylene blue is reduced into a colorless leuco compound. Methylene blue and formic aldehyde can be replaced by other substances (for example, other aldehydes, amino acids, purine bases on the one hand, and various dyes, nitrates, thio-compounds on the other). The nature of the reaction does not change from this. Bach, on the basis of a number of considerations, named this enzyme of milk "perhydridase." Similar enzymes were also found in other plant and animal materials. At present, it can be assumed that oxidoreductases are present in all living cells without exception. The biological role of these enzymes is enormous. It is precisely thanks to them that the primary oxidation of various organic substances (in particular respiratory materials) occurs in the living cell. Carbohydrates, fats, proteins and their decomposition products cannot be directly oxidized by atmospheric O₂. This oxidation proceeds according to the scheme indicated above due to the hydroxyl of water. However, as we have already seen, such oxidation occurs only when at the same time a reduction process occurs, with the help of which the H of water is removed. In the living cell, this role of hydrogen acceptors can be played by a variety of compounds. In particular, during fermentation, various decomposition products of carbohydrates (for example aldehydes) are reduced. As a result of this, reduction products-alcohols, methane, and even in some cases H-are necessarily formed in the process mentioned. Atmospheric O₂ plays no role here, and therefore a number of fermentations can occur even in its complete absence. The situation is different in the process of respiration. Here, the acceptance of hydrogen is carried out by a special mechanism in which oxidases play by no means the last role. As numerous studies by V. Palladin and other authors have shown, in the cells of plants and animals, along with oxidases, there are also those substances that can be oxidized with the help of these enzymes-polyphenols. Thus, aerobic oxidation and in particular cellular respiration occur according to the following scheme: the oxygen of the air under the action of oxidases oxidizes "respiratory chromogens" - substances of the type of polyhydric phenols. In this case, quinone-like compounds, the so-called "respiratory pigments," are formed. The latter have the ability to bind the H of water and to be reduced back to chromogen, while the hydroxyl of water goes to oxidize various organic substances (carbohydrates, amino acids, etc.). The oxidation of these substances and the associated reduction of pigments occurs in the living cell only thanks to the presence of oxidoreductases. Apparently, a much greater role than the polyphenols indicated by V. Palladin is played as an acceptor and carrier of oxygen both in plant (yeast, bacteria) and especially in animal cells by derivatives of hemin - histohematin discovered by Mac Munn, or cytochrome (Keilin), contained in all cells without exception that have aerobic metabolism. Cytochrome easily passes from the oxidized form to the reduced form and back. Since these forms differ in their spectral properties, their mutual transition-and thus to a certain extent cellular respiration-can be followed directly by the eye without even violating the integrity of the cell. Cytochrome is easily reduced by hydrogen of organic molecules activated by dehydrogenases (see below), or, if one adheres to the scheme of hydrolytic oxidation, by hydrogen of water under the influence of oxidoreductases.

In turn, the reduced cytochrome, under the influence of a special oxidase differing in some properties from ordinary phenolases, is again oxidized by atmospheric oxygen into its original form. An artificially composed system of cytochrome + oxidase, in Keilin's experiments, showed complete similarity with the natural respiratory enzyme. Consequently, in the process of respiration, two categories of enzymes participate: first, oxidase enzymes that cause the formation of H acceptors (respiratory pigments), and second, oxidoreductase enzymes that accelerate the oxidation-reduction processes going at the expense of the elements of water. This division of oxidative enzymes into two groups appears quite justified from both chemical and biological points of view. However, not all authors agree with the scheme of respiration outlined above. In particular, Wieland (H. Wieland) completely denies the activation of atmospheric O₂. In his opinion, every oxidation of organic substances in a living cell begins with the hydration of these substances, with the attachment of an entire water molecule. -Further, from the thus obtained hydrates, hydrogen is removed, dehydration occurs, as a result of which we obtain compounds more oxidized than the original substances. As an example of such a process, Wieland cites the reaction of oxidation of CO to CO₂ in the presence of palladium black as a hydrogen acceptor. CO + H₂O = H·CO → CO₂ + H₂ (acceptor). The main moment of the entire process as a whole is, in Wieland's opinion, dehydration, proceeding at a noticeable rate only in the presence of specific catalyst-enzymes, the action of which is directed toward the liberation of active H. If hydrogen is thus detached from the hydrate molecule, it can easily be captured by any acceptor, whether it be methylene blue, aldehyde, or even gaseous O₂. Consequently, no activation of atmospheric O₂ by oxidative enzymes is required or occurs. The action of oxidases, as well as of oxidoreductases, is directed at the H of hydrates, at their dehydration. Therefore, Wieland denies the existence of oxidases as an independent group and proposes to unite all oxidative enzymes under one name 'dehydrases.' To confirm his opinion, Wieland conducted a series of experiments aimed at showing that typical oxidases promote the oxidation of the substrate even in the absence of gaseous O₂ with methylene blue as the H acceptor. Conversely, oxidoreductases produce oxidation associated with the absorption of atmospheric O₂. However, in his experiments, Wieland did not deal with physiologically pure enzymes, and in the preparations he used, representatives of both groups could be present simultaneously. Further research has shown that when using thoroughly purified enzyme preparations, it is not possible to repeat the mentioned experiments, which of course significantly reduces the persuasiveness of Wieland's arguments. The noted disagreement in the basic views on the nature and biological significance of oxidative enzymes creates a very strong difficulty in the study of this branch of enzymology. Diametrically opposite to Wieland's views is the standpoint of Warburg, who sees the primary moment of the action of respiratory enzymes in the activation of oxygen. This activation is carried out under the influence of catalytically acting organic compounds (and perhaps also free) compounds of heavy metals, mainly iron, to a lesser extent also copper, manganese, and others. Biologically dominant in significance as respiratory enzymes (in the sense in which this term is used by Warburg, i.e., activators of oxygen) are the complexes of iron with pyrrole compounds of the type of hematin, in turn connected with certain organic bases (similarly, for example, in hemoglobin, hematin is connected with the basic protein-globin). By exceptionally elegant experiments, Warburg was even able to obtain the absorption spectrum of the respiratory enzyme without isolating it as a chemical individual from the cell (which is still not possible). This spectrum, giving the main absorption band in the region of about 4,300 Å, almost exactly coincides with the spectra of artificially obtained hematin a compounds (only slightly shifted toward the red side). Undoubtedly, the explanation of the true mechanism of oxidation processes in the cell should be sought in the synthesis of the extreme views of Wieland (exclusive activation of hydrogen) and Warburg (exclusive activation of oxygen). The overwhelming majority of modern researchers incline toward this synthesis, and Warburg himself in one of his most recent works also recognizes 'activation of the substrate' as one of the possible factors of biological oxidations. Such a synthetic view amounts essentially to the fact that the main pathway of oxidation processes in the cell is the following: activated hydrogen of organic molecules (or their hydrates, according to Wieland, or simply hydrogen of water in the scheme of hydrolytic oxidation) through the intermediate participation of hydrogen acceptors or carriers (of the type of respiratory pigments or cytochrome) reacts with oxygen activated by oxidases or Warburg's respiratory enzyme. However, this path is not the only one. In a number of cases, biological oxidations have shown the formation of hydrogen peroxide. This can happen only if the hydrogen acceptor is molecular, non-activated oxygen: 2H + O₂ = H₂O₂, whereas active, atomic oxygen would give the formation of water. Apparently, the formation of hydrogen peroxide must be taken into account in most biological oxidations. At the same time, the oxidative enzymes themselves are very sensitive to hydrogen peroxide, rapidly being destroyed by it. Hence the importance of the universally distributed enzyme catalase (see), which decomposes hydrogen peroxide into water and molecular oxygen, which can again be used by the cell. In addition, peroxide can be decomposed by peroxidase with the removal of active oxygen, which also goes to new oxidations. Recent works (1931) have established the fact of extremely great importance: it turned out that these last two enzymes, catalase and peroxidase, which are components of the general oxidative system of the cell, are, like Warburg's respiratory enzyme, iron-containing pyrrole derivatives, close to hematin and giving a characteristic absorption spectrum with a main band at 4,200 Å. At the same time, the intensity of this band runs approximately parallel to the activity of the enzyme preparation. The content of hematin is even in the purest preparations extremely small, of the order of 0.1-0.6%. Obviously, if the enzymatic action is inherent in these hematin derivatives (and this can now be stated with almost complete certainty), then their enzymatic activity (which is also distinguished by high specificity) is approximately 10⁷ times higher than that of ordinary iron-containing pigments (hemoglobin and its derivatives). The nomenclature and classification of oxidative enzymes are extremely confused and contradictory. This is mainly due to two circumstances: first, the nomenclature of this group of enzymes is not subject to any definite principle and is rather random. Therefore, often the same enzyme appears in the literature under several different names. Secondly, only a very limited number of works on oxidative enzymes have been carried out with physiologically pure preparations. In most cases, researchers dealt with complex autolytic mixtures, tissue pulps, cells of microorganisms killed with acetone, or even simply with freshly excised animal organs. It is clear that in such objects the oxidation of one or another substance may depend on the joint action of a whole complex of enzymes. However, it was usually attributed to a particular enzyme specific for a given substrate. For a number of cases, it has already been possible to show at the present time that this is incorrect. The mentioned 'enzymes' are not individual, but represent a mixture of oxidases and oxidoreductases, the joint action of which causes one or another oxidative reaction. Therefore, in the following presentation, the classification of oxidative enzymes is based on their division into two groups: 1) oxidases and 2) oxidoreductases. But since from a number of mixtures it has not yet been possible to isolate individual enzymes and we know only their cumulative action for the time being, in some cases it is necessary to temporarily preserve the old terminology and to single out these enzymes into a special mixed third group. Group I, oxidases (true oxidases, direct oxidases, aero-oxidases, chromo-oxidases)—enzymes that cause the formation of respiratory pigments. The basis of the action of these enzymes is the activation of oxygen. This group includes: 1. Phenolases—enzymes that oxidize various polyatomic phenols, aromatic amines, and hydroiodic acid with atmospheric oxygen. These enzymes are very widely distributed in both animal and plant cells, where they can be detected by the reaction with pyrogallol.

However, the isolation of phenolases in the form of purified preparations is associated with significant difficulties due to their instability. Therefore, obtaining active preparations is only possible when using certain, mainly plant, objects (Russula delica mushrooms). On the basis of a number of data, Bach and Schoda suggested that phenolase consists of two parts, one of which activates the O₂ molecule with the formation of peroxide, and the other accelerates the oxidative action of the resulting hydrogen peroxide. The first component was named oxygenase, and the second, which activates hydrogen peroxide, peroxidase. The action of phenolase can be schematically represented by the following equations: 2) R-/\ - OH H-O R-/\ - O +

| ->

| +2H_aO KJ-он н-o \J-o Peroxidase has been found in a number of plant and animal objects and exists as an independent enzyme in the absence of oxygenase. 2. Peroxidases are the most thoroughly studied oxidative enzymes. Their action is quite similar to the action of phenolases. But while the latter activate atmospheric O₂, peroxidases carry out the oxidation of phenols, amines, etc. only at the expense of hydrogen peroxide. Peroxidases are very stable with respect to various external influences and therefore can be easily obtained from various plant and animal objects. Particularly large amounts of peroxidase are contained in the roots and sprouts of higher plants. From the horseradish root, Willstätter (R. Willstätter) and his students (R. К1ш) obtained the purest preparations of peroxidase, which made it possible to approach the elucidation of the chemical nature of this enzyme. It is very interesting that the blood pigment oxyhemoglobin possesses peroxidase activity. But even when it is isolated in chemically pure form, its action is incomparably weaker than that of plant peroxidases. 3. Tyrosinase is an enzyme that oxidizes, with the help of free oxygen, one of the products of protein breakdown - tyrosine (O-oxiphenyl-α-aminopropionic acid). Under the action of tyrosinase, a colorless aqueous solution of tyrosine in the presence of atmospheric oxygen turns red, then brown, purple, and black. After this, an ink-black precipitate of melanin falls out, and the liquid becomes colorless again. The reaction proceeds with the elimination of ammonia and CO₂ (deamination of the alanine group). Tyrosinase is less widespread than the first two enzymes mentioned. However, it can be found in a number of plant and animal objects. The peel of potatoes is particularly rich in tyrosinase. The oxidation of tyrosine and the formation of melanin from it represent a combination of successive reactions. Therefore, tyrosinase is hardly an individual enzyme. More likely, it is a mixture of enzymes, but since oxidases play the dominant role here, it can be classified in the first group of O. f. In addition to what has been said, it is necessary to note another group of oxidases, named by Dubois (R. Dubois) luciferases. The action of these enzymes is responsible for the luminescence of a number of lower marine animals, insects, and bacteria. According to the data of the mentioned author, here there is an enzymatic oxidation by atmospheric oxygen of a special substance - luciferin. However, this reaction is far from being studied (see Luminescence). II group - oxidoreductases (reductases, dehydrogenases) - enzymes that accelerate oxidative-reductive processes occurring simultaneously at the expense of the elements of water. 1. Peroxydase (Schardinger's enzyme) is an enzyme of milk, from which it can be obtained in the form of a purified preparation by precipitation with acetone. Peroxydase simultaneously oxidizes and reduces various organic and inorganic substances. In particular, in its presence various aldehydes, amides, and amino acids are oxidized. This must necessarily be associated with the reduction of such substances as, for example, methylene blue, nitrates, thio compounds, etc. Oxidation and reduction proceed at the expense of the elements of water, and therefore the reaction can occur in the absence of gaseous O₂. Atmospheric O₂, on the contrary, even slows down this reaction, as it gradually destroys the enzyme. Because peroxidase can simultaneously oxidize and reduce aldehydes (Cannizzaro reaction), some authors consider this enzyme as a special case of aldehydeases. 2. Aldehydeases (aldehydrazymes) - enzymes that catalyze the Cannizzaro reaction. The action of these enzymes was first discovered in the oxidation of salicylaldehyde to salicylic acid. Therefore, this enzyme was initially named salicylase. However, it was later established that salicylase can also act on other aldehydes. In this case, along with the oxidation of one aldehyde molecule to the corresponding acid, another molecule is reduced to alcohol. The reaction proceeds successfully in the absence of atmospheric O₂, since it proceeds at the expense of the elements of water. Along with the reduction of the second aldehyde molecule, other substances can also be reduced, in particular, as shown by Neuberg (C. Neuberg), ketones, nitro compounds, thio compounds, etc. Aldehydeases are extremely widespread in both the animal and plant worlds. They are particularly abundant in the liver of animals and in yeast. These enzymes play an exceptionally important role in the metabolism of substances in the living cell. Unfortunately, they are still very poorly studied. - 3. Aldehydemutases - enzymes that catalyze the reaction called 'internal Cannizzaro' (Neuberg). In this reaction, the simultaneous oxidation and reduction of two atomic groups of the same molecule occurs, proceeding at the expense of the elements of water. The best-studied example of this reaction can be the transformation of methylglyoxal into lactic acid: CHa

CHa I

I co+н₂->снон , сно o сооы According to Neuberg, this reaction is carried out by the action of an enzyme which he called ketone-aldehydemutase (glyoxalase Deakin). III group O. f. (mixed group). This includes: 1. Alcoholoxidases (alcoholdehydrogenases) - enzymes that oxidize alcohols to the corresponding aldehydes. These are then converted to acids by aldehydeases. Oxidation occurs at the expense of atmospheric O₂, but since these enzymes contain oxidoreductases, oxidation can also proceed in the presence of such H acceptors as, for example, methylene blue. Alcoholoxidases were first obtained by Buchner (E. Buchner) in the form of acetone-killed acetic acid bacteria. Later, this enzyme was found by Battelli (F. Battelli) and Stern in animal tissues. The liver of horses is particularly rich in it. 2. Acidoxidases (acidodehydrogenases) - enzymes that oxidize hydroxy acids. They were first discovered by Battelli and Stern in the muscles and other tissues of animals. The enzyme acting on succinic acid (succinoxidase of Battelli and Stern or succinodehydrogenase T.Thunberg's) has been studied in the greatest detail. 3. Purineoxidases (purinedehydrogenases) - enzymes that oxidize the breakdown products of nucleic acids - purine bases. Adenine and guanine, formed during the hydrolysis of nucleic acid, under the influence of hydrolytic enzymes are converted to hypoxanthine and xanthine. These are the substances that purineoxidases act on. For example, xanthine oxidase oxidizes hypoxanthine to xanthine, and the latter to uric acid. Uric acid is further oxidized to allantoin under the influence of uricase. Purineoxidases have been found only in animal organisms.

A. Oparin, E. Engelhardt. Investigation of cells for the presence of oxidative enzymes in their protoplasm. Oxidative enzymes have great importance both in the clinical laboratory and in the pathological-histological study of tissues. It is used mainly to identify bone marrow (myeloid) type cells, which, unlike elements of a lymphoid and histoid nature, contain oxidative enzymes in their protoplasm. The need to differentiate cells by means of oxidative enzyme reactions in clinical practice arises when it is necessary to determine the type of leukemia (see) in cases where the latter is based on an increase in the number of white elements, morphologically unclear in terms of their nature. Reactions for oxidative enzymes are performed on smears, for example of blood, best fixed in formalin vapor, and on sections made on a freezing microtome; tissue pieces are also best fixed in formalin. Of the reactions for oxidase, the Winkler-Schultze reaction is most commonly used, which is based on the action of α-naphthol and dimethyl-para-phenylenediamine solutions on the tissue. This reaction, on the proposal of Graff, is commonly abbreviated as the Nadi reaction. According to Winkler-Schultze, solutions of naphthol and dimethyl-para-phenylenediamine are used separately, according to Graff-in a single mixture. According to the Winkler-Schultze method, the α-naphthol solution is prepared by dissolving 1 g of α-naphthol in 100 cm³ of distilled water, bringing the latter to a boil and adding drop by drop pure caustic potash until the naphthol dissolves (usually about 1 cm³ of caustic potash is sufficient). Dimethyl-para-phenylenediamine is dissolved in 1% concentration in distilled water at room temperature. Smears or sections are immersed for several minutes first in the naphthol solution, and then directly in the dimethyl-para-phenylenediamine solution; then-washing in distilled water. Smears are dried and examined with an immersion system; sections are examined in water or glycerin or, through alcohol and xylene, are mounted in Canadian balsam. Nuclei can be stained with carmine. According to Graff's method, sections are immersed for 10-15 minutes in a mixture of equal parts of 1% aqueous solution of α-naphthol and 0.2% aqueous solution of dimethyl-para-phenylenediamine. After rinsing the sections in water, they are placed for 2-3 minutes in Lugol's iodine solution. Washed in water, to which 5 drops of saturated lithium carbonate solution are added per 10 cm³. After this, the nuclei are stained with alum carmine, the sections are washed and mounted in glycerin-gelatin. In the Nadi reaction, the presence of oxidase is revealed by the formation of indophenol blue in the form of dark blue grains (in Graff's method, brownish from iodine). The reagents* for the Nadi reaction are unstable; they must be stored in dark bottles and frequently renewed. The reaction for peroxidase is carried out using benzidine. 1 g of benzidine is dissolved in 100 cm³ of tap water with shaking; into this solution, a little (1-2 drops) of commercial hydrogen peroxide is added at the time of use. Smears and sections are kept in this mixture for several minutes, after which they are washed in water, the nuclei are stained with hematoxylin or Giemsa stain; after washing, smears are dried, and sections, through alcohol and xylene, are mounted in balsam. Peroxidase grains are yellowish-brown. It should be noted that for practical purposes, the Nadi reaction for oxidase and the benzidine reaction for peroxidase can be considered equivalent, since in tissue elements, during the breakdown of phenols under the influence of hydrogen peroxide, products are formed that are the same as those given by the oxidase reactions. a. Abricosov.