H2O2 Oxidoreductase Research Articles

Studies on hog intestine mucosa peroxidase.

A method has been developed for isolating and purifying peroxidase from hog intestinal mucosa. The enzyme exhibits an absorbance ratio A417 nm/A280 nm of 0.906.Studies on the homogeneity of intestinal peroxidase by electrophoresis on polyacrylamide gel and cellulose acetate strips showed its high degree of purification; the enzyme showed a single band at pH 8.45 with the cathodic mobility.Intestinal peroxidase is a haemoprotein. Its absorption spectrum, as well as that of its derivatives (CN‐peroxidase, reduced peroxidase, CN‐reduced peroxidase and pyridine haemochromogen) is of the same type as the spectrum of lactoperoxidase and its corresponding derivatives.Kinetic studies on the reaction of intestinal peroxidase with hydrogen peroxide and guaiacol showed that this haemoprotein has the properties of true peroxidase (donor: H2O2 oxidoreductase). The k1 value was determined as 5.9 × 106 M−1× sec−1, and Km (H2O2) was 260 μM. Optimum pH of the studied peroxidase was in the range pH 7.5–8.Intestinal peroxidase, with respect to deamination and decarboxylation of amino acids, does not resemble the myeloperoxidase.

Oxidation of Thiocyanate to Cyanide Catalyzed by Hemoglobin

Thiocyanate ion is oxidized at acid pH by hydrogen peroxide to sulfate and cyanide. The reaction is catalyzed in the erythrocyte by hemoglobin acting as a peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7). Kinetic studies show thiocyanate ion fulfills criteria for a substrate for the peroxidase. The peroxidase activity is enhanced by haptoglobin and inhibited by azide, aminotriazole, fluoride, iodide, and cyanide. No other enzyme catalyzing this reaction was found in hemolysates of bovine blood. Oxyhemoglobin was a more active catalyst than methemoglobin but was rapidly converted to the latter in the incubation system. Equivalent amounts of sulfate and cyanide were produced initially, but the cyanide was converted to cyanate and ammonia. The conversion was not rapid nor catalyzed by hemoglobin except in the presence of thiocyanate. The enzymic reaction resembles the acid-catalyzed oxidation of thiocyanate by hydrogen peroxide. Oxidation of sulfur dicyanide, a proposed intermediate product, was catalyzed also by hemoglobin. Cyanide produced in vivo is converted in part to thiocyanate by sulfur transferase systems. The thiocyanate-cyanide cycle probably accounts for some of the physiological effects of thiocyanate.

Theory of oscillations in peroxidase catalyzed oxidation reactions in open system

1. The applicability of the (1910) Lotka6 model to the horseradish peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7) catalyzed oxidation reactions in an open system is investigated.2. The input of reactant by diffusion in stead of a constant input flux does not prevent oscillations of low damping. The gain of 3 in the branched chain reaction in the peroxidase system in stead of 2 in the Lotka model causes an increased damping, but the low damping observed in the experiments is still possible.3. The combination of linear branching and quadratic termination, which is commonly believed to prevail in the peroxidase system, leads to a damping which is much higher than observed in the experiments.4. A critical lower concentration of donor is known to exist below which the peroxidase catalyzed oxidation does not take place. This phenomenon is predicted by models of the Lotka type where the branching and the termination are both linear or both quadratic. It does not occur if the branching is linear and the termination is quadratic.5. Compound III is not assumed to play any significant role in relation to the origin of the damped oscillation.

Compound III kinetics and chemiluminescence in oscillatory oxidation reactions catalyzed by horseradish peroxidase

1. The peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7) catalyzed oxidation of reduced nicotinamide adenine dinucleotides, dihydroxyfumaric acid or indole-3-acetic acid, in a reaction system open to O2, exhibit damped oscillations in the reaction rate.2. All known aerobic oxidation reactions catalyzed by peroxidase alone are autocatalytic. The damped oscillation is a consequence of the autocatalysis.3. Compound III is observed spectroscopically in the NADH and the dihydroxyfumaric acid oxidation but not in the indole-3-acetic acid oxidation. Large differences exist in the Compound III kinetics in the NADH and the dihydroxyfumaric acid oxidation. Compound III is not essential in the mechanism of the oscillation.4. The oxidation of the three donors is accompanied by chemiluminescence whose intensity increases in the order NADH < dihydroxyfumaric acid < indole-3-acetic acid. Except in the NADH oxidation the chemiluminescence is strong enough to be used for kinetic measurements and possibly also for spectroscopic identification of intermediates.

Preparation of some iron-binding proteins and α-lactalbumin from bovine milk

The preparation of the iron-binding milk proteins— “red protein,” blood transferrin, and lactoperoxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7) — by chromatography on DEAE-cellulose and phosphocellulose is described. The “red protein” is distributed in the casein and whey fractions of milk. In some fractions it forms complexes with other proteins which drastically change its chromatographic behavior. The transferrins found in the milk and blood of an individual cow are shown to be the same by gel electrophoresis. The preparation of an electrophoretically homogeneous α-lactalbumin by chromatography on DEAE-cellulose is also described.

Reaction of peroxidase with reduced nicotinamide-adenine dinucleotide and reduced nicotinamide-adenine dinucleotide phosphate

Summary 1. Peroxidase (donor:H2O2 oxidoreductase, EC 1.11.1.7) catalyzes the aerobic oxidation of NADH and NADPH at acidic pH in the absence of cofactors such as Mn2+ and monophenols. 2. Peroxidase Compound III appears during the reaction and disappears when almost all the oxygen in the reaction solution has been consumed. 3. A part of peroxidase is rapidly reduced with consumption of H2O2 or O2 in the presence of NADH and NADPH. 4. It is suggested that the free radicals derived from NADH and NADPH are active intermediates in the formation of Compound III and the reduction of peroxidase. 5. The different types of peroxidase-oxidase reaction possible when dihydroxy-fumarate, triose reductone, indole acetate and NADH are used as hydrogen donors are discussed.

Studies on rat eosinophil peroxidase

Summary 1. Eosinophils, substantially free from neutrophils, have been obtained from the peritoneal cavity of rats. A haemoprotein has been extracted from the eosinophils at pH 3.7 and partially purified. 2. Kinetic studies of the catalysed reaction of hydrogen peroxide with guaiacol show that the haemoprotein is a true peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7), k1 being approx. 5.0 · 106 M−1 sec−1 and Km (H2O2) approx. 4.5 · 10−4 M. 3. The oxidized and reduced forms of eosinophil peroxidase have spectra of the same type as horse-radish and milk peroxidases; the spectrum of the reduced form is different from that of reduced myeloperoxidase. 4. It is concluded that myeloperoxidase is present in blood only in neutrophils, and might be better termed “neutrophil peroxidase”, and that eosinophil peroxidase is a different enzyme. 5. The position of the a band of the pyridine haemochrome (567 mμ) formed from eosinophil peroxidase suggests that the prosthetic group is not protohaem.

COMPARISON OF THE ENZYME OXIDIZING THYROID HORMONE WITH L-AMINO ACID OXIDASE.

An oxidase, which catalyzes mainly the deamination of thyroxine, 3,3′,5-triiodothyronine and 3,5-diiodothyronine, has been partially purified from rat-kidney mitochondrial extracts by successive (NH4)2SO4 fractionation and diethylaminoethyl-cellulose-column treatment. The enzyme preparation was contaminated with catalase (H2O2: H2O2 oxidoreductase, EC 1.11.1.6) in relatively high concentration, but contained no thyroid-hormone transaminase. The enzyme preparation had no effect on either diiodotyrosine or tyrosine, leucine and lactic acid were well oxidized with the formation of keto acids. The elution pattern of the partially purified oxidase on diethylaminoethyl-cellulose column indicates that the enzyme oxidizing thyroid hormone is identical to the mammalian l-amino acid oxidase (l-amino acid: O2 oxidoreductase, EC 1.4.3.2) which catalyzes chiefly the deamination of leucine and the dehydrogenation of lactic acid. Furthermore, the prosthetic group of the purified enzyme oxidizing thyroid hormone was identified as riboflavin 5′-phosphate, which is well known as the prosthetic group of the mammalian l-amino acid oxidase.

INFLUENCE OF SOME INHIBITING AND ACTIVATING SUBSTANCES ON THE LIGHT REACTION IN VITRO OF PHOTOBACTERIUM PHOSPHOREUM.

Addition of H2O2 to a bacterial luciferase preparation resulted in the formation of a compound with a fluoresence maximum at about 470 mμ. This compond was probably the same as the one that is formed as a result of ultraviolet irradiation of luciferase preparations after addition of FMNH2. It is assumed to be the luminescent molecule in bacterial luminescence. The effect of a number of substances (K3Fe(CN)6, K4Fe(CN)6, ascorbic acid, peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7), tryptophan and p-chloromecuribenzoate) on both the light reaction and fluorescence increase reaction was investigated. Substances inhibiting the fluorescence increase reaction (K3Fe(CN)6, K4Fe(CN)6, ascorbic acid) likewise could inhibit the light reaction. Both effects are, at least partly, ascribed to an effect on the fluorescent group of luciferase. Light reaction inhibitors affecting other groups at the enzyme surface (p-chloromercuribenzoate) did not influence the fluorescence increase effect. Activators of the light reaction (cystine, K4Fe(CN)6) are suggested to act by their protective action on sulfhydryl groups or on dissociable groups, presumed to be attached to these sulfhydryl groups. A discussion of the results leads to the conclusion that the light reaction consists of at least two consecutive reactions, involving two different groups of the luciferase molecule.

The alteration of intracellular enzymes: I. Yeast catalase and the Euler effect

Two forms of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) activity, soluble and lipid-bound, exist in the total homogenate as well as in mitochondria, microsomes and plasma membranes of mice liver, rat liver and hepatomas.The lipid-bound catalase activity is revealed by the treatment of the tissue with n-butanol.An inverse correlation between morphological, immunochemical characteristics (reflecting the extent of malignancy) of the hepatomas studied and the activity of the two forms of catalase was found.The nature of two forms of catalase activity is discussed. One infers that experimental data available do not favour the view of their being isozymes.