Catalase, peroxidase and metmyoglobin as catalysts of coupled peroxidatic reactions

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Abstract
A manometric method is described for the study of peroxidatic activities of hemoproteins. The catalytic activities of catalase, peroxidase and metmyoglobin are compared in coupled or peroxidatic oxidations of different hydrogen donors by means of H2O2 generated by the glucose oxidase (notatin) system under standardized conditions. The catalytic efficiencies of the 3 hemoproteins are expressed as pC; the negative logarithm of the molar concentration at which they utilize 50% of generated H2O2 for coupled oxidation of a given donor. The following donors were examined: H2O2, ethanol, aldehydes, formate, nitrite, ascorbic acid, ferrocyanide, phenols and azide. All 3 hemoproteins catalyze the coupled oxidation of H2O2 i.e. the catalatic decomposition of generated H2O2 to O2 and water. However, in this reaction the efficiencies of peroxidase and especially of metmyoglobin are much lower than that of catalase. Ethanol is oxidized peroxidatically only by catalase. At no concentration are peroxidase, metmyoglobin or free hematin able to promote this reaction which appears to be the only absolutely specific catalytic property of catalase. Cyclic oxidation of ethanol to acetic acid is described, whereby H2O2 generated by pure xanthine oxidase and a little acetaldehyde is used by pure catalase to oxidize ethanol to acetaldehyde thus regenerating the substrate for xanthine oxidase. In this reaction both H2O2 and acetaldehyde are utilized as fast as they are generated. An almost specific requirement for catalase was demonstrated for formate although a very low activity could be detected at high metmyoglobin concentrations. Coupled oxidation of pyrogallol and certain other phenols is catalyzed not only by peroxidase, and to a much lesser degree by metmyoglobin, but also by catalase. The peroxidatic activity of catalase towards pyrogallol, although about 10000 times lower than that of peroxidase, is somewhat higher than the activity of metmyoglobin and is of the same magnitude as its peroxidatic activity towards nitrite, formate and ethanol. This militates against the view that catalase iron is accessible only to very small donor molecules. Study of the coupled oxidation of ascorbic acid must be carried out under the best attainable metal-free conditions as well as in the presence of a chelating agent such as ethylenediaminetetraacetic acid. Peroxidase shows marked activity in catalysis of coupled oxidations of ascorbic acid and ferrocyanide, metmyoglobin is about 1000 times less active, whereas catalase is completely inactive. Observations that azide in presence of catalase and peroxide undergoes peroxidatic oxidation are confirmed and their bearing upon the mechanism of catalytic decomposition of H2O2 is discussed.