Histochemical demonstration of hydroperoxidase activity by two improved methods different in their mechanisms.

Abstract

Catalase and peroxidases have been investigated for a long time especially in enzyme-chemical analysis. However, their physiological roles have not been fully solved yet. The classification of catalase and peroxidases is based on the differences of specificity for hydrogen donators, but it is not always clear. These enzymes have been generalized under the name of “ hydroperoxidase ” proposed by Theorell (1951) . The conclusions to be drawn from the work of Theorell (1948, 1951) and Chance (1951) on catalase and peroxidases are that their mechanisms of action are similar enough to classify them under a single name, the hydroperoxidase. Moreover, catalase and peroxidases are iron-protoporphyrin IX-protein, and react with the same substrate such as hydrogen peroxide or alkyl monohydrogen peroxide. In fact, catalase activity has been characterized by production of oxygen gas when catalase reacts upon hydrogen peroxide, while, it is generally thought that the enzyme may act like the peroxidase in vivo. This assumption was demonstrated in vitro by Tauber (1952) . He found that crystalline catalase and dilute hydrogen peroxide could oxidize alpha-naphthol and p-phenylene diamine into indophenol purple, and I have also found crystalline catalase and dilute hydrogen peroxide or monoethyl hydrogen peroxide could oxidize alpha-naphthol and dimethyl p-phenylene diamine into indophenol blue. In other words, crystalline catalase has catalase activity and also has peroxidase activity according to the combination of substrate and hydrogen donator. It can be thought that catalase does not act as an antidotal enzyme but as oxidative enzyme in the living tissues. Therefore, there is no reason which catalase and peroxidases should be distinguished in histochemistry. Many histochemical techniques concerning hydroperoxidase have been reported, (Osgood, 1937; Graham, 1916; Lison, 1953; Nishiyama and Kobayashi, 1953a, 1953b) and recently Takamatsu and lizima (1953) introduced the name of “ hydroperoxidase ” in their method with the same concept as Theorell's, but these techniques don't seem to be satisfactory as discussed later.In this paper, several defects of the histochemical techniques hitherto reported were reinvestigated and several staining conditions were newly devised to cover the insufficiencies. Using hydrogen peroxide and monoethyl hydrogen peroxide as substrate, respectively, two modified methods different from each other in their mechanisms were given for the purpose of demonstrating accurately the strength and localization of hydroperoxidase activity.