Studies on the oxidative inactivation of enzymes

Abstract

The activity of ribonuclease after treatment with hydrogen peroxide in a peroxide/enzyme ratio of 55,000 was determined at pH 5.00. Very little loss of activity was found under these conditions. Using a modified Kunitz method, the slope obtained in the presence of H 2O 2 was −0.072 ± 0.014 compared with a control of −0.086 ± 0.016 (The absolute value of the slope is a measure of enzyme activity.) The presence of 3-aminotriazole caused little effect on the activity in the presence of peroxide (slope −0.080 ± 0.016). These data, together with other information in the literature, suggest that denaturation of the enzyme is a necessary first step for complete inactivation of the enzyme by peroxide. A similar situation may prevail in the case of catalase. 3-Aminotriazole, a known inhibitor of catalase, was found to be unreactive to a series of twelve amino acids. 3-Aminotriazole (3-AT), however, did react with the oxidized form of cystine and cysteine. 3-AT was also found to react strongly with basic hemin alone (especially in the presence of hydrogen peroxide) and in the presence of histidine and acetate. From these data and other information in the literature, a mechanism for the inactivation of catalase caused by 3-AT and H 2O 2 is suggested.