Measurement of Catalase Activity Using Catalase Inhibitors

Abstract

Catalase is an enzyme that scavenges hydrogen peroxide in the body and has the role of protecting the organism from oxidative stress. Since catalase activity is associated with various diseases, including diabetes, skin diseases like vitiligo, renal failure, and heart failure, it is important to measure its activity. However, it has been difficult to accurately evaluate catalase activity alone, because there are other substances in vivo , such as iron ions, that decompose hydrogen peroxide in addition to catalase. To solve this problem, we conducted a study to develop a method to correctly measure catalase activity from samples containing impurities with hydrogen peroxide removal activity. In this study, catalase inhibitors were added to bovine catalase solution, ferric chloride solution, cell lysates of control cells and experimentally generated catalase knockdown cells (CAT KD), and these mixtures were reacted with hydrogen peroxide to determine the percentage of hydrogen peroxide remaining in the reaction solution after a certain time. The catalase inhibitors used, 3-amino-1H-1,2,4-triazole (3-AT) and sodium azide (NaN 3 ), inhibited the removal of hydrogen peroxide by bovine catalase at a high rate in in - vitro experiments. However, these catalase inhibitors did not inhibit hydrogen peroxide removal in the Fenton reaction of iron ion and hydrogen peroxide in in - vitro experiments. On the other hand, hydrogen peroxide removal by cell lysate was inhibited by the addition of 3-AT or NaN 3 . The inhibitory effect was equivalent or superior to that of CAT KD cells, in which catalase was experimentally knocked down. These results suggested that 3-AT and NaN 3 specifically inhibit hydrogen peroxide removal of catalase. Through these studies, we found that when cell lysate with a catalase inhibitor was mixed with hydrogen peroxide, hydrogen peroxide that was not removed by catalase inhibition remained in the test tube after a certain time, and this residual hydrogen peroxide reflected the hydrogen peroxide removal activity of catalase. By measuring this unremoved hydrogen peroxide, it was possible to evaluate catalase activity from samples containing impurities that have hydrogen peroxide removal properties.