Hydrogen peroxide, which has a relatively long half-life among reactive oxygen species generated in the body, is mainly removed by catalase and other enzymes. Currently, catalase activity is generally measured by adding hydrogen peroxide to cell lysate (or tissue lysate) and measuring the amount of hydrogen peroxide remaining after a certain time. However, since hydrogen peroxide removal factors other than catalase such as glutathione peroxidase, peroxiredoxin, and Fe2+ are present in the cell lysate, it is difficult to say that the enzyme activity (=catalase activity) is accurately measured. Therefore, in order to evaluate hydrogen peroxide removal enzyme activity in living cells, we selected culture medium that does not affect intracellular hydrogen peroxide removal, treatment protocol for enzyme inhibitors that do not affect hydrogen peroxide detection, and combinations of various hydrogen peroxide removal enzyme inhibitors to evaluate each enzyme separately, and we developed a method to evaluate enzyme activity in detail, which cannot be determined by measuring cell lysate. The results suggest that the activity of various hydrogen peroxide removal enzymes in living cells can be evaluated in more detail than with conventional methods.
