Cu,Zn-superoxide dismutase is an intracellular catalyst for the H2O2-dependent oxidation of dichlorodihydrofluorescein

초록

Dichlorodihydrofluorescein (DCFH2) is a widely used probe for intracellular H2O2, However, H2O2 can oxidize DCFH2 only in the presence of a catalyst, whose identity in cells has not been clearly defined. We compared the peroxidase activity of Cu,Zn-superoxide dismutase (CuZnSOD), cytochrome c, horseradish peroxidase (HRP), Cu2+, and Fe3+ under various conditions to identify an intracellular catalyst. Enormous increase by bicarbonate in the rate of DCFH2 oxidation distinguished CuZnSOD from cytochrome c and HRP. Cyanide inhibited the reaction catalyzed by CuZnSOD but accelerated that by Cu2+ and Fe3+. Oxidation of DCFH2 by H2O2 in the presence of a cell lysate was also enhanced by bicarbonate and inhibited by cyanide. Confocal microscopy of H2O2-treated cells showed enhanced DCF fluorescence in the presence of bicarbonate and attenuated fluorescence for the cells pre-incubated with KCN. Moreover, DCF fluorescence was intensified in CuZnSOD-transfected HaCaT and RAW 264.7 cells. We propose that CuZnSOD is a potential intracellular catalyst for the H2O2-dependent oxidation of DCFH2.

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