Cu,Zn-superoxide dismutase is an intracellular catalyst for the H(2)O(2)-dependent oxidation of dichlorodihydrofluorescein

• Published in: Molecules and cells 2006-02, Vol.21 (1), p.161
• Main Authors: Kim, Young-Mi, Lim, Jung-Mi, Kim, Byung-Chul, Han, Sanghwa
• Format: Article
• Language: English
• Subjects: Animals; Catalysis; Cattle; Copper - metabolism; Fluoresceins - metabolism; Gene Expression; Humans; Hydrogen Peroxide - metabolism; Hydrogen Peroxide - pharmacology; Keratinocytes - cytology; Keratinocytes - enzymology; Macrophages - cytology; Macrophages - enzymology; Mice; Oxidation-Reduction - drug effects; Peroxidase - metabolism; Superoxide Dismutase - metabolism; Zinc - metabolism
• ISSN: 1016-8478

Dichlorodihydrofluorescein (DCFH(2)) is a widely used probe for intracellular H(2)O(2). However, H(2)O(2) can oxidize DCFH(2) 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), Cu(2+), and Fe(3+) under various condi-tions to identify an intracellular catalyst. Enormous increase by bicarbonate in the rate of DCFH(2) oxidation distinguished CuZnSOD from cytochrome c and HRP. Cyanide inhibited the reaction catalyzed by CuZnSOD but accelerated that by Cu(2+) and Fe(3+). Oxidation of DCFH(2) by H(2)O(2) in the presence of a cell lys-ate was also enhanced by bicarbonate and inhibited by cyanide. Confocal microscopy of H(2)O(2)-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 H(2)O(2)-dependent oxidation of DCFH(2).