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Cu,Zn-Superoxide Dismutase Is an Intracellular Catalyst for the H2O2-dependent Oxidation of Dichlorodihydrofluorescein  

Kim, Young-Mi (Department of Biochemistry, Kangwon National University)
Lim, Jung-Mi (Department of Biochemistry, Kangwon National University)
Kim, Byung-Chul (Department of Biochemistry, Kangwon National University)
Han, Sanghwa (Department of Biochemistry, Kangwon National University)
Publication Information
Molecules and Cells / v.21, no.1, 2006 , pp. 161-165 More about this Journal
Abstract
Dichlorodihydrofluorescein ($DCFH_2$) is a widely used probe for intracellular $H_2O_2$. However, $H_2O_2$ 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 conditions 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 $H_2O_2$ in the presence of a cell lysate was also enhanced by bicarbonate and inhibited by cyanide. Confocal microscopy of $H_2O_2$-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 $H_2O_2$-dependent oxidation of $DCFH_2$.
Keywords
Cu,Zn-Superoxide Dismutase; Dichlorodihydrofluorescein; Hydrogen Peroxide; Peroxidase;
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