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

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), cytochr...

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Bibliographic Details
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
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Summary: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).
ISSN:1016-8478