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), 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).
Kim
Young-Mi
YM
Department of Biochemistry, Kangwon National University, Chunchon 200-701, Korea.
Lim
Jung-Mi
JM
Kim
Byung-Chul
BC
Han
Sanghwa
S
D000818:Animals; D002384:Catalysis; D002417:Cattle; D003300:Copper / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology; D008264:Macrophages / Q000166:cytology / Q000201:enzymology; D051379:Mice; D010084:Oxidation-Reduction / Q000187:drug effects; D009195:Peroxidase / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D005452:Fluoresceins; C065013:2',7'-dichlorodihydrofluorescein; D003300:Copper; D006861:Hydrogen Peroxide; D009195:Peroxidase; D013482:Superoxide Dismutase; D015032:Zinc