Assay of superoxide dismutase: cautions relevant to the use of cytochrome c, a sulfonated tetrazolium, and cyanide.
Two commonly used assays for superoxide dismutase (SOD) activity have been compared, one using cytochrome c and the other using XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) as the indicating scavenger of superoxide. The use of cyanide to selectively suppress Cu,Zn-SOD and thus to allow assay of both Cu,Zn-SOD and Mn-SOD in mixtures of the two was also explored, as was the influence of pH. The XTT assay became more sensitive at elevated pH, because the rate of the superoxide/XTT reaction declines with increasing pH. This was clearly seen with the Cu,Zn-SOD but barely with Mn-SOD because the former retains full activity from pH 5 to 10 while the latter does not. Cyanide reacted with cytochrome c, but not XTT, in a concentration- and time-dependent manner and thus diminished its reducibility by superoxide. Cytochromes endogenous to tissue fractions were reduced by the xanthine oxidase reaction and this caused a decrease in absorbance 470 nm which interfered with the XTT assay. The alkalinizing effect of cyanide salts and the problems encountered in neutralizing cyanide stock solutions are discussed.
Okado-Matsumoto
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Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Fridovich
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D003486:Cyanides / Q000494:pharmacology*; D003574:Cytochrome c Group / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism*; D007700:Kinetics; D013482:Superoxide Dismutase / Q000032:analysis*; D013778:Tetrazolium Salts / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000494:pharmacology
D003486:Cyanides; D003574:Cytochrome c Group; D007202:Indicators and Reagents; D013778:Tetrazolium Salts; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase