Assay of Superoxide Dismutase: Cautions Relevant to the Use of Cytochrome c, a Sulfonated Tetrazolium, and Cyanide
✍ Scribed by Ayako Okado-Matsumoto; Irwin Fridovich
- Publisher
- Elsevier Science
- Year
- 2001
- Tongue
- English
- Weight
- 226 KB
- Volume
- 298
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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-4nitro-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 reduceability 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.