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 s cavenger of superoxide. The use of cyanide to selectively suppress Cu,Zn-SO D and thus to allow assay of both Cu,Zn-SOD and Mn-SOD in mixtures of the t wo 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 b arely 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 reduc eability by superoxide. Cytochromes endogenous to tissue fractions were red uced by the xanthine oxidase reaction and this caused a decrease in absorba nce 470 nm which interfered with the XTT assay. The alkalinizing effect of cyanide salts and the problems encountered in neutralizing cyanide stock so lutions are discussed. (C) 2001 Academic Press.