The tetrazolium dyes MTS and XTT provide new quantitative assays for superoxide and superoxide dismutase.
The tetrazolium dyes MTS and XTT were reduced to their soluble formazans by superoxide radical anions (O2-) produced by the oxidation of xanthine by xanthine oxidase under standard conditions. These reactions were compared to the well-known reductions of NBT and cytochrome c by the xanthine/xanthine oxidase system. Reduction of the dyes was completely inhibited by superoxide dismutase (SOD). Rate constants for the reaction of MTS and XTT with O2- were estimated at 1.3 +/- .1 x 10(5) M-1S-1 and 8.6 x 10(4) M-1S-1 respectively. The stable MTS and XTT formazans have high extinction coefficients in the visible range which enable sensitive detection and quantification of superoxide radicals, avoiding some of the problems inherent in assays based on production of the insoluble NBT formazan. MTS and XTT have considerable potential both for the quantitative assay of radical production in living tissues and for the assay of superoxide dismutase activity in tissue extracts. Implications for the interpretation of cell culture growth assays which employ these dyes are discussed.
Sutherland
M W
MW
Biology Department, Faculty of Sciences, University of Southern Queensland, Toowoomba, Australia. marksuth@usq.edu.au
Learmonth
B A
BA
D003574:Cytochrome c Group; D007202:Indicators and Reagents; D007700:Kinetics; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013482:Superoxide Dismutase / Q000032:analysis* / Q000378:metabolism; D013481:Superoxides / Q000032:analysis; D013778:Tetrazolium Salts; D013844:Thiazoles; D014969:Xanthine Oxidase / Q000494:pharmacology
D003574:Cytochrome c Group; D007202:Indicators and Reagents; D013778:Tetrazolium Salts; D013844:Thiazoles; D013481:Superoxides; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide; C070380:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase