Spectrophotometric assay for superoxide dismutase based on tetrazolium salt 3'--1--(phenylamino)-carbonyl--3, 4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate reduction by xanthine-xanthine oxidase.
XTT (3'-{1-[(phenylamino)-carbonyl]-3, 4-tetrazolium}bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate) was reduced to a water-soluble product with an absorbance maximum at about 470 nm by superoxide anion generated by xanthine-xanthine oxidase (XO). The rate of XTT reduction was linearly related to XO activity and the reduction was inhibited by superoxide dismutase (SOD). A perfect inhibition of the reduction of XTT by SOD was achieved, suggesting that XTT does not interact with XO. The present XTT-based assay had a higher sensitivity than a conventional nitroblue tetrazolium-based assay by a factor of 2.5 at pH 10.2. This method was applicable to the SOD assay in the pH range 8.0-10.2.
Ukeda
H
H
Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku, 783, Japan. hukeda@cc.kochi-u.ac.jp
Maeda
S
S
Ishii
T
T
Sawamura
M
M
D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000378:metabolism*; D010084:Oxidation-Reduction; D013482:Superoxide Dismutase / Q000032:analysis*; D013481:Superoxides / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000378:metabolism*
D007202:Indicators and Reagents; D013778:Tetrazolium Salts; D013481:Superoxides; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase