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.