Flow-injection assay of superoxide dismutase based on the reduction of highly water-soluble tetrazolium

A flow-injection analysis (FIA) for superoxide dismutase (SOD) activity was developed based on the use of tetrazolium salt, WST-1 (4-[3-(4-iodophenyl) -2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt) an d an enzyme reactor packed with Sepharose 4B on which xanthine oxidase (XO) and catalase were co-immobilized. WST-1 is highly water-soluble, and no ad hesion of the reduced form to the FIA line was observed during continuous o peration for 3 months. As optimized conditions, a sample (9 vol) was mixed with a reagent solution (1 vol) containing 3 mM hypoxanthine and 2 mM WST-1 , and the mixed solution (20 mu l) was injected into a carrier stream of 50 mM carbonate buffer (pH 10.2) at a flow rate of 0.4 ml/min. Under the cond itions, the concentration of the SOD preparation giving 50% inhibition (IC5 0) was 2.7 mu g/ml and the sampling frequency was 30 samples/h. When the SO D activity in erythrocytes of rats was determined by the present FIA method , the values were linearly related to those obtained by the conventional ni troblue tetrazolium (NBT) assay (r=0.975; n=18). The enzyme reactor was sta ble for at least 200 repetitive injections.