Flow-Injection Assay of Superoxide Dismutase Based on the Reduction of Highly Water-Soluble Tetrazolium

Abstract

Hiroyuki UKEDA, Ashok K. SARKER, Daisuke KAWANA and Masayoshi SAWAMURA Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku 783–8502, Japan 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) and 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 adhesion of thereduced form to the FIA line was observed during continuous operation for 3 months. As optimized conditions, a sample (9 vol) was mixed with areagent solution (1 vol) containing 3 mM hypoxanthine and 2 mM WST-1, and the mixed solution (20 μl) 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 conditions, the concentration of the SOD preparation giving 50% inhibition (IC50) was 2.7 μg/ml and the sampling frequency was 30 samples/h. When the SOD activity in erythrocytes of rats was determined by the present FIA method, the values were linearlyrelated to those obtained by the conventional nitroblue tetrazolium (NBT) assay (r=0.975; n=18). The enzymereactor was stable for at least 200repetitive injections