Microplate superoxide dismutase assay employing a nonenzymatic superoxide generator

The antioxidant enzyme superoxide dismutase (EC 1.15.1.1) (SOD) catalyzes the conversion of superoxide anion radical (O-2(.-)) to hydrogen peroxide and molecular oxygen. SOD helps prevent tissue damage by O-2(.-) and its metabolites, and augmentation of tissue SOD is a useful therapeutic strategy in certain diseases having an oxidative-injury component. Routine application of direct SOD assays is not technically facile, since the short half-life of the O-2(.-) substrate and its free radical nature necessitate specialized analytical equipment to detect and measure O-2(.-) chemically. Consequently, indirect SOD assays which monitor some change in an indicator substance reacting with O-2(.-) are routinely used, particularly for biological samples. Limitations of indirect test systems utilizing heme-based indicators for the presence of O-2(.-) and/or enzymatic O-2(.-) generators led us to develop a SOD microassay based on spectrophotometric assessment of O-2(.-)-mediated nitro blue tetrazolium reduction by an aerobic mixture of NADH and phenazine methosulfate, which produces superoxide chemically at nonacidic pH (Rao, Free Radical Biol. Med. 7, 513-519, 1989). The proposed SOD assay system is formatted for use in an automated 96-well microplate reader and has the virtues of a nonheme indicator, a nonenzymatic O-2(.-) source, physiological pH, and economy of time and materials. The assay has been applied to measure purified and tissue SOD (Cu,Zn- and Mn-types) activity as well as O-2(.-) turnover by small-molecule ''SOD mimetics.'' (C) 1995 Academic Press, Inc.