Microplate Superoxide Dismutase Assay Employing a Nonenzymatic Superoxide Generator

Publisher： Elsevier

ISSN： 0003-2697

Source： Analytical Biochemistry, Vol.232, Iss.2, 1995-12, pp. : 243-248

Disclaimer: Any content in publications that violate the sovereignty, the constitution or regulations of the PRC is not accepted or approved by CNPIEC.

Previous Menu Next

Abstract

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."