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 (O2.-) to hydrogen peroxide and molecular oxygen. SOD helps prevent tissue damage by O2.- 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 O2.- substrate and its free radical nature necessitate specialized analytical equipment to detect and measure O2.- chemically. Consequently, indirect SOD assays which monitor some change in an indicator substance reacting with O2.- are routinely used, particularly for biological samples. Limitations of indirect test systems utilizing heme-based indicators for the presence of O2.- and/or enzymatic O2.- generators led us to develop a SOD microassay based on spectrophotometric assessment of O2.- 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 O2.- 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 O2.- turnover by small-molecule "SOD mimetics."
Ewing
J F
JF
NitroMed, Inc., NitroMed Research Laboratories, Boston, Massachusetts 02118, USA.
Janero
D R
DR
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D002277:Carcinoma / Q000201:enzymology / Q000473:pathology; D002417:Cattle; D003124:Colorimetry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000145:classification; D008175:Lung Neoplasms / Q000201:enzymology / Q000473:pathology; D008297:Male; D008773:Methylphenazonium Methosulfate; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods*; D009243:NAD; D009363:Neoplasm Proteins / Q000032:analysis; D009580:Nitroblue Tetrazolium; D009928:Organ Specificity; D010084:Oxidation-Reduction; D011190:Potassium Cyanide / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013053:Spectrophotometry; D013482:Superoxide Dismutase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000145:classification; D013481:Superoxides / Q000138:chemical synthesis*; D014407:Tumor Cells, Cultured
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009363:Neoplasm Proteins; D009243:NAD; D013481:Superoxides; D009580:Nitroblue Tetrazolium; D008773:Methylphenazonium Methosulfate; D013482:Superoxide Dismutase; D011190:Potassium Cyanide