Use of nitro blue tetrazolium in the reaction of adrenaline autooxidation for the determination of superoxide dismutase activity

Abstract

Addition of nitro blue tetrazolium (NBT) into the reaction of adrenaline autooxidation allows to identify directly superoxide anion formation (О · ) in this superoxide-generating system. The kinetics of formation of adrenochrome and О · were compared under the same conditions. Three possible approaches to the use the adrenaline autooxidation reaction for determining the activity of superoxide dismutase (SOD) and revealing the antioxidant properties of various compounds are discussed. Two of these approaches have been described previously: the spectrophotometric method of the registration of adrenochrome, the final product of adrenaline autoxidation, at 347 nm (Sirota, 1999) and the polarographic method, based on determination of oxygen consumption for О · formation (Sirota, 2011). Here, a novel approach to this problem is presented; it consists in the spectrophotometric determination of О · using NBT. The application of this approach enables one to lower the pH of carbonate buffer from 10.5 to 9.7 and to decrease (4-fold) the amount of added adrenaline, i.e., to generate milder and more effective conditions for revealing and studying the antioxidant properties of examined materials.