Source of superoxide anion radical in aerobic mixtures consisting of NAD[P]H, 5-methylphenazinium methyl sulfate and nitroblue tetrazolium chloride

The source of superoxide anion radical (O 2 ⪰) in aerobic mixtures consisting of NAD[P]H, 5-methylphenazinium methyl sulfate (or its 1-methoxy derivative) and tetrazolium salt was investigated using superoxide dismutase (SOD), Mn(II), ferricytochrome-C, and epinephrine as probes. NAD[P]H + phenazine + O 2 was found to reduce nitroblue tetrazolium, iodonitrotetrazolium, and thiazolyl blue in a manner sensitive to agents that dismutate O 2 ⪰, iviz., SOD and Mc(II). It also mediated the reduction of ferricytochrome-C, and augmented the autooxidation of epinephrine to the adrenochrome, without a tetrazolium salt present in the medium. The autooxidation ofepinephrine, but not the reduction of ferricytochrome-C, was found to be sensitive to SOD. Nitroblue tetrazolium, either singly or in combination with SOD, did not stimulate the reduction of ferricytochrome-C. The oxidation of NADH, mediated by a catalytically low concentration of phenazine(+O 2) was augmented two-fold by SOD. These observations are consistent with, and lend support to a scheme of redox events (Scheme-3) wherein it is proposed that the source of O 2 ⪰ in the NAD[P]H + phenazine + tetrazolium(+O 2) system is the reduced phenazine, that the tetrazoinyl radical (a one-electron reduction product of tetrazolium) may not reduce O 2 to O 2 ⪰, that the redox reaction between semiquinone radicals of phenazine and O 2 is reversible, and that the disproportionation of semiquinone radicals constitutes an important rate-limiting reaction in the expression of phenazine redox couple.