EVALUATION OF ACTIVITY OF PUTATIVE SUPEROXIDE-DISMUTASE MIMICS - DIRECT ANALYSIS BY STOPPED-FLOW KINETICS

By stopped-flow kinetic analysis, we have directly evaluated the super oxide dismutase (SOD) activity of a number of organic nitroxides and i ron- and manganese-based complexes that have been attributed with havi ng SOD activity based upon competition experiments with cytochrome c. In 60 mM HEPES buffer, pH 8.1, or 50 mM potassium phosphate buffer, pH 7.8, Mn(II) and manganese complexes of desferal had no detectable SOD activity by stopped-flow analysis (catalytic rate constant (k(cat) < 10(5.5) M-1 s-1), whereas Mn(II) and manganese complexes of desferal i nhibited the reduction of cytochrome c by superoxide generated by the xanthine/xanthine oxidase system. )-N,N',N'-tetrakis(2-pyridylmethyl)e thylenediamine (FeTPEN) was eight times more active than ris[N-(2-pyri dylmethyl)-2-aminoethyl]amine(FeTPAA) in the cytochrome c assay, but o nly FeTPAA catalyzed the first-order decay of superoxide (k(cat) = 2.1 5 x 10(6) M-1 s-1) by stopped-flow. Fe(III)-tetrakis(4-N-methylpyridyl )porphine ( FeTMPP) was active at low micromolar concentrations in bot h the cytochrome c and stopped-flow assays. At high micromolar concent rations, the organic nitroxides 2,2,6,6-tetramethylpiperidin-1-yloxy ( TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPOL) wer e inhibitory in the cytochrome c assay, but showed no detectable SOD a ctivity by stopped-flow. None of the tested compounds inhibited xanthi ne oxidase activity as shown by the measurement of urate production. U nder the conditions of the cytochrome c assay, FeTPEN, TEMPO, and TEMP OL oxidized reduced cytochrome c which rationalizes the false positive s for these compounds in this assay. The inhibitory activities of Mn(I I) and the manganese desferal complexes in the cytochrome c assay appe ar to be due to a stoichiometric, not catalytic, reaction with superox ide as catalytic amounts of these agents do not induce a first-order d ecay of superoxide as shown by stopped-flow.