Kinetic studies on spin trapping of superoxide and hydroxyl radicals generated in NADPH-cytochrome P-450 reductase-paraquat systems. Effect of iron chelates.
Electron spin resonance (ESR) studies on spin trapping of superoxide and hydroxyl radicals by 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were performed in NADPH-cytochrome P-450 reductase-paraquat systems at pH 7.4. Spin adduct concentrations were determined by comparing ESR spectra of the adducts with the ESR spectrum of a stable radical solution. Kinetic analysis in the presence of 100 microM desferrioxamine B (deferoxamine) showed that: 1) the oxidation of 1 mol of NADPH produces 2 mol of superoxide ions, all of which can be trapped by DMPO when extrapolated to infinite concentration; 2) the rate constant for the reaction of superoxide with DMPO was 1.2 M-1 s-1; 3) the superoxide spin adduct of DMPO (DMPO-OOH) decays with a half-life of 66 s and the maximum level of DMPO-OOH formed can be calculated by a simple steady state equation; and 4) 2.8% or less of the DMPO-OOH decay occurs through a reaction producing hydroxyl radicals. In the presence of 100 microM EDTA, 5 microM Fe(III) ions nearly completely inhibited the formation of the hydroxyl radical adduct of DMPO (DMPO-OH) as well as the formation of DMPO-OOH and, when 100 microM hydrogen peroxide was present, produced DMPO-OH exclusively. Fe(III)-EDTA is reduced by superoxide and the competition of superoxide and hydrogen peroxide in the reaction with Fe(II)-EDTA seems to be reflected in the amounts of DMPO-OOH and DMPO-OH detected. These effects of EDTA can be explained from known kinetic data including a rate constant of 6 x 10(4) M-1 s-1 for reduction of DMPO-OOH by Fe(II)-EDTA. The effect of diethylenetriamine pentaacetic acid (DETAPAC) on the formation of DMPO-OOH and DMPO-OH was between deferoxamine and EDTA, and about the same as that of endogenous chelator (phosphate).
Yamazaki
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Department of Chemistry and Biochemistry, Utah State University, Logon 84322-0300.
Piette
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Grover
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D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003579:Cytochrome Reductases / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006878:Hydroxides / Q000378:metabolism*; D017665:Hydroxyl Radical; D007502:Iron Chelating Agents; D007700:Kinetics; D009249:NADP / Q000378:metabolism*; D010269:Paraquat / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*
D005609:Free Radicals; D006878:Hydroxides; D007502:Iron Chelating Agents; D013481:Superoxides; D017665:Hydroxyl Radical; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D003579:Cytochrome Reductases; D010269:Paraquat