Direct electron spin resonance detection of free radical intermediates during the peroxidase catalyzed oxidation of phenacetin metabolites.
The oxidation of the phenacetin metabolites p-phenetidine and acetaminophen by peroxidases was investigated. Free radical intermediates from both metabolites were detected using fast-flow ESR spectroscopy. Oxidation of acetaminophen with either lactoperoxidase and hydrogen peroxide or horseradish peroxidase and hydrogen peroxide resulted in the formation of the N-acetyl-4-aminophenoxyl free radical. Totally resolved spectra were obtained and completely analyzed. The radical concentration was dependent on the square root of the enzyme concentration, indicating second-order decay of the radical, as is consistent with its dimerization or disproportionation. The horseradish peroxidase/hydrogen peroxide-catalyzed oxidation of p-phenetidine (4-ethoxyaniline) at pH 7.5-8.5 resulted in the one-electron oxidation products, the 4-ethoxyaniline cation free radical. The ESR spectra were well resolved and could be unambiguously assigned. Again, the enzyme dependence of the radical concentration indicated a second-order decay. The ESR spectrum of the conjugate base of the 4-ethoxyaniline cation radical, the neutral 4-ethoxyphenazyl free radical, was obtained at pH 11-12 by the oxidation of p-phenetidine with potassium permanganate.
D000082:Acetaminophen / Q000378:metabolism*; D000627:Aminophenols / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D066298:In Vitro Techniques; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D010615:Phenacetin / Q000378:metabolism*; D010628:Phenetidine / Q000378:metabolism*
D000627:Aminophenols; D005609:Free Radicals; D010628:Phenetidine; D000082:Acetaminophen; D010544:Peroxidases; D010615:Phenacetin