Peroxyl Radical-Scavenging Activity of Plasma Determined by Electron Paramagnetic Resonance.

Abstract

To determine the peroxyl radical-scavenging activity of several biological substances, we developed a spin trapping assay using electron paramagnetic resonance (EPR). Peroxyl radicals, generated in a mixture of tert-butyl hydroperoxide and methemoglobin in sodium phosphate buffer, were trapped by 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO). Scavenging activities were calculated from DMPO-OOR (tert-butyl peroxyl radical spin adduct) signal by a kinetic competition study. The scavenging activity, R, of a given substance was expressed as the relative second-order rate constant of the reaction with peroxyl radical as compared with that obtained for DMPO. Of the various biological substances evaluated, ascorbic acid (R=227), uric acid (R=28), glutathione (R=24), and α-tocopherol (R=14) were major peroxyl radical scavengers in phosphate buffer. This method will be useful for determining peroxyl radical-scavenging activities of biological materials.