Measurements of Superoxide Anion Radical and Superoxide Anion Scavenging Activity by Electron Spin Resonance Spectroscopy Coupled with DMPO Spin Trapping

Abstract

Abstract A quantitative analysis of superoxide anion radical (·O2−) and hydroperoxyl radical (·OOH) generated in the hypoxanthine–xanthine oxidase (HPX–XOD) reaction system in the presence of dimethyl sulfoxide (DMSO) was explored by a spin-trapping method using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) combined with electron spin resonance spectroscopy (ESR). ·O2− and/or ·OOH was detected by ESR spectra of the spin adduct, DMPO–O2− (or DMPO–OOH). The concentration of DMPO–O2− was increased up to three times by the addition of DMSO. The half-life of DMPO–O2−, which is the time period to reduce to one-half of the initial intensity, also became about 70 times longer than that in the system without DMSO. These results suggest that the short half-life of DMPO–O2− that has been reported is attributable to the partial reaction of hydroxyl radical (·OH) with DMPO–O2−. Consequently quantitative analysis of ·O2− was possible in the presence of DMSO (>0.35 M). Under these conditions, kinetic approaches show that the generation of ·O2− in the HPX-XOD reaction is a first-order reaction and that its rate constant is 6.9 × 10−8 M s−1. Finally, the competitive reaction of DMPO and SOD toward ·O2− was shown to be one unit of superoxide dismutase (Cu/Zn– SOD) scavenging ·O2− by the rate constant of 7.0 × 10−6 M min−1. This method, which can be used for measurement of SOD-like and SOD-minic activity, should be called the superoxide anion scavenging activity method.