MEASUREMENTS OF SUPEROXIDE ANION-RADICAL AND SUPEROXIDE ANION SCAVENGING ACTIVITY BY ELECTRON-SPIN-RESONANCE SPECTROSCOPY COUPLED WITH DMPO SPIN-TRAPPING

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 x 10(-8) Ms-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 x 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.