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

• Published in: Bulletin of the Chemical Society of Japan Vol. 67; no. 4; pp. 1085 - 1090
• Main Authors: Kohno, Masahiro, Mizuta, Yukio, Kusai, Masako, Masumizu, Toshiki, Makino, Keisuke
• Format: Journal Article
• Language: English
• Published: Tokyo The Chemical Society of Japan 01.04.1994; Chemical Society of Japan
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Oxidoreductases; Hyperoxides; Enzymatic activity; Enzyme; Hypoxanthine; Superoxide dismutase; Xanthine oxidase; Oxidoreductases; Radical scavenger; Biological activity; Free radical
• ISSN: 0009-2673; 1348-0634
• DOI: 10.1246/bcsj.67.1085

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.