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

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.

Interfacial mechanisms of heterogeneous Fenton reactions catalyzed by iron-based materials: A review

“…Some rate constants of O 2U − with selected substances are listed in Table 2. In free radical-dominated chain reactions, organic compounds are initially transformed to organic radicals by free radicals, and (Kohno et al, 1994) or in organic-rich media . On one hand, the generation rate of DMPO-OOH/ DMPO-O 2 − adducts is lower than that of DMPO-OH adduct.…”

“…Alternatively, DMPO-OOH/DMPO-O 2 − adducts readily decompose to DMPO-OH adduct in aqueous medium (Kohno et al, 1994;Zhao et al, 1998). Hence, the interference from the co-existing UOH in solution should be eliminated before HO 2U /O 2U − detection.…”

Characterization of Superoxide Anion Scavenging Compounds in Roselle (Hibiscus sabdariffa L.) Extract by Electron Spin Resonance and LC/MS

“…As reported by Kohno et al (1994), DMSO reacts selectively with hydroxyl radical that may compete with the formation of superoxide anion-DMPO adduct. Therefore, it was suggested to add DMSO in order to suppress the side reaction and increase selectivity and sensitivity.…”

Electron Spin Resonance Analysis of Superoxide Anion Radical Scavenging Activity with Spin Trapping Agent, Diphenyl-PMPO

“…This technique has been verified to be useful to detect the short live superoxide anion radical at room temperature, and in fact it has been widely used for the investigations of the SOD activity and the SOSA of natural antioxidants. 4,5 There are many spin-trapping agents for superoxide anion radical, including 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), and α-phenyl-N-tert-butylnitrone (PBN). 6 Although DMPO is the most common agent in the ESR analysis, it has some limitations.…”