MEASUREMENTS OF SUPEROXIDE ANION-RADICAL AND SUPEROXIDE ANION SCAVENGING ACTIVITY BY ELECTRON-SPIN-RESONANCE SPECTROSCOPY COUPLED WITH DMPOSPIN-TRAPPING
M. Kohno et al., MEASUREMENTS OF SUPEROXIDE ANION-RADICAL AND SUPEROXIDE ANION SCAVENGING ACTIVITY BY ELECTRON-SPIN-RESONANCE SPECTROSCOPY COUPLED WITH DMPOSPIN-TRAPPING, Bulletin of the Chemical Society of Japan, 67(4), 1994, pp. 1085-1090
A quantitative analysis of superoxide anion radical (.O2-) and hydrope
roxyl radical (.OOH) generated in the hypoxanthine-xanthine oxidase (H
PX-XOD) reaction system in the presence of dimethyl sulfoxide (DMSO) w
as 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, DMP
O-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, als
o became about 70 times longer than that in the system without DMSO. T
hese results suggest that the short half-life of DMPO-O2- that has bee
n reported is attributable to the partial reaction of hydroxyl radical
(.OH) with DMPO-O2-. Consequently quantitative analysis of .O2- was p
ossible in the presence of DMSO (>0.35 M). Under these conditions, kin
etic approaches show that the generation of .O2- in the HPX-XOD reacti
on 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) scavengi
ng .O2- by the rate constant of 7.0 x 10(-6) M min-1. This method, whi
ch can be used for measurement of SOD-like and SOD-minic activity, sho
uld be called the superoxide anion scavenging activity method.