The SOD-like activity of several biological substances was evaluated by an ESR spin-trapping technique. Superoxide radicals (O2\ewdot) were supplied enzymatically from a hypoxanthine–xanthine oxidase reaction to the evaluating system. By using a spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), the generated O2\ewdot was trapped stoichiometrically (1:1) as the spin adduct of O2\ewdot (DMPO–O2−). When biological substances were added to the system, a decrease in the ESR signal intensities of the adducts was observed. This phenomenon could be explained as being an inhibition of adduct formation, and related to the reactivity of added biological substances with O2\ewdot, called an SOD-like activity. By the method of kinetic competition with a 50% inhibitory dose (ID50), the second-order rate constant for the reaction between O2\ewdot and biological substance was determined. These rate constants can be used as a measure of the reactivity.

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.

SUMMARYA quantitative analysis of hydroxyl radical (-OH) generated in the Fe 2+-hydrogen peroxide reaction system was explored by a spin-trapping method us!rig 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) combined with electron spin resonance spectroscopy. Based on the numerical analysis of Fentonrelated reactions, the reduction of DMPO-OH adduct from t:1 stoichiometry prominent at high concentrations of Fe 2+ was consistent with a reaction model in which a molar amount of hydrogen peroxide was reduced by two molar amounts of Fe 2+. Furthermore, time-dependent decrease in DMPO-OH tqhuantity, apparent at much higher concentration of Fe 2+, was proved due to e reaction not with Fe 2+ but with Fe 3+.

The primary propagating radicals in photo-initiated polymerization of isoprene and MMA were directly detected and assigned for the first time by use of time-resolved ESR spectroscopy. Based on the kinetic analysis including the spin-lattice relaxation, the rate constant of the formation and the relaxation time were determined for the primary propagating radicals by simulating time-profile of the FT-ESR intensity.

By employing EPR spectrometry with the aid of a spin-trapping agent, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), the generation of superoxide anion and hydroxyl radical was reevaluated during the respiratory burst of porcine and human neutrophils. Properly prepared resting neutrophils did not generate any spin-trapped radical, and, when the cells were stimulated with phorbol myristate acetate, only DMPO-OOH, the spin-trapped adduct of superoxide anion, was detected. No formation of DMPO-OH, the spin-trapped adduct of the hydroxyl radical, was observed. DMPO-OOH was also detected principally when the neutrophils were stimulated with opsonized zymosan, a particulate stimulus. In the latter case, however, the formation of DMPO-OOH ceased shortly after the addition of zymosan and subsequent production of DMPO-OH was observed. The production of DMPO-OH was found to be associated with cell injury. DMPO at the concentration usually used for the experiment (0.045-0.09 M) injured phagocytizing neutrophils, causing lysis of the cells. On the other hand, an addition of cell homogenate or glutathione-glutathione peroxidase system to the suspension of intact cells which were producing DMPO-OOH resulted in the formation of DMPO-OH. Thus, DMPO-OH was probably derived from DMPO-OOH by the action of enzymes and/or factor(s) which were released from the lysed cells.