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By employing electron spin resonance spectroscopy, we examined the free radicals scavenging effects of hepatic metallothionein (MT) isoforms I and II (MTs-I and II) on four types of free radicals. Solutions of 0.15 mM of MT-I and 0.3 mM of MT-II were found to scavenge the 1,1-diphenyl-2-picrylhydrazyl radicals (1.30 x 10(15) spins/ml) completely. In addition, both isoforms exhibited total scavenging action against the hydroxyl radicals (1.75 x 10(15) spins/ml) generated in a Fenton reaction. Similarly, 0.3 mM of MT-I scavenged almost 90% of the superoxide (2.22 x 10(15) spins/ml) generated by the hypoxanthine and xanthine oxidase system, while a 0.3 mM MT-II solution could only scavenge 40% of it. By using 2,2,6,6-tetramethyl-4-piperidone as a "spin-trap" for the reactive oxygen species (containing singlet oxygen, superoxide and hydroxyl radicals) generated by photosensitized oxidation of riboflavin and measuring the relative signal intensities of the resulting stable nitroxide adduct, 2,2,6,6-tetramethyl-4-piperidine-1-oxyl, we observed that MT-II (0.3 mM) could scavenge 92%, while MT-I at 0.15 mM microl/ml concentrations could completely scavenge all the reactive species (2.15 x 10(15) spins/ml) generated. The results of these studies suggest that although both isoforms of MT are able to scavenge free radicals, the MT-I appears to be a superior scavenger of superoxide and 1,1 diphenyl-2-picrylhydrazyl radicals.

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.

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