Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical

Abstract

To enhance the sensitivity of EPR spin trapping for radicals of limited reactivity, high concentrations (10–100 mM) of spin traps are routinely used. We noted that in contrast to results with other hydroxyl radical detection systems, superoxide dismutase (SOD) often increased the amount of hydroxyl radical-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) produced by the reaction of hypoxanthine, xanthine oxidase and iron. One possible explanation for these results is that high DMPO concentrations (∼ 100mM) inhibit dismutation of superoxide (O 2−) to hydrigen peroxide (H 2O 2). Therefore, we examined the effect of DMPO on O 2− dismutation to H 2O 2. Lumazine ± 100mM DMPO was placed in a Clark oxygen electrode following which xanthine oxidase was added. The amount of H 2O 2 formed in this reaction was determined by introducing catalase and measuring the amount of dioxygen regenerated. Lumazine was used as the xanthine oxidase substrate to increase the percentage of H 2O 2 generated via O 2− dismutation as compared to direct divalent O 2 reduction. In the presence of 100 mM DMPO, H 2O 2 generation decreased 43%. DMPO did not scavenge H 2O 2 nor alter the rate of O 2− production. The effect of DMPO was concentration-dependent with inhibition of H 2O 2 production observed at [DMPO] > 10 mM. Inhibition of H 2O 2 production of DMPO was not observed if SOD was present or if the rate of O 2− formation increased. The spin trap 2-methyl-2-nitroso-propane (MNP, 10 mM) also inhibited H 2O 2 formation (81%). However, α-phenyl-N-tert-butylnitrone (PBN, 10 mM), 3,3,5,5 tetramethyl-1-pyrroline N-oxide (M 4PO, 100 mM), α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN, 100 mM) had no effect. These data suggest that in experimental systems in which the rate of O 2− generation is low, formation of H 2O 2 and thus other H 2O 2-derived species (e.g., OH) may be inhibited by commonly used concentrations of some spin traps. Thus, under some experimental conditions spin traps may potentially prevent production of the very free radical species they are being used to detect.