ESR spin trapping investigation of radical formation from the reaction between hematin and tert-butyl hydroperoxide

Various mechanisms have been proposed for the reaction between heme proteins and organic hydroperoxides, including a peroxidase-type mechanism and homolytic cleavage. We used electron spin resonance (ESR) spectroscopy to investigate the formation of radicals in a hematin/tert-butyl hydroperoxide system. Spin trapping studies, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO), showed the formation of peroxyl and alkoxyl radicals in this system. At higher hematin concentrations an alkyl radical adduct could also be detected, which was identified as the methyl radical by using the spin trap 2-methyl-2-nitrosopropane. Furthermore, the relative contribution of the peroxyl and alkoxyl radical adducts was determined at various DMPO concentrations using computer simulation. It was found that at low DMPO concentrations both the alkoxyl and the peroxyl radical adducts could be detected. At higher DMPO concentrations, on the other hand, the alkoxyl radical dominated, whereas the peroxyl radical adduct decreased to a small portion of the total radical adduct population. Thus, the alkoxyl radical was the initial radical, produced by homolytic scission of the O—O bond of the hydroperoxide by ferric hematin. Although some tert-butyl peroxyl radicals could be detected by direct ESR, the majority of the peroxyl radicals detected by spin trapping were methyl peroxyl radicals, formed in the reaction between methyl radicals (formed by β-scission of the alkoxyl radicals) and oxygen.