Spin trapping study of superoxide production in ferrous ion oxidation

The oxidation of Fe2+ was investigated by electron paramagnetic resonance (EPR) spin trapping techniques with N-t-butyl-alpha-phenylnitrone (PBN) and dimethyl sulfoxide. Under pure oxygen, the spin adduct PBN/.OCH3 was rapidly generated by the addition of Fe2+ (0.2-1.2 mM) into phosphate buffer containing ethylenediaminetetraacetate (EDTA), dimethyl sulfoxide and PBN at pH 7.4, but it decayed. The decay process of PBN/.OCH3 consists of two components. The fast decay was dependent on Fe2+ concentration. Another was due to destruction of the spin adduct by superoxide anion (.O2-), because superoxide dismutase (SOD) markedly prevented the decay. Catalase decreased the yield of PBN/.OCH3. When Fe(3+)-EDTA and ascorbate were used instead of Fe(2+)-EDTA, similar phenomena were detected. These results demonstrate that Fe2+ reacts with O2 to generate .O2-, then H2O2, which produces .CH3 by the reaction with Fe2+ and dimethyl sulfoxide. The .OCH3 radical results from the reaction between .CH3 and O2. The adduct PBN/.OCH3 decays by the reaction with Fe2+ and .O2-.