Oxidation of spin trap 5,5-dimethyl-1-pyrroline-1-oxide in an electron paramagnetic resonance study of the reaction of methemoglobin with hydrogen peroxide.

The possibility that methemoglobin (metHb) may function as a biological Fenton reagent to produce hydroxyl radical from hydrogen peroxide is investigated by electron paramagnetic resonance (EPR) spin-trapping techniques. The spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gives a nine-line EPR spectrum and no hydroxyl radical or superoxide spin adduct signals for the metHb/H2O2 system. From the known hyperfine splitting constants, the spectrum is assigned to 5,5-dimethylpyrrolidone-2(2)-oxyl-(1) (DMPOX), an oxidized derivative of DMPO. The likely involvement of the peroxidase activity of metHb in this reaction is suggested by the oxidation of DMPO to DMPOX by horseradish peroxidase as well. Furthermore, peroxidase inhibitors prevent the formation of DMPOX. Spectrophotometric assays confirm the peroxidase activity of metHb toward typical phenolic and nonphenolic substrates under the conditions used for the EPR experiments. The visible absorption spectra indicate the formation of a ferrylHb intermediate and its reduction by DMPO. Glutathione and ascorbic acid compete with DMPO as electron donors in the reaction to form thiyl and ascorbate radicals. Neither hydroxyl radical nor any other signal is observed when N-tert-butyl-alpha-phenylnitrone (PBN) is used as the spin trap in the metHb/H2O2 system. It is concluded that methemoglobin-bound iron may not catalyze the Fenton reaction forming hydroxyl radical, but can oxidize a variety of substrates, including DMPO, in a peroxidase-type reaction.