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 biologica l Fenton reagent to produce hydroxyl radical from hydrogen peroxide is investigated by electron paramagnetic resonance (EPR) spin-trapping t echniques. The spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gives a nine-line EPR spectrum and no hydroxyl radical or superoxide spin a dduct signals for the metHb/H2O2 system. From the known hyperfine spli tting constants, the spectrum is assigned to 5,5-dimethylpyrrolidone-2 (2)-oxyl-(1) (DMPOX), an oxidized derivative of DMPO. The likely invol vement of the peroxidase activity of metHb in this reaction is suggest ed by the oxidation of DMPO to DMPOX by horseradish peroxidase as well . Furthermore, peroxidase inhibitors prevent the formation of DMPOX. S pectrophotometric assays confirm the peroxidase activity of metHb towa rd typical phenolic and nonphenolic substrates under the conditions us ed for the EPR experiments. The visible absorption spectra indicate th e formation of a ferrylHb intermediate and its reduction by DMPO. Glut athione and ascorbic acid compete with DMPO as electron donors in the reaction to form thiyl and ascorbate radicals. Neither hydroxyl radica l nor any other signal is observed when N-tert-butyl-alpha-phenylnitro ne (PBN) is used as the spin trap in the metHb/H2O2 system. It is conc luded that methemoglobin-bound iron may not catalyze the Fenton reacti on forming hydroxyl radical, but can oxidize a variety of substrates, including DMPO, in a peroxidase-type reaction.