Detection of free radicals produced from the reaction of cytochrome P-450 with linoleic acid hydroperoxide

The ESR spin-trapping technique was employed to investigate the reaction of rabbit cytochrome P-450 1A2 (P450) with linoleic acid hydroperoxide. This system was compared with chemical systems where FeSO4 or FeCl3 was used in place of P450. The spin trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) Was employed to detect and identify radical species. The DMPO adducts of hydroxyl, O-2(-.), peroxyl, methyl and acyl radicals were detected in the P450 system. The reaction did not require NADPH-cytochrome P-450 reductase or NADPH. The same DMPO-radical adducts were detected in the FeSO4 system. Only DMPO-(OH)-O-. radical adduct and carbon-centred radical adducts were detected in the FeCl3 system. Peroxyl radical production was completely O-2-dependent. We propose that polyunsaturated fatty acids are initially reduced to form alkoxyl radicals, which then undergo intramolecular rearrangement to form epoxyalkyl radicals. Each epoxyalkyl radical reacts with O-2, forming a peroxyl radical. Subsequent unimolecular decomposition of this peroxyl radical eliminates O-2(-.) radical.