NITRIC-OXIDE TRAPPING OF TYROSYL RADICALS GENERATED DURING PROSTAGLANDIN ENDOPEROXIDE SYNTHASE TURNOVER - DETECTION OF THE RADICAL DERIVATIVE OF TYROSINE-385

Tyrosyl radicals have been detected during turnover of prostaglandin e ndoperoxide H synthase (PGHS), and they are speculated to participate in cyclooxygenase catalysis, Spectroscopic approaches to elucidate the identity of the radicals have not been definitive, so we have attempt ed to trap the radical(s) with nitric oxide (NO), NO quenched the EPR signal generated by reaction of purified ram seminal vesicle PGHS with arachidonic acid, suggesting that NO coupled with a tyrosyl radical t o form inter alia nitrosocyclohexadienone, Subsequent formation of nit rotyrosine was detected by Western blotting of PGHS incubated with NO and arachidonic acid or organic hydroperoxides using an antibody again st nitrotyrosine. Both arachidonic acid and NO were required to form n itrotyrosine, and tyrosine nitration was blocked by the PGHS inhibitor indomethacin. The presence of superoxide dismutase had no effect on n itration, indicating that peroxynitrite was not the nitrating agent, T o identify which tyrosines were nitrated, PGHS was digested with tryps in, and the resulting peptides were separated by high pressure liquid chromatography and monitored with a diode array detector, A single pep tide was detected that exhibited a spectrum consistent with the presen ce of nitrotyrosine. Consistent with Western blotting results, both NO and arachidonic acid were required to observe nitration of this pepti de, and its formation was blocked by the PGHS inhibitor indomethacin, Peptide sequencing indicated that the modified residue was tyrosine 38 5, the source of the putative catalytically active tyrosyl radical.