Oxidative modification of glutamine synthetase by 2,2 '-azobis(2-amidinopropane) dihydrochloride

In the present study, we examined the pattern of protein modification elici ted by alkylperoxyl radicals and alkylperoxides. To this end, we exposed gl utamine synthetase (GS) and the peptide melittin to solutions containing 2, 2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which is known to decom pose in aqueous, aerobic solutions to yield alkyl radicals and alkylperoxid es. Under our conditions, pH 7.4, 37 degrees C, the AAPH-dependent formatio n of alkylhydroperoxide increased linearly with time and led to 40% inactiv ation of GS in 1 h and to complete inactivation in 4 h. Complete inactivati on was associated with the loss of 2 of 16 histidine residues, 6 of 17 tyro sine residues, 5 of 16 methionine residues, and all of the tryptophan resid ues (2 residues) per subunit, Inactivation of GS was associated also with s ome protein fragmentation and the formation of some higher molecular weight aggregates. Exposure of GS to AAPH led also to the generation of protein c arbonyl derivatives (0.34 mol/mol subunit) and to formation of a significan t amount (0.038 mol/mol subunits) of quinoprotein derivatives. To investiga te the mechanism of tryptophan modification, the 26-amino-acid peptide, mel ittin, which contains one tryptophan but no histidine, tyrosine, or methion ine residues, was treated with AAPH, N-Formylkynurenine was identified as t he major product of tryptophan oxidation in melittin. (C) 1999 Academic Pre ss.