Oxidative dimerization of proteins: Role of tyrosine accessibility

Purpose. To investigate the importance of two possible mechanisms of tyrosi ne oxidation on the yield of protein dimerization, The model chosen is hen and turkey egg-white lysozymes, which differ by seven amino acids, among wh ich one tyrosine is in the 3 position, Materials and methods. Aqueous solut ions of proteins were oxidized by OH. or N-3(.) free radicals produced by g amma or pulse irradiation in an atmosphere of N2O. Protein dimers were quan tified by SDS-PAGE and reverse-phase HPLC, Dityrosines were identified by a bsorption and fluorescence. Results. Using N-3(.) free radicals, the initia l yields of dimerization are equal to (8.6 +/- 0.7) x 10(-9) mol J(-1) for both proteins. Using OH. free radicals, they become equal to (1.23 +/- 0.1) x 10(-8) and (4.42 +/- 0.1) x 10(-8) mol J(-1) for hen and turkey egg-whit e lysozymes, respectively (gamma radiolysis), Discussion. N-3(.) radicals r eact primarily with tryptophan residues only, Tyrosine gets oxidized by int ramolecular long-range electron migration, whereas OH. may react directly w ith tyrosines, We propose a low participation of Tyr3 in turkey protein in the intramolecular process, because Tyr3 is far from all tryptophans, On th e other hand, Tyr3 is very accessible to solvent and in a flexible area; th us collisions with OH. could easily be followed by intermolecular dimerizat ion, (C) 2000 Academic Press.