Oxidative Dimerization of Proteins: Role of Tyrosine Accessibility

Purpose. To investigate the importance of two possible mechanisms of tyrosine oxidation on the yield of protein dimerization. The model chosen is hen and turkey egg-white lysozymes, which differ by seven amino acids, among which one tyrosine is in the 3 position. Materials and methods. Aqueous solutions of proteins were oxidized by OH• or N•3 free radicals produced by γ or pulse irradiation in an atmosphere of N2O. Protein dimers were quantified by SDS–PAGE and reverse-phase HPLC. Dityrosines were identified by absorption and fluorescence. Results. Using N•3 free radicals, the initial yields of dimerization are equal to (8.6 ± 0.7) × 10−9 mol J−1 for both proteins. Using OH• free radicals, they become equal to (1.23 ± 0.1) × 10−8 and (4.42 ± 0.1) × 10−8 mol J−1 for hen and turkey egg-white lysozymes, respectively (γ radiolysis). Discussion. N•3 radicals react primarily with tryptophan residues only. Tyrosine gets oxidized by intramolecular long-range electron migration, whereas OH• may react directly with tyrosines. We propose a low participation of Tyr3 in turkey protein in the intramolecular process, because Tyr3 is far from all tryptophans. On the other hand, Tyr3 is very accessible to solvent and in a flexible area; thus collisions with OH• could easily be followed by intermolecular dimerization.