Oxidative Dimerization of Proteins: Role of Tyrosine Accessibility

Abstract

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.