Aggregation of α- and β- caseins induced by peroxyl radicals involves secondary reactions of carbonyl compounds as well as di-tyrosine and di-tryptophan formation

Abstract

The present work examined the role of Tyr and Trp in oxidative modifications of caseins, the most abundant milk proteins, induced by peroxyl radicals (ROO^•). We hypothesized that the selectivity of ROO^• and the high flexibility of caseins (implying a high exposure of Tyr and Trp residues) would favor radical-radical reactions, and di-tyrosine (di-Tyr) and di-tryptophan (di-Trp) formation. Solutions of α- and β-caseins were exposed to ROO^• from thermolysis and photolysis of AAPH (2,2'-azobis(2-methylpropionamidine)dihydrochloride). Oxidative modifications were examined using electrophoresis, western blotting, fluorescence, and chromatographic methodologies with diode array, fluorescence and mass detection. Exposure of caseins to AAPH at 37 °C gave fragmentation, cross-linking and protein aggregation. Amino acid analysis showed consumption of Trp, Tyr, Met, His and Lys residues. Quantification of Trp and Tyr products, showed low levels of di-Tyr and di-Trp, together with an accumulation of carbonyls indicating that casein aggregation is, at least partly, associated with secondary reactions between carbonyls and Lys and His residues. AAPH photolysis, which generates a high flux of free radicals increased the extent of formation of di-Tyr in both model peptides and α- and β- caseins; di-Trp was only detected in peptides and α-casein. Thus, in spite of the high flexibility of caseins, which would be expected to favor radical-radical reactions, the low flux of ROO^• generated during AAPH thermolysis disfavours the formation of dimeric radical-radical cross-links such as di-Tyr and di-Trp, instead favoring other O₂-dependent crosslinking pathways such as those involving secondary reactions of initial carbonyl products.

Keywords: Caseins; Di-tryptophan; Di-tyrosine; Peroxyl radicals; Protein carbonyls; Protein crosslinking; Protein fragmentation; Tryptophan; Tyrosine.