Reaction of cysteine residues with oxidized tyrosine residues mediates cross-linking of photo-oxidized casein proteins

Photo-oxidation of casein proteins is commonplace during milk processing and storage. A major consequence of such light exposure is protein cross-linking and aggregation. Although caseins are key milk components, the nature of the cross-links and the mechanisms involved are poorly characterized, with most previous work having been focused on detecting and quantifying di-tyrosine formed on dimerization of two tyrosine-derived phenoxyl radicals. However, this is only one of a large number of possible cross-links that might be formed. In this study, we have investigated the potential involvement of secondary reactions between oxidized protein side-chains and the thiol group of cysteine (Cys) residues in casein cross-linking. Casein proteins were subjected to photo oxidation using visible light in the presence of a sensitizer (riboflavin or rose Bengal) and O-2, then incubated with a Cys-containing peptide (glutathione, GSH) or protein (kappa-casein), and subsequently analyzed by SDS-PAGE, immunoblotting and LC-MS. Our data indicate that that photo-oxidized (but not parent) caseins react efficiently with the Cys-containing species, likely via Michael addition to quinones formed from tyrosine residues to give glutathionylated species or protein adducts. Thus, oxidized alpha-casein reacts with native kappa-casein to give high molecular mass aggregates. This adduct formation was prevented by alkylation of the Cys thiol group. The crosslink site and the residues involved have been confirmed by liquid chromatography-mass spectrometry (LC-MS) proteomic analysis. Together, these data extend our knowledge of the mechanisms involved in casein oxidation and aggregation.