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, then incubated with a Cys-containing peptide (glutathione, GSH) or protein (κ-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 α-casein reacts with native κ-casein to give high molecular mass aggregates. This adduct formation was prevented by alkylation of the Cys thiol group. The cross-link 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.