Oxidation of α- and β-Caseins Mediated by AAPH-Derived Free Radicals

Proteins are one of the most relevant components of bovine milk reaching concentrations close to 35 g/L. Amongst milk proteins, α- and β-caseins highlight for their high concentration, their functional properties and also for their nutritive value. It has been reported that as consequence of lipid-peroxidation, and also photo-oxidation processes, both kinds of caseins are oxidized generating carbonyl groups, tryptophan-related products and di-tyrosine bridges. The latter being implied in the formation of aggregates of β-casein during the photosensitization mediated by riboflavin. In the present work we studied the oxidative modifications on α- and β-caseins induced by free radicals derived from the thermolysis of AAPH (2,2′-Azobis(2-methylpropionamidine) dihydrochloride). SDS-PAGE results showed that, depending on the experimental conditions (AAPH concentrations; 10-100 mM) and the type of casein (α- or β-), fragments or aggregates can be formed. In particular, the exposition of α-casein (3 mg/mL) to AAPH (10 or 100 mM) at 37°C, triggered exclusively the formation of aggregates with high molecular weight. This result is in agreement with the presence of a high number (ten) of tyrosine residues in this protein. On the other hand, the kinetic of the β-casein (3 mg/mL) oxidation, mediated by the free radicals generated from AAPH (20 mM), revealed a continuous formation of two fragments of the protein along the time of the experiments (3 hours). This result was in agreement with the kinetic profile of tryptophan consumption as well as with the formation of N-formyl-kynurenine and kynurenine (both followed by fluorescence technique). By contrast, at a high AAPH concentration (100 mM) no fragments of β-casein were evidenced, the SDS-PAGE results showing only the formation of aggregates. This behavior was compatible with the formation of di-tyrosine as was evidenced by fluorescence spectroscopy. Acknowledgements This work was supported by FONDECYT (grant n°1141142) and CONICYT/Doctorado Nacional (grant n°21150130).