Effect of Oxidation and Protein Unfolding on Cross-Linking of ?-Lactoglobulin and ?-Lactalbumin

Oxidation and heat treatment can initiate changes in the amino acid composition, structure, solubility, hydrophobicity, conformation, function, and susceptibility to proteolysis of proteins. These can result in adverse consequences for mammals, plants, foodstuffs, and pharmaceuticals. This study investigated whether and how individual or combined treatment with heat, a commonly encountered factor in industrial processing, and HO alters the structure and composition of two major milk whey proteins, ?-lactalbumin and ?-lactoglobulin, and mixtures of these. Thermal treatment induced reducible cross-links in isolated ?-lactoglobulin, but not isolated ?-lactalbumin under the conditions employed. Cross-linking occurred at lower temperatures and to a greater extent in the presence of low concentrations of HO. HO did not induce cross-linking in the absence of heat. Mixtures of ?-lactalbumin and ?-lactoglobulin showed similar behavior, except that mixed ?-lactalbumin-?-lactoglobulin dimers were detected. Cross-linking was associated with formation of sulfenic acids (RS-OH species), oxidation of methionine residues, cleavage of disulfide bonds in ?-lactalbumin, altered conformation of disulfide bonds in ?-lactoglobulin, alterations in the fluorescence intensity and maximum emission wavelength of endogenous tryptophan residues, and binding of the hydrophobic probe 8-anilinonaphthalenesulfonate. These data are consistent with increased unfolding and subsequent aggregation of the protein, with these changes being maximized in the presence of both heat and HO. The enhanced aggregation detected with HO is consistent with additional pathways to aggregation above that induced by heat alone. These mechanistic insights provide potential strategies for modulating the extent and nature of protein modification induced by thermal and oxidant treatment.