Modifications of high-order structures upon heating of β-Lactoglobulin : dependence on the protein concentration

A study on the concentration dependence of the modifications ensuing from thermal treatment of bovine β-lactoglobulin was carried out by using a combination of techniques. Heat-induced changes in tertiary structure were monitored by intrinsic tryptophan fluorescence, while modifications in protein surface hydrophobicity were studied both during their occurrence and at equilibrium by using the fluorescent hydrophobic probe 1,8-anilinonaphthalenesulfonate. The association equilibria in the heated and cooled protein and the stabilization of aggregates by intermolecular disulfides were studied by gel permeation chromatography and nonreducing, denaturing electrophoresis. Results indicate that irreversible modification of the tertiary structure is not concentration dependent, while the temperature required for the occurrence of protein swelling, the initial step in the formation of associated forms of the protein, increases with the protein concentration. Stabilization of aggregates by intermolecular disulfides was dependent on concentration only at temperatures below 75 °C.