Changes in Structure and in Interactions of Heat-Treated Bovine β-Lactoglobulin

Heat stress on structure and ligand binding of β-LG has been studied by fluorescence, circular dichroism and gel electrophoresis at pH 6.5. Native PAGE gel electrophoresis shows that denaturation of β-LG is reversible up to 75°C then it becomes irreversible due to aggregation of β-LG. Formation of aggregated β-LG is completed at 95°C. Circular dichroism results indicate that formation of aggregated β-LG is accompanied by the scrambling of disulfide bonds (creation of new intramolecular and intermolecular disulfide bridges and rearrangement of old intramolecular disulfide bridges). Addition of ethanolic retinol causes a change in polarity of the solution and favors transformation of the β↔α structure. In the presence of retinol, the β-helix content of the secondary structure of heat-treated β-LG is increased and the major portion of its secondary structure is helical. Fluorescence results show that heat-treated β-LG at 95°C can still bind retinol. The refolding of the tertiary structure of β-LG heat-denatured at 95°C may recreate a retinol binding site. Surprisingly, the affinity of the new site for retinol is higher than that of native β-LG; however, the apparent molar ratio is lower than one. The binding properties of β-LG for terpenoids have been measured after its heat treatment at 20, 75 and 95°C. The intensity of tryptophan emission at 330 nm was changed only in the case of the interaction with β-ionone. Other ligands probably cannot bind to β-LG or they bind in a binding site far from the tryptophan residues, hence not affecting its fluorescence.