Energetics of β-lactoglobulin-flavor compounds interactions

Interaction of bovine β-lactoglobulin (BLG) with several flavor compounds (FC) (2-methylpyrazine, vanillin, 2-acetylpyridine, 2- and 3-acetylthiophene, methyl isoamyl ketone, heptanone, octanone, and nonanone) was studied by high-sensitivity differential scanning calorimetry. The denaturation temperature, enthalpy, and heat capacity increment were determined at different FC concentrations. It was found that the denaturation temperature and heat capacity increment do not depend on the FC concentration, while the denaturation enthalpy decreases linearly with the FC concentration. These thermodynamic effects disclose the preferential FC binding to the unfolded form of BLG. By the obtained calorimetric data, the free energies of FC binding vs. the FC concentrations were calculated. These dependences were shown to be linear. Their slope relates closely to the overall FC affinity for the unfolded BLG in terms of the Langmuir binding model. The overall BLG affinity for FC varies from 20 M^-1 (2-methylpyrazine) up to 360 M^-1(nonanone). The maximal stoichiometry of the BLG-FC complexes was roughly estimated as a ratio of the length of the unfolded BLG to the molecular length of FC. Using these estimates, the apparent BLG-FC binding constants were determined. They are in the range of 0.3-8.0 M^-1 and correlated strictly with the FC lipophilicity descriptor (logP).