Effects of pH on the Molecular Binding between β-Lactoglobulin and Bixin

Abstract

Binding between β-lactoglobulin (β-Lg), the major whey protein, and bixin, the major carotenoid in annatto, was studied at pH 3.0-10.0. The Fourier transform infrared spectroscopy and UV-vis absorption spectroscopy results showed that the binding involved a complex formation. The tryptophan quenching fluorescence and analytical ultracentrifugation data showed that there were two specific binding sites and that the binding affinity increased significantly with an increase in pH. A higher efficiency of energy transfer from tryptophan fluorescence to bixin was observed at higher pH. Thermodynamic parameters and the number of specific binding sites obtained from isothermal titration calorimetry and analytical ultracentrifugation suggested that binding involved mostly hydrophobic interactions for the two specific binding sites. The impacts of pH on binding were correlated to the conformation of β-Lg, the hydrophobic pocket of which becomes more available at higher pH and ionic strength.