Characterization of pH-induced transitions of β-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies

Abstract

Depending on solution conditions, β-lactoglobulin can exist in one of its six pH-dependent structural states. We have characterized the acid and basic-induced conformational transitions between these structural states over the pH range of pH 1 to pH 13. To this end, we have employed high-precision ultrasonic and densimetric measurements coupled with fluorescence and CD spectroscopic data. Our combined spectroscopic and volumetric results have revealed five pH-induced transitions of β-lactoglobulin between pH 1 and pH 13. The first transition starts at pH 2 and is not completed even at pH 1, our lowest experimental pH. This transition is followed by the dimer-to-monomer transition of β-lactoglobulin between pH 2.5 and pH 4. The dimer-to-monomer transition is accompanied by decreases in volume, v° (−0.008(±0.003) cm3 g−1), and adiabatic compressibility, k°S (−(0.7(±0.4)) × 10−6 cm3 g−1 bar−1). We interpret the observed changes in volume and compressibility associated with the dimer-to-monomer transition of β-lactoglobulin, in conjunction with X-ray crystallographic data, as suggesting a 7 % increase in protein hydration, with the hydration changes being localized in the area of contact between the two monomeric subunits. The so-called N-to-Q transition of β-lactoglobulin occurs between pH 4.5 and pH 6 and is accompanied by increases in volume, v° (0.004(±0.003) cm3 g−1), and compressibility, k°S ((0.7(±0.4)) × 10−6 cm3 g−1 bar−1). The Tanford transition of β-lactoglobulin is centered at pH 7.5 and is accompanied by a decrease in volume, v° (−0.006(±0.003) cm3 g−1), and an increase in compressibility, k°S ((1.5(±0.5)) × 10−6 cm3 g−1 bar−1). Based on these volumetric results, we propose that the Tanford transition is accompanied by a 5 to 10 % increase in the protein hydration and a loosening of the interior packing of β-lactoglobulin as reflected in a 12 % increase in its intrinsic compressibility. Finally, above pH 9, the protein undergoes irreversible base-induced unfolding which is accompanied by decreases in v° (−0.014(±0.003) cm3 g−1) and k°S (−(7.0(±0.5)) × 10−6 cm3 g−1 bar−1). Combining these results with our CD spectroscopic data, we propose that, in the base-induced unfolded state of β-lactoglobulin, only 80 % of the surface area of the fully unfolded conformation is exposed to the solvent. Thus, in so far as solvent exposure is concerned, the base-induced unfolded states of β-lactoglobulin retains some order, with 20 % of its amino acid residues remaining solvent inaccessible.