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

Depending on solution conditions, beta-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 beta-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 beta-lactoglobulin between pH 2.5 and pH 4. The dimer-to-monomer transition is accompanied by decreases in volume, v(o) (-0.008(+/-0.003) cm(3) g(-1)), and adiabatic compressibility, k(s)(o) (-(0.7(+/-0.4)) x 10(-6) cm(3) g(-1) bar(-1)). We interpret the observed changes in volume and compressibility associated with the dimer-to-monomer transition of beta-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 beta-lactoglobulin occurs between pH 4.5 and pH 6 and is accompanied by increases in volume, v(o) (0.004(+/-0.003) cm(3) g(-1)), and compressibility k(s)(o) ((0.7(+/-0.4)) x 10(-6) cm(3) g(-1) bar(-1)). The Tanford transition of beta-lactoglobulin is centered at pH 7.5 and is accompanied by a decrease in volume, v(o) (-0.006(+/-0.003) cm(3) g(-1)), and an increase in compressibility, k(s)(o) ((1.5(+/-0.5)) x 10(-6) cm(3) 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 beta-lactoglobulin as deflected 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(o) (-0.014(+/-0.003) cm(3) g(-1)) and k(s)(o)(-(7.0(+/-0.5)) x 10(-6) cm(3) g(-1) bar(-1)). Combining these results with our CD spectroscopic data, we propose that, in the base-induced unfolded state of beta-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 beta-lactoglobulin retains some order, with 20% of its amino acid residues remaining solvent inaccessible. (C) 2001 Academic Press.