Pressure denaturation of beta-lactoglobulin. Different stabilities of isoforms A and B, and an investigation of the Tanford transition.

Beta-lactoglobulin, the main whey protein in bovine milk, exists in several isoforms of which the most abundant are isoforms A and B. We have previously reported the denaturation of beta-lactoglobulin A by hydrostatic pressure [Valente-Mesquita, V.L., Botelho, M.M. & Ferreira, S.T. (1998) Biophys. J. 75, 471-476]. Here, we compare the pressure stabilities of isoforms A and B. These isoforms differ by two amino-acid substitutions: Asp64 and Val118 in isoform A are replaced by glycine and alanine, respectively, in isoform B. Replacement of the buried Val118 residue by the smaller alanine side-chain is not accompanied by significant structural rearrangements of the neighbouring polypeptide chain and creates a cavity in the core of beta-lactoglobulin. Pressure denaturation experiments revealed different stabilities of the two isoforms. Standard volume changes (DeltaVunf) of - 49 +/- 8 mL.mol-1 and -75 +/- 3 mL.mol-1, and unfolding free energy changes (DeltaGunf) of 8.5 +/- 1.3 kJ.mol-1 and 11.3 +/- 0.4 kJ.mol-1 were obtained for isoforms A and B, respectively. The volume occupied by the two methyl groups of Val118 removed in the V118A substitution is approximately 40 A3 per monomer of beta-lactoglobulin, in excellent agreement with the experimentally measured difference in DeltaVunf for the two isoforms (DeltaDeltaVunf = 26 mL.mol-1, corresponding to approximately 43 A3 per monomer). Thus, the existence of a core cavity in beta-lactoglobulin B may explain its enhanced pressure sensitivity relative to beta-lactoglobulin A. beta-Lactoglobulin undergoes a reversible pH-induced conformational change around pH 7, known as the Tanford transition. We have compared the pressure denaturation of beta-lactoglobulin A at pH 7 and 8. Unfolding free energy changes of 8.5 +/- 1.3 and 8.3 +/- 0.3 kJ.mol-1 were obtained at pH 7 and 8, respectively, showing that the thermodynamic stability of beta-lactoglobulin is identical at these pH values. Interestingly, DeltaVunf was dependent on pH, and varied from -49 +/- 8 mL.mol-1 to -68 +/- 2 mL.mol-1 at pH 7 and 8, respectively. The large increase in DeltaVunf at pH 8 relative to pH 7 appears to be associated with an overall expansion of the protein structure and could explain the increased pressure sensitivity of beta-lactoglobulin at alkaline pH.