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 previous ly reported the denaturation of beta-lactoglobulin A by hydrostatic pressur e [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-ch ain is not accompanied by significant structural rearrangements of the neig hbouring polypeptide chain and creates a cavity in the core of beta-lactogl obulin. Pressure denaturation experiments revealed different stabilities of the two isoforms. Standard volume changes (Delta V-unf) of - 49 +/- 8 mL.m ol(-1) and -75 +/- 3 mL.mol(-1), and unfolding free energy changes (Delta G (unf)) 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 g roups of Val118 removed in the V118A substitution is approximate to 40 Angs trom(3) per monomer of beta-lactoglobulin, in excellent agreement with the experimentally measured difference in Delta V-unf for the two isoforms (Del ta Delta V-unf = 26 mL.mol(-1), corresponding to approximate to 43 Angstrom (3) per monomer). Thus, the existence of a core cavity in beta-lactoglobuli n B may explain its enhanced pressure sensitivity relative to beta-lactoglo bulin A. beta-Lactoglobulin undergoes a reversible pH-induced conformationa l 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 fre e 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, Delta V-unf was dependent on pH, and varied from -49 +/- 8 mL.mol(-1) to -68 +/- 2 mL.m ol(-1) at pH 7 and 8, respectively. The large increase in Delta V-unf at pH 8 relative to pH 7 appears to be associated with an overall expansion of t he protein structure and could explain the increased pressure sensitivity o f beta-lactoglobulin at alkaline pH.