Pressure denaturation of β‐lactoglobulin

Abstract

β‐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 β‐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 β‐lactoglobulin. Pressure denaturation experiments revealed different stabilities of the two isoforms. Standard volume changes (Δ V unf ) of – 49 ± 8 mL·mol −1 and −75 ± 3 mL·mol −1 , and unfolding free energy changes (Δ 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 groups of Val118 removed in the V118A substitution is ≈ 40 Å 3 per monomer of β‐lactoglobulin, in excellent agreement with the experimentally measured difference in Δ V unf for the two isoforms (ΔΔ V unf = 26 mL·mol −1 , corresponding to ≈ 43 Å 3 per monomer). Thus, the existence of a core cavity in β‐lactoglobulin B may explain its enhanced pressure sensitivity relative to β‐lactoglobulin A. β‐Lactoglobulin undergoes a reversible pH‐induced conformational change around pH 7, known as the Tanford transition. We have compared the pressure denaturation of β‐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 β‐lactoglobulin is identical at these pH values. Interestingly, Δ V unf 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 Δ V unf 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 β‐lactoglobulin at alkaline pH.