Salt‐dependent monomer–dimer equilibrium of bovine β‐lactoglobulin at pH 3

Although bovine β‐lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer–dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO₄ was more effective than other salts by about 20‐fold, suggesting that anion binding is responsible for the salt‐induced dimer formation, as observed for acid‐unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO₄, the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (ΔH_D) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric β‐lactoglobulin at pH 3.0 with NaClO₄. The net heat effects after subtraction of the heat of salt dilution, corresponding to ΔH_D, were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the ΔH_D value at 20°C and the heat capacity change (ΔC_p) of dimer formation. In both NaCl and NaClO₄, the obtained ΔC_p value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed ΔC_p values were consistent with the calculated value from the X‐ray dimeric structure using a method of accessible surface area. These results indicated that monomer–dimer equilibrium of β‐lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.