Calorimetric studies of interactions between low molecular weight salts and bovine serum albumin in water at pH values below and above the isoionic point.

Isothermal titration calorimetry was used to determine the temperature and salt concentration dependence of the enthalpy of mixing, Δmix H, of bovine serum albumin (BSA) in aqueous buffer solutions with several low molecular weight salts. Three buffers were used: acetate (pH = 4.0), MOPS (7.2), and borate (9.2). Since the isoionic point of BSA is at pI ≈ 4.7, the net charge of BSA in acetate buffer was positive (≈ +20), while in the other two buffer solutions it was negative (≈ -15 in MOPS and ≈ -25 in borate). The majority of the recorded heat effects were exothermic, while only at pH = 9.2 a weak endothermic effect upon mixing BSA with LiCl, NaCl, and KCl was observed. For all buffer solutions the absolute values of Δmix H of sodium salts followed the order: NaCl < NaBr < NaNO3 < NaI < NaSCN, which is the reverse Hofmeister series for anions. The magnitude of the effects was the largest in acetate buffer and decreased with an increasing pH value of the solution. While the effect of varying the anion of the added salts was strongly pronounced at all pH values, the effect of the cation (LiCl, NaCl, KCl, RbCl and CsCl salts) was weak. The most interesting feature of the results obtained for pH > pI was the fact that Δmix H were considerably more sensitive to the anion (co-ion to the net BSA charge) than to the cation species. This indicated that anions interacted quite strongly with the BSA even at pH values where the net charge of the protein was negative. We showed that Δmix H at high addition of salts correlated well with the enthalpy of hydration of the corresponding salt anion. This finding suggested, consistently with some previous studies, that a part of the exothermic contribution to Δmix H originated from the hydration changes upon the protein-salt interaction. Theoretical analysis, based on the primitive model of highly asymmetric electrolyte solutions solved within the mean spherical approximation, was used to estimate Coulomb effects upon mixing.