Thermodynamic characterization of interactions of native bovine serum albumin with highly excluded (glycine betaine) and moderately accumulated (urea) solutes by a novel application of vapor pressure osmometry

The thermodynamic consequences of interactions of native bovine serum albumin (BSA) with two smaller solutes (glycine betaine or urea) in aqueous solution are characterized by a novel application of vapor pressure osmometry (VPO), which demonstrates the utility of this method of investigating preferential interactions involving solutes that are either accumulated or excluded near the surface of a protein. From VPO measurements of osmolality (water activity) as a function of the solute concentration in the presence and absence of BSA, we determine the dependence of the solute molarity (C-3) On that of BSA (C-2) at fixed temperature (37 degrees C), pressure (similar to 1 atm), and osmolality (over the range 0-1.6 molal). After some thermodynamic transformations, these results yield values of (m) Gamma(mu 3)(o) = lim(m2-->0)(partial derivative m(3)/partial derivative m(2))(T,P,mu 3,) which characterizes the interdependence of solute molalities when temperature, pressure, and the chemical potential of solute 3 are fixed. This form of the preferential interaction coefficient can be interpreted directly in terms of the molecular exclusion or accumulation of the solute (relative to water) near the protein surface. Within experimental uncertainty, (m) Gamma(mu 3)(o) is proportional to m(3) both for glycine betaine (0-0.9 m) and for urea (0-1.6 m). For glycine betaine partial derivative(m) Gamma(mu 3)(o)/partial derivative m(3) = -49 +/- 4, a value consistent with the interpretation that this solute is completely excluded from the hydrated surface of BSA, whereas for urea partial derivative(m)<Gamma(mu 3)/partial derivative m(3) = 6 +/- 1, which indicates a moderate extent of accumulation at the surface of native BSA. The preferential accumulation of solutes (e.g., urea) that have some binding affinity for a protein can be quantified and interpreted using the two-domain model if the extent of hydration of the protein has been determined using a completely excluded solute (e.g., glycine betaine). Complete exclusion from the local hydration domain surrounding proteins, if general, justifies the use of glycine betaine as a thermodynamic probe of the changes ill hydration that accompany protein folding, protein association, and protein-ligand binding interactions.