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 aq ueous solution are characterized by a novel application of vapor press ure osmometry (VPO), which demonstrates the utility of this method of investigating preferential interactions involving solutes that are eit her accumulated or excluded near the surface of a protein. From VPO me asurements of osmolality (water activity) as a function of the solute concentration in the presence and absence of BSA, we determine the dep endence of the solute molarity (C-3) On that of BSA (C-2) at fixed tem perature (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)(partia l derivative m(3)/partial derivative m(2))(T,P,mu 3,) which characteri zes the interdependence of solute molalities when temperature, pressur e, and the chemical potential of solute 3 are fixed. This form of the preferential interaction coefficient can be interpreted directly in te rms 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 consis tent with the interpretation that this solute is completely excluded f rom the hydrated surface of BSA, whereas for urea partial derivative(m )<Gamma(mu 3)/partial derivative m(3) = 6 +/- 1, which indicates a mod erate extent of accumulation at the surface of native BSA. The prefere ntial accumulation of solutes (e.g., urea) that have some binding affi nity for a protein can be quantified and interpreted using the two-dom ain model if the extent of hydration of the protein has been determine d using a completely excluded solute (e.g., glycine betaine). Complete exclusion from the local hydration domain surrounding proteins, if ge neral, justifies the use of glycine betaine as a thermodynamic probe o f the changes ill hydration that accompany protein folding, protein as sociation, and protein-ligand binding interactions.