Confirmation of Free Solvent Model Assumptions in Predicting the Osmotic Pressure of Concentrated Globular Proteins

Abstract

Previously the osmotic pressure of the concentrated globular proteins immuno-γ-globulin, bovine serum albumin, hen egg lysozyme, and ovalbumin in moderate-ionic-strength solutions was successfully modeled using a free solvent model (Yousef et al., J. Colloid Interface Sci.197, 108, 1998; 207, 273, 1998; AIChE J., 2001). This model considered the average solute–solvent and microion–solute interactions, represented the hydrated macromolecule and the cooperative interacting water and salts as a single species, and used a mole fraction concentration variable. Although the model assumed no fitted parameters, the hydration number was regressed on due to its sensitivity and compared with the 1 g H2O/g BSA estimate determined from H217O magnetic resonance studies of other globular proteins. The resulting average hydration numbers were in agreement with the reported estimates. These results suggest that solvent–solute interactions are dominant factors when analyzing nonidealities associated with the osmotic pressure data of concentrated protein solutions in moderate-ionic-strength solutions. However, the condition for the applicability of the free solvent model is that long-range interactions between hydrated macromolecules are screened to reduce solute–solute interaction. This condition requires a moderately high salt concentration. The objective of this study is to confirm this requirement for a successful model representation of the osmotic pressure of these protein solutions at high concentrations. To accomplish this, studies of concentrated solutions of ovalbumin at low ionic strength 0.01 M at pH 7.0 were carried out and modeled using the free solvent approach. The results showed that no reasonable free solvent model solution could be obtained. These results strengthen the argument that the free solvent model has a physically realizable representation for concentrated protein solutions in moderate salt concentrations.