Molecular thermodynamics of protein interactions and phase behavior in aqueous electrolyte solution

Despite wide-spread use of salting-out precipitation for concentration and purification of proteins, the fundamental underlying mechanisms are not well understood. This work is concerned primarily with experimental determination of protein salting-out phase behavior and of protein interactions in aqueous electrolyte solutions.; Salting-out phase equilibria are reported for four proteins (lysozyme, chymotrypsin, ovalbumin, and hemoglobin) alone and in three binary mixtures over a wide range of pH and ionic strength. Protein concentrations were measured in the supernatant and dense phases. Results are compared with the expected salting-out behavior based on known protein physico-chemical properties. The effects of protein-protein and protein-ion association on the observed phase behavior are discussed. For salting out from protein mixtures, protein size and protein-protein association influence selectivity.; Specific ion-protein binding has been examined using chloride ion binding to lysozyme. {dollar}sp{lcub}35{rcub}{dollar}Cl NMR experiments quantitatively measured tight chloride binding to lysozyme. Low concentrations of chloride ions were shown to enhance salting out of lysozyme in ammonium sulfate solutions, illustrating the importance of specific ion-protein interactions.; Osmotic second virial coefficients measured by low-angle laser-light scattering (LALLS) are reported for chymotrypsin for a range of pH and ionic strength. Protein osmotic second virial coefficients are related to protein potentials of mean force through McMillan-Mayer theory. At low ionic strength, electrostatic interactions, including dipole interactions, are used to describe pH-dependent osmotic second virial coefficients. An effective Hamaker constant regressed from experimental osmotic second virial coefficients indicates that attractive interactions such as hydrophobic interactions and specific protein-protein association need to be considered.; Potentials of mean force are calculated for interactions of dipoles with a finite length. Results calculated for chymotrypsin potentials of mean force show that significant errors are introduced in describing protein dipole interactions unless finite-size dipolar effects are included in the potential of mean force. The length of charge separation in the dipole affects the potential of mean force most strongly at protein-protein distances near contact.