Molecular thermodynamics of protein phase behavior in aqueous electrolyte solutions

Understanding protein-protein interactions is of fundamental importance for design of efficient protein recovery and purification processes. Development and optimization of protein-separation processes requires knowledge of the behavior of proteins in complex aqueous solutions, where salts, polymers, organic solvents, or other solutes may be present at high concentrations. The focus of this research is on salt-induced protein precipitation, a frequent first step recovery technique used in the laboratory and industry. The objective of this work is twofold. First, protein interactions in aqueous electrolyte solutions are quantified as a function of solution conditions through experiment. Second, molecular thermodynamic models are developed to account for measured protein interactions.; Protein interactions are governed by protein properties (size, shape, and surface chemistry) and by solution conditions (salt type and concentration, pH, and temperature). In this work, three experimental methods were used to quantify protein interactions as a function of protein properties and solution conditions. First, dynamic light-scattering (DLS) measurements of lysozyme in aqueous MgCl₂ solutions provided the hydrodynamic radius (18.6 Å) and an interaction parameter as a function of salt concentration and temperature. A maximum in attractive interactions is observed at an intermediate salt concentration (0.8 M). Second, the temperature at which a liquid-liquid phase separation occurs, or cloud-point temperature (CPT), was determined for lysozyme in aqueous electrolyte solutions as a function of salt type, salt concentration, and pH. The CPT increased as salt concentration increased for monovalent salts and a maximum in the CPT is observed for divalent cationic salts. Finally, low-angle laser-light scattering (LALLS) measurements of poly-L-lysine (PLL) in aqueous NaCl solutions provided the osmotic second virial coefficient and weight-average molecular weight as a function of salt concentration and PLL secondary structure.; Protein interactions in solution are described on the molecular level in terms of a two-body potential of mean force (PMF). The protein-protein PMF is a measure of the overall interactions of two protein molecules as a function of the physicochemical properties of the protein and the solvent. PMF models are formulated to account for interactions of protein molecules measured by experiment. For CPT measurements, a square-well potential is used to account for the specific interactions of various ions on the CPT. Using the Random Phase Approximation (RPA) in conjunction with the square-well PMF, depths of the square well were calculated. For LALLS measurements, DLVO theory in conjunction with a square-well potential (for specific interactions) is used to account for experimental observations. Attractive interactions are roughly 2.5 times greater for poly-L-lysine in the β-sheet conformation compared to the α-helix conformation.