Salting out of aqueous proteins: Phase equilibria and intermolecular potentials

Abstract

Abstract Salting‐out phase equilibria are reported for lysozyme and α‐chymotrypsin from concentrated ammonium‐sulfate solutions. Supernatant and dense‐phase protein concentrations and the resulting protein partition coefficients are given as a function of solution pH and ionic strength. Phase equilibria with a trivalent salt (sodium citrate) confirm that ionic strength, rather than salt concentration, is the appropriate variable describing phase equilibria. The salting‐out behavior of a mixture of an aqueous lysozyme and α‐chymotrypsin is independent of the presence of the other protein. Parameters for a molecular‐thermodynamic description of salting‐out behavior are obtained from low‐angle laser‐light scattering (LALLS). Osmotic second virial coefficients from LALLS are reported over a range of pH for dilute chymotrypsin concentrations in aqueous electrolyte solutions at 0.01 and 1.0 M ionic strengths. Effective Hamaker constants, regressed from experimental osmotic second virial coefficients, are determined for models of the protein – protein potential of mean force. In addition to excluded volume, dispersion, and shielded charge – charge potentials, the description of protein ‐ protein interactions includes attractive charge – dipole and dipole – dipole potentials as well as an osmotic‐attraction potential that becomes important at high salt concentrations. Protein dipole – dipole potentials are required to account for the observed pH dependence of osmotic second virial coefficients, especially at low ionic strength.