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.