Some characteristics of protein precipitation by salts

The solubilities of lysozyme, alpha-chymotrypsin and bovine serum albumin (BSA) were studied in aqueous electrolyte solution as a function of ionic strength, pH, the chemical nature of salt, and initial protein concentration. Compositions were measured for both the supernatant phase and the precipitate phase at 25 degrees C. Salts studied were sodium chloride, sodium sulfate, and sodium phosphate. For lysozyme, protein concentrations in supernatant and precipitate phases are independent of the initial protein concentration; solubility can be represented by the Cohn salting-out equation. Lysozyme has a minimum solubility around pH 10, close to its isoelectric point (pH 10.5). The effectiveness of the three salts studied for precipitation were in the sequence sulfate > phosphate > chloride, consistent with the Hofmeister series. However, for alpha-chymotrypsin and BSA, initial protein concentration affects the apparent equillibrium solubility. For these proteins, experimental results show that the compositions of the precipitate phase are also affected by the initial protein concentration. We define a distribution coefficient kappa(e) to represent the equilibrium ratio of the protein concentration in the supernatant phase to that in the precipitate phase. When the salt concentration is constant, the results show that, for lysozyme, the protein concentrations in both phases are independent of the initial protein concentrations, and thus kappa(e) is a constant. For alpha-chymotrypsin and BSA, their concentrations in both phases are nearly proportional to the initial protein concentrations, and therefore, for each protein, at constant salt concentration, the distribution coefficient kappa(e) is independent of the initial protein concentration. However, for both lysozyme and alpha-chymotrypsin, the distribution coefficient falls with increasing salt concentration. These results indicate that care must be used in the definition of solubility. Solubility is appropriate when the precipitate phase is pure, but when it is not, the distribution coefficient better describes the phase behavior.