Some characteristics of protein precipitation by salts

Abstract

The solubilities of lysozyme, α‐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°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 α‐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 κe 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 κe is a constant. For α‐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 κe is independent of the initial protein concentration. However, for both lysozyme and α‐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. © 1992 John Wiley & Sons, Inc.

Initial protein concentration effects on precipitation by salt

“…Lysozyme, ␣-chymotrypsin, and bovine albumin had a protein content of approximately 95% or better and thus were used without further purification. ␣-Chymotrypsin is known to self-digest in aqueous solution, which was avoided by using phenylmethane sulfonyl fluoride (PMSF) according to Shih et al (1992). The protein solutions of lysozyme, ␣-chymotrypsin, and bovine albumin were prepared with phosphate buffer, whereas amyloglucosidase was prepared with acetate buffer.…”

“…Moreover, because protein precipitation has usually been regarded as phase separation resulting in a solid phase of protein precipitate and a saturated protein liquid phase, the influence of the initial protein concentration on precipitation is often ignored. However, there is evidence showing that protein solubility can vary with the initial protein concentration; nevertheless, only a handful of articles in the literature have been concerned with this factor (Chiew et al, 1995;Iyer and Przybycien, 1994;Shih et al, 1992).…”

Solubilidade De Imunoglobulina G Em Soluções De Sais

“…Além disso, segundo Shih et al (1992) O parâmetro β', varia independentemente da natureza do sal usado, mas é influenciado pela proteína, pH e temperatura. Apesar do uso da imunoglobulina G na determinação da solubilidade em todas as soluções de sais na mesma temperatura de 5°C, o parâmetro β' apresentou valores relativamente próximos, mas não totalmente iguais.…”

Some new frontiers in chemical engineering thermodynamics

“…Solubilities of proteins in salt solutions have received attention from various authors (Cohn and Edsall, 1943;Shih et al, 1992) but many questions remain. A possible method for developing an appropriate theory is provided by the Random-Phase Approximation as discussed, for example, by Vlachy et al (1993), following similar efforts for aqueous protein-polymer mixtures by Hall (1990, 1992).…”