LIQUID LIQUID-PHASE SEPARATION OF AQUEOUS LYSOZYME SOLUTIONS - EFFECTS OF PH AND SALT IDENTITY

We have studied the properties of liquid-liquid phase separation in aqueous solutions of the protein lysozyme. The system under investigation was a lysozyme solution in sodium phosphate buffer of high ionic strength and of pH far below the isoelectric pH of lysozyme. Liquid-liquid phase separation was marked by opacification of the solution as its temperature was lowered, at fixed concentration, to a well-defined value called Tcloud. Below this temperature the solutions separated macroscopically into two coexisting liquid phases. We have conducted measurements of Tcloud as a function of ionic strength of the sodium phosphate buffer, its pH, and the identity and concentration of added salts. We have observed the following: (1) Tcloud was insensitive to changes in the total ionic strength of the sodium phosphate buffer within the range of the (rather high) ionic strengths studied (0.300-0.600 M), while the buffer pH was held constant. (2) However, when salts (NaCl, NaBr, KCl, KBr) were added to the sodium phosphate buffer keeping the total ionic strength of the buffer constant (by decreasing the contents of sodium phosphate correspondingly), Tcloud was observed to increase roughly linearly with salt concentration. The identity of the salt, at constant total ionic strength and pH of the buffer, had a marked effect on the magnitude of that increase. (3) Finally, Tcloud increased approximately linearly with pH between pH values of 5.8 and 8.0 while the total ionic strength of the buffer was held constant. Our data suggest that the pH and ionic strength alone are not sufficient to describe the conditions for liquid-liquid phase separation in our solutions. Each ionic species present in the solution must be individually assayed for its effect on phase separation. A thermodynamic Gibbs free energy for hard spheres with an attraction is used to model the phase separation phenomenon in aqueous lysozyme solutions. © 1990 American Chemical Society.