Measurement of Cloud-Point Temperatures for Lysozyme in Electrolyte Solutions with Thermooptical AnalysisMeasurement of Cloud-Point Temperatures for Lysozyme in Electrolyte Solutions with Thermooptical Analysis

초록

Liquid-liquid phase-separation data are obtained for aqueous solutions of lysozyme and α-chymotrypsin. Thermooptical analysis (TOA) overcomes many defects of the light scattering method, which is most commonly used for this purpose, and provides a simple, rapid and reliable experimental method to determine cloud-point curves of aqueous protein solution systems. The TOA apparatus described here uses very small samples, and cloud-point curves can easily obtained in very short time. The cloud-point temperature (CPT) is measured as a function of protein concentration, salt type and salt concentration at pH 4.0, 7.0, 10.0. Salts used includes those from mono and divalent cations and anions. For the monovalent cations studied, as salt concentration increases, the CPT increases. For divalent cations, as salt concentration rises, a maximum in the CPT is observed and attributed to ion binding to the protein surface and subsequent water structuring. For anions at fixed salt concentration, the CPT decreases with rising anion kosmotropic character. Comparison of CPTs for pH 4.0, 7.0 and 10.0 revealed two trends. At low ionic strength for a given salt, differenced in CPT can be explained in terms of repulsive electrostatic interactions between protein molecules, while at higher ionic strength, differences can be attributed to hydration forces. A more efficient isolation method of protein solutions would be based on predicting the solution conditions, including the cloud-point temperature as a function of protein concentration, salt type, salt concentration and pH, that favor selective precipitation of the target protein.