Mechanism of precipitation of proteins by polyethylene glycols. Analysis in terms of excluded volume.
The apparent solubilities of various proteins (14,000 to 670,000 daltons) were measured in the presence of polyethylene glycols (PEGs) of different sizes. All of the solubility curves, determined by measuring the protein concentration in the supernate of centrifuged mixtures, exhibited the characteristic linear dependence of log S (g/liter) on PEG concentration (%, w/v). For human albumin in PEG-4000 at pH 4.5, this linearity extended over a 1,000-fold range of solubility, even though the appearance of the sedimented phase changed from a viscous fluid to a white amorphous solid. The slope, beta, decreased from 0.27 to 0.09 with decreasing Mr of PEG from 20,000 to 400, but was insensitive to changes in solution conditions (pH, T, salts), suggesting the absence of specific chemical interactions between protein and polymer. This conclusion was supported by the observation that concentrations of PEG up to 30% (w/v) had no significant effect on the melting temperature of ribonuclease A. Furthermore, equilibrium dialysis measurements, as well as various spectral measurements, provided no evidence for such interactions. Using a steric exclusion model (Edmond, E., and Ogston, A. G. (1968) Biochem. J. 109, 569-576) and assuming that the chemical potential of the solid phase is constant, beta can be related to interaction coefficients calculated from co-volumes using the equivalent sphere radii of PEG (r2) and protein (r3). Although good agreement was obtained for albumin in PEG-20,000, the predicted dependence of beta on r3 was greater than observed and the predicted dependence of beta on r2 was of opposite direction to that observed. However, the interaction coefficient determined from the equilibrium dialysis measurements of albumin and PEG-1000 agreed with the predicted value. Thus, the exclusion of low concentrations of PEG by albumin can be explained by a simple excluded volume model, whereas the exclusion of protein out of solution by PEG appears to be more complex.
D000818:Animals; D002417:Cattle; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008970:Molecular Weight; D011092:Polyethylene Glycols; D011489:Protein Denaturation; D011506:Proteins / Q000302:isolation & purification*; D012995:Solubility; D013816:Thermodynamics
D011506:Proteins; D011092:Polyethylene Glycols