Abstract

Only recently have advances in colloid physics allowed development of a complete picture of the phase behavior of colloids interacting with very short-range interaction potentials so as to allow the phase behavior of proteins to be described quantitatively. The correlation between protein interactions and phase behavior is addressed here using this framework.

First, the second osmotic virial coefficients (b₂) of seven proteins (ovalbumin, ribonuclease A, bovine serum albumin, α-lactalbumin, myoglobin, cytochrome c and catalase) are measured in salt solutions. Comparison of the interaction trends shows that at low salt concentrations protein-protein interactions can be either attractive or repulsive, possibly due to the anisotropy of protein charge distributions. At high salt concentrations, the behavior depends on the salt: in sodium chloride, protein interactions generally show little salt dependence up to very high salt concentrations, whereas in ammonium sulfate there is a sharp drop in b₂ with increasing salt concentration beyond a particular threshold.

The relation between protein interactions and phase behavior in salt solutions is then studied by investigating salting-out of six proteins (ovalbumin, ribonuclease A, soybean trypsin inhibitor, lysozyme, and β-lactoglobulin A and B). When interpreted within the framework of the theoretical phase diagram, the results show that the formation of aggregates can be interpreted as a gas-liquid phase separation for both amorphous and gel-like aggregates. A notable additional feature is the existence of a second aggregation line for some proteins, interpreted as the spinodal. The formation of aggregates can be interpreted, in the light of theoretical results from mode-coupling theory, as a kinetically trapped state or a gel phase that occurs through the intermediate of a gas-liquid phase separation.

The effects of pH on protein interactions and protein phase behavior were investigated by measuring b₂ for ovalbumin, and the aggregate and crystal solubilities for ovalbumin, β-lactoglobulin A and B, ribonuclease A and lysozyme. For both acidic and basic proteins, the aggregate solubility during protein salting-out decreases with decreasing pH. Contrary to what is commonly believed, the isoelectric point was a minimum for neither aggregate nor crystal solubility.

The effect of another common additive for protein crystallization, polyethylene glycol (PEG), was studied in terms of the value of b₂ for three proteins (ovalbumin, ribonuclease A, and myoglobin) and four PEG molecular weights (PEG 400, 1000, 3350 and 8000). The physical origin of the effect of PEG on protein interactions was examined by comparing the theoretical prediction of the Asakura-Oosawa potential to the results of the m-PRISM derived potential for depletion forces. Because PEG is almost always used in combination with a salt, their effects on PEG/protein phase behavior were investigated by measuring PEG 8000/ovalbumin liquid-liquid (L-L) phase separation as a function of sodium chloride and ammonium sulfate. At low salt concentration, L-L phase separation occurs with increasing protein concentration, presumably due to repulsive electrostatic interactions between protein molecules. At high salt concentration, the behavior depends on the nature of the salt, presumably because of the effects of critical fluctuations on depletion forces.

Finally, the effects of salts and polymers were compared to the effects of organic precipitants, which share the ability to induce attractive protein-protein interactions. Ovalbumin phase behavior was investigated in ammonium sulfate, poly(ethylene glycol) 8000 and 2-methyl-2,4-pentanediol (MPD) at pH 7. The similarities in the shapes of the phase diagrams suggest independence of the global shape of the phase diagram of the physical origin of the attraction between proteins. The phase diagrams of ovalbumin in ammonium sulfate at pH 6 and ribonuclease A in MPD at pH 6, conditions under which these two proteins crystallize, are also presented. Comparisons to the results of vapor diffusion experiments under similar solution conditions for these two proteins illustrate how crystallization conditions can be designed based on knowledge of the experimental phase diagram of proteins.