Importance of protein-protein interactions on protein crystallisation

There is a strong link between solubility, and thus crystallisation, and the molecular interactions of proteins in dilute salt solutions. Such molecular interactions are governed by the weak interaction forces (electrostatic, hydration and hydrophobic). Such forces can be quantitatively estimated in terms of a second virial self-coefficient (B22) and a second virial cross-coefficient (B23) for a single and a binary protein system, respectively. Previous studies confirmed the relation between a value of the second virial coefficient and a type of interaction (attractive or repulsive). The aim of this thesis is to correlate the second virial coefficient with the solubility and nucleation for single and binary protein systems. Model proteins used in this work are lysozyme and ovalbumin from egg-white, and α-amylase from Bacillus Licheniformis (BLA). The measurements are performed for sodium chloride and ammonium sulphate solutions in an acidic pH at 20 oC. Interaction chromatography is used in this work to estimate the B22 and B23 values for the model proteins in salt solutions. From the measured values of B22 and B23, the type of interaction is generalised as a function of the salt type, salt concentration, pH and protein type. For the single protein systems, in ammonium sulphate solutions (0.1 - 2.4 M) at pH 4.0 and 7.0, repulsion or no interactions are observed below 0.8 M and, as the salt concentrations are increased attractive self-interactions are observed for the model proteins. However, for the sodium chloride solutions (0.1 - 2.0 M) at pH 4.0 and 7.0, the interaction patterns vary with the salt concentration, the pH and the type of protein studied. A common feature of the self-interaction for all the model proteins is the attractive interactions close to the isoelectric point. For the binary protein systems, three distinct regions are observed in the ammonium sulphate solutions (0.1 - 1.6 M) at pH in the range 4.0 - 7.0. Attractive or no cross-interactions are observed at low salt concentrations (< 0.5 M). At the intermediate salt concentrations (0.5 - 1.0 M), the cross-interactions are constant and near zero. This is followed by a sharp increase in the attractive interactions above 1.0 M ammonium sulphate concentrations. However, for sodium chloride solutions (0.1 - 1.6 M) at pH 4.0 - 7.0, two distinct regions are observed. Attraction or no interactions are observed at low salt concentrations (< 0.5 M) and above 0.5 M concentrations of sodium chloride, negligible cross-interactions are observed between model proteins. For the single protein system, an overall increase in the solubility of three model proteins is observed with an increase in the concentrations of ammonium sulphate and also for sodium chloride solutions except for BLA, where a salting-in behaviour is observed. Linear regression is used on the solubility data to determine the parameters of the Cohn equation (β and Ks) where the values of β vary with solution pH, protein type and salt type. The values of Ks vary with protein type and salt type. However, it is insensitive to the solution pH for lysozyme in ammonium sulphate, ovalbumin in sodium chloride and BLA in ammonium sulphate solutions. For the binary protein system, the presence of ovalbumin had a measurable effect on lysozyme solubility at pH < 5.0 in both salts. In low concentration sodium chloride solutions (< 0.3 M), a decrease in the solubility of lysozyme was observed with the presence of ovalbumin at acidic pH < 5.0. However, in ammonium sulphate solutions, the lysozyme solubility increases with the addition of ovalbumin in the salt concentration range 1.6 - 2.0 M and at pH < 4.0. The primary nucleation threshold values are also determined for lysozyme in sodium chloride and ammonium sulphate solutions. In sodium chloride solutions (0.2 - 1.0 M), the critical supersaturation values increase as the solution pH is raised from 4.0 to 7.0; however in ammonium sulphate solutions (1.0 - 2.0 M), the reverse effect is observed. The critical supersaturation required to nucleate lysozyme in ammonium sulphate solutions is approximately three times higher than in sodium chloride solutions. For the single protein systems, the measured values of solubility and B22 were correlated using published models (RSL and HDW). For each protein-salt combination, a reasonable single correlation between solubility and B22 is possible as the salt concentrations and pH are varied. There are separate correlations for sodium chloride and ammonium sulphate solutions. Based on the correlation curve of solubility and B22, it is proposed that the acidic pH range (4.0 - 5.0) is better for crystallising and precipitating globular proteins from these salt solutions. If the values of solubility and B22 are converted into a non-dimensional quantity, the data derived from the different protein-salt systems collapse onto a single curve for the same salt type. The B22 values are also correlated with the critical supersaturation (ln(c*/S)) for the primary nucleation of lysozyme in salt solutions. The values of the critical supersaturation increase as the values of the second virial coefficient become negative or reduce. The ideal critical supersaturation required to create nuclei of lysozyme in salt solutions is between 0.1 and 1.4. For the binary protein systems, B23 values were related to the slope of the lysozyme and ovalbumin plot at same salt concentration and solution pH. Further work is required for binary protein systems to generalise such correlations as a function of the salt concentration and pH. The correlations derived in this thesis are useful generally to predict the solubility and primary nucleation of globular protein in salt solutions. This work reinforces the importance of the second virial coefficient in predicting the crystallisation of protein in salt solutions