| dc.description.abstract | Understanding the proteins self-interactions in solution system has great potential value due to its relevance in several research and applications fields, such as bio-separation、protein conformational disease and biopharmaceutical development. Particularly in several stages of biopharmaceutical process, protein aggregation has been observed frequently. Therefore, a priori prediction of protein pair potentials will provide crucial information for analyzing or screening suitable solution conditions. The second virial coefficient (B22), a thermodynamic parameter that characterizes the non-ideal solution behavior arising from two body solute-solute interactions, is a potential predictive tool. Determinations of B22 proteins have been extensively and mostly done by self-interaction chromatography (SIC). In this study, a novel platform, isothermal titration calorimetry (ITC), is established to describe the B22 of proteins with polarized surface charges distribution in aqueous solutions by means of measure the dilution enthalpy of protein solution. With the aim, B22 of Myoglobin and Ribonuclease A are measured by ITC in different solution conditions(pH、salt concentration、salt type), and compared to results obtained from self-interaction chromatography (SIC). Furthermore, size-exclusion chromatography and crystallization results offer the efficient way to let us verify whether or not proteins are stable in solution. Detailed discussion as following: in the research of Myoglobin, the protein-protein interactions are repulsive with increasing salt concentration in ITC and SIC system. Moreover, different kinds of salt in solution affect the proteins’ behavior, which are consistent with Hofmeister Series. On the other hand, the research of Ribonuclease A shows that protein-protein interactions change from repulsive to attractive with increasing NaCl concentration in ITC system. Furthermore, comparing the experimental results from ITC and SIC, we discover the adverse results as the pH value of solution is close to the pI value of protein and at lower salt concentration. Under this circumstance, the charge distribution on protein surface is anisotropic. Meanwhile, B22 measurement in SIC system is not convincing because the immobilized proteins can’t move freely. On the contrary, we can describe protein behaviors in solution precisely by ITC measurement, because of the degrees of freedom of protein are not restricted. Additionally, at the aforementioned conditions, the less aggregation of Ribonuclease A is detected from the size-exclusion chromatography measurements. Consequently, we successfully establish a platform to measure B22 of proteins with isotropic or anisotropic surface charges distribution in ITC system. | en_US |