Title

The second virial coefficient of proteins in aqueous solutions and its relationship to protein solubility and long-term aggregation stability

Date of Completion

January 2005

Keywords

Health Sciences, Pharmacy|Biophysics, General

Degree

Ph.D.

Abstract

The relationship of second virial coefficient, B22 with solubility is reviewed and its relationship to protein self-association and aggregation is investigated. A novel method was developed for the measurement of B 22 that utilized a novel fabricated dual-source dual-detector cell for simultaneous measurement of protein concentration and the corresponding scattered light intensity in conjunction with SEC. This method provided reliable determination of the B22 values for such proteins as lysozyme, chymotrypsinogen and chymotrypsin. This methodology was further used to characterize protein self-association using β-lactoglobulin A (βLg). The Debye plots of βLg showed curvature at pH 3.0, for varying NaCl concentrations (0.02--0.5 M), indicative of the association behavior of this protein. Modified Debye light scattering equation that included the monomer-dimer equilibrium model yielded Ka values ranging from 102 to 10 5 M-1. The Ka values thus obtained followed similar trend to those reported in literature. Finally, the relationship of B22 to the extent of irreversible protein aggregation upon storage was investigated. A monoclonal antibody and ovalbumin were incubated at 37°C for a period of 3 months under various solution conditions to monitor the extent of physical aggregation and B22 values were determined under similar solution conditions. Both proteins readily aggregated at pH 4.0 in the presence of various co-solutes, however, were completely resistant to aggregation at pH 7.4. Debye plots of the monoclonal antibody showed moderate attractive interactions at pH 7.4, whereas, at pH 4.0, nonlinear Debye plots were obtained, indicating self-association of this protein. CD studies showed partially unfolded structure of antibody at pH 4.0 compared to that at pH 7.4. In case of ovalbumin, similar B22 values were obtained in all solution conditions irrespective of the fact whether the protein aggregated or not. CD studies of ovalbumin indicated presence of unfolded species at pH 4.0. Thus, formation of a structurally-altered state is must for irreversible aggregation to proceed. Since this "aggregation-prone" species is generally present in a small fraction, it is unlikely that B22 will correlate with aggregation. Nevertheless, Debye plots in conjunction with spectroscopic studies can provide important insights into the mechanism of irreversible protein aggregation in solution. ^