Determination of Second Virial Coefficient of Proteins Using a Dual-Detector Cell for Simultaneous Measurement of Scattered Light Intensity and Concentration in SEC-HPLC

AbstractA method is proposed for the measurement of the B22 value of proteins in aqueous solutions in flow-mode that utilizes a novel fabricated dual-detector cell, which simultaneously measures protein concentration and the corresponding scattered light intensity at 90°, after the protein elutes from a size-exclusion column. Each data point on the chromatograms obtained from the light scattering detector and the concentration (ultraviolet) detector is converted to Rayleigh’s ratio, Rθ, and concentration, c, respectively. The B22 value is calculated from the slope of the Debye plot (Kc/Rθ versus c) generated from a range of concentrations obtained from these chromatograms for a single protein injection. It is shown that this method provides reliable determination of the B22 values for such proteins as lysozyme, chymotrypsinogen, and chymotrypsin in various solution conditions that agree well with those reported in literature

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

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.

Proceedings of International Conference on Women Researchers in Electronics and Computing

This proceeding contains articles on the various research ideas of the academic community and practitioners presented at the international conference, “Women Researchers in Electronics and Computing” (WREC’2021). WREC'21 was organized in online mode by Dr. B R Ambedkar National Institute of Technology, Jalandhar (Punjab), INDIA during 22 – 24 April 2021. This conference was conceptualized with an objective to encourage and motivate women engineers and scientists to excel in science and technology and to be the role models for young girls to follow in their footsteps. With a view to inspire women engineers, pioneer and successful women achievers in the domains of VLSI design, wireless sensor networks, communication, image/ signal processing, machine learning, and emerging technologies were identified from across the globe and invited to present their work and address the participants in this women oriented conference. Conference Title: International Conference on Women Researchers in Electronics and ComputingConference Acronym: WREC'21Conference Date: 22–24 April 2021Conference Location: Online (Virtual Mode)Conference Organizers: Department of Electronics and Communication Engineering, Dr. B. R. Ambedkar National Institute of Technology, Jalandhar, Punjab, INDI