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Originally published as Biophys J. BioFAST on October 1, 2004.
doi:10.1529/biophysj.104.048686
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Biophysical Journal 87:4048-4055 (2004)
© 2004 The Biophysical Society

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

Harminder Bajaj, Vikas K. Sharma and Devendra S. Kalonia

Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut

Correspondence: Address reprint requests to Dr. Devendra S. Kalonia, U-2092, School of Pharmacy, University of Connecticut, Storrs, CT 06269. Tel.: 860-486-3655; Fax: 860-486-4998; E-mail: kalonia{at}uconn.edu.

A 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, R{theta}, and concentration, c, respectively. The B22 value is calculated from the slope of the Debye plot (Kc/R{theta} 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.




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A. Saluja, A. V. Badkar, D. L. Zeng, S. Nema, and D. S. Kalonia
Ultrasonic Storage Modulus as a Novel Parameter for Analyzing Protein-Protein Interactions in High Protein Concentration Solutions: Correlation with Static and Dynamic Light Scattering Measurements
Biophys. J., January 1, 2007; 92(1): 234 - 244.
[Abstract] [Full Text] [PDF]


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