Section on Physical Biochemistry, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, US Department of Health and Human Services, Building 8, Bethesda, MD 20892, USA.
A method and apparatus for automated measurement of the concentration dependence of static light scattering of protein solutions over a broad range of concentrations is described. The gradient of protein concentrations is created by successive dilutions of an initially concentrated solution contained within the scattering measurement cell, which is maintained at constant total volume. The method is validated by measurement of the concentration dependence of light scattering of bovine serum albumin, ovalbumin, and ovomucoid at concentrations up to 130 g/L. The experimentally obtained concentration dependence of scattering obtained from all three proteins is quantitatively consistent with the assumption that no significant self-association occurs over the measured range of concentrations.
"Rather it initially rises as expected from the ideal LS equation, then plateaus, and eventually declines with higher concentration (see high-concentration case study, Section 5.5). Many non-associating proteins exhibit light scattering behavior which is fit well by the effective hard sphere assumption including scattering that eventually decreases with concentration (Fernández & Minton, 2008). The same work described CG-SLS apparatus suitable for high concentrations. "
[Show abstract][Hide abstract] ABSTRACT: This chapter contains sections titled: Introduction Overview Multiangle Light Scattering Dynamic Light Scattering Case Study: MALS and DLS Measurements of an Aggregating Protein Conclusion Acknowledgments References
Formulation and Process Development Strategies for Manufacturing of a Biopharmaceutical, Edited by Feroz Jameel and Susan Hershenson, 01/2009: chapter 21: pages M. Larkin and P. J. Wyatt; Wiley & Sons, New York.
[Show abstract][Hide abstract] ABSTRACT: Using an experimental technique recently developed in this laboratory (Fernández C. and A. P. Minton. 2008. Anal. Biochem. 381:254-257), the Rayleigh light scattering of solutions of bovine serum albumin, hen egg white ovalbumin, hen egg white ovomucoid, and binary mixtures of these three proteins was measured as a function of concentration at concentrations up to 125 g/L. The measured concentration dependence of scattering of both pure proteins and binary mixtures is accounted for nearly quantitatively by an effective hard particle model (Minton A. P. 2007. Biophys. J. 93:1321-1328) in which each protein species is represented by an equivalent hard sphere, the size of which is determined by the nature of repulsive interactions between like molecules under a given set of experimental conditions. The light scattering of solutions of chymotrypsin A was measured as a function of concentration at concentrations up to 70 g/L at pH 4.1, 5.4, and 7.2. At each pH, the measured concentration dependence is accounted for quantitatively by an effective hard particle model, according to which monomeric protein may self-associate to form an equilibrium dimer and, depending upon pH, an equilibrium pentamer or hexamer.
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