Binding of proteins to polyelectrolytes studied by capillary electrophoresis

Yang Gao, Purdue University

Abstract

This study is focused on the quantitative characterization of soluble complexes between proteins and polyelectrolytes in aqueous solution. Model systems are comprised of acidic proteins, such as $\beta$-lactoglobulin (BLG) or bovine serum albumin (BSA), complexing with polyanions, such as sodium polystyrenesulfonate (NaPSS) or sodium poly(2-acrylamido-2-methylpropanesulfonate) (PAMPS) in phosphate buffers in which the proteins are carrying net negative charge. A novel capillary electrophoresis based method, Frontal Analysis Continuous Capillary Electrophoresis (FACCE), was developed for quantitative equilibrium study of the binding. The method has been proved to be simple, efficient, reproducible, sample-saving, and free from effects due to slow binding kinetics. Quantitative measurement of the binding for BLG/NaPSS and BSA/NaPSS complexes indicate that these systems are consistent with the 'overlapping binding site model'. The large difference in binding affinities between NaPSS and PAMPS for the same proteins probably arises from the difference in flexibility and hydrophobicity of the two polymers. Hydrophobic interactions between proteins and polymers are more systematically studied in the measurements of the binding of BLG and BSA to a series of copolymers (maleic acid and vinyl alkyl ether) of various hydrophobicity. It was found that the longer the alkyl side-chain in the copolymer, the stronger the tendency of the copolymer to form intrapolymer micelles as well as to interact with proteins hydrophobically; competition of the two processes determines the outcome of binding. In addition, the results indicate that a minimum alkyl side-chain length of 3-4 carbons must be reached before the polymers can interact hydrophobically with proteins. Information on the structure and cooperativity of BLG/NaPSS and BSA/NaPSS complexes in phosphate buffer was also obtained by establishing the relationship between the stoichiometry of the complexes and their properties, such as diffusion coefficients and electrophoretic mobilities. It was found that the formation of BSA/NaPSS complexes is anti-cooperative while the formation of BLG/NaPSS complexes is not. The structures of both complexes were consistent with a free-draining model.

Degree

Ph.D.

Advisors

Dubin, Purdue University.

Subject Area

Analytical chemistry|Chemistry|Polymers

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