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  • Title: Effects of solute multivalency in quantitative affinity chromatography: evidence for cooperative binding of horse liver alcohol dehydrogenase to blue Sepharose.
    Author: Hogg PJ, Winzor DJ.
    Journal: Arch Biochem Biophys; 1985 Jul; 240(1):70-6. PubMed ID: 3160307.
    Abstract:
    In an affinity chromatographic study designed to examine the validity of considering successive interactions between an affinity matrix and a multivalent partitioning solute to be governed by a single intrinsic binding constant, a recycling partition equilibrium procedure has been used to investigate the interaction of horse liver alcohol dehydrogenase with Blue Sepharose in imidazole-chloride buffer, pH 7.5, I = 0.154. A value of 6000 M-1 has been obtained for the initial binding of this bivalent enzyme to matrix, an interaction which leads to a three- to fourfold enhancement of the subsequent interaction of that molecule with Blue Sepharose. Although this evidence of positive cooperativity in the enzyme-matrix interaction points to a deficiency in quantitative affinity chromatography theory based on equivalence and independence of these interactions [L. W. Nichol, L. D. Ward, and D. J. Winzor (1981) Biochemistry 20, 4856-4860], it is shown that such treatment leads to a much better description of the experimental situation than that provided by an alternative analysis based on cooperativity of enzyme-matrix interactions to the extent that only a single enzyme-matrix complex exists [P. Kyprianou and R. J. Yon (1982) Biochem. J. 207, 549-556]. Moreover, since the characterization of solute-ligand interactions by affinity chromatography is shown to be not unduly dependent upon mechanistic correctness of the thermodynamic model used for the solute-matrix interaction, the technique continues to have great potential for quantitative studies of ligand binding.
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