title: Effects of solute multivalency in quantitative affinity chromatography: evidence for cooperative binding of horse liver alcohol dehydrogenase to blue Sepharose.
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.
authors.forename: P J
authors.initials: PJ
authors.lastname: Hogg
authors.forename: D J
authors.initials: DJ
authors.lastname: Winzor
mesh_terms: D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D004396:Coloring Agents; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D008956:Models, Chemical; D011485:Protein Binding; D012685:Sepharose / Q000031:analogs & derivatives; D012997:Solvents
chemical_list: D004396:Coloring Agents; D012997:Solvents; C029483:Blue-Sepharose; D012685:Sepharose; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase