Effects of solute multivalency in quantitative affinity chromatography: evidence for cooperative binding of horse liver alcohol dehydrogenase to blue Sepharose.
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.
Hogg
P J
PJ
Winzor
D J
DJ
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
D004396:Coloring Agents; D012997:Solvents; C029483:Blue-Sepharose; D012685:Sepharose; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase