Evidence for negative cooperativity in the adsorption of phosphorylase b on hydrophobic agaroses

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Abstract
The adsorption of phosphorylase b occurs through the positive cooperative interaction of a critical number of approximately 3-5 butyl and a higher number of methyl residues with corresponding sites on the enzyme. The amount of adsorbed phosphorylase b [rabbit muscle] per ml of packed gel (methyl-, butyl-Sepharose) in the absence and presence of 1.1 M ammonium sulfate at temperatures between 0 and 34.degree. C is a power function of the free solute equilibrium concentration (Freundlich isotherm). The adsorption of cyanmyoglobin to phosphocellulose is described by the Langmuir equation. The surface coverage dependent isosteric heats of adsorption for phosphorylase b indicate an endothermic reaction only on the butyl-Sepharose in the presence of high salt concentrations. Scatchard plots of the Freundlich isotherms of phosphorylase b are concave upwards, typical of negative cooperativity. Hill plots of these isotherms (5-70% saturation) yield coefficients between nH = 0.39 and 0.71. At high surface coverages, the Hill coefficients approach 1. Apparent association constants (K0.5) of 4-39 .times. 104 M-1 are calculated for the adsorption of phosphorylase b, as compared to 2-9 .times. 104 M-1 for the adsorption of cyanmyoglobin. In general, negative cooperativity of binding may be explained by changes in the affinity of the ligand for the matrix, due to the sequential, multivalent adsorption and competition of phosphorylase b molecules for the critical number of alkyl residues (nonindependence of binding) on 1 side and to variations in the configuration of binding and entropy on the other.