Measurement of cooperativity of binding of calcium to neutral sodium pectate

Using ion-specific electrodes, we have measured isotherms of binding of calcium to a purified and well characterized sample of pectic acid in aqueous solutions adjusted initially to neutral conditions with sodium hydroxide. Binding isotherms were obtained for several 'buffer' concentrations of excess sodium chloride. Non-linear least squares analysis of the combined isotherms, on the basis of a theoretical model by Record et al, (15), has provided estimates for the stoichiometry and mass action constant of the ion exchange between sodium and calcium ions adsorbed to the pectin. The best fit to the data suggests approximately an average one-to-one displacement of sodium by calcium as would be expected on the basis of reported single counterion activity measurements. Simple tests for cooperativity, e. g. Scatchard and Hill plots, on the combined isotherms suggest a net anti-cooperative binding of calcium. The reason for this at first sight surprising behaviour is the well established anti-cooperative nature of ion binding to polyelectrolytes which in the present case appears to 'mask' the positive element of the cooperativity associated with conformational change. To reveal the latter by binding studies alone, a very detailed and precise definition of the isotherm is required. We have attempted to obtain such data for one 'buffer' concentration of sodium chloride. Representation of these results by a Tanford plot and subsequent curve-fitting using the statistical mechanic al model of Zimm and Rice (13), for the situation of combined conformational transition and ion binding to a polymer, provides unambiguous evidence for positive cooperativity, i.e. a small value of the Zimm Bragg cooperativity parameter sigma (<10(-4)) emerges from the treatment indicating that the cooperativity associated with conformational change is substantial.