Binding isotherms of nucleosides and polynucleotides measured by continuous ultrafiltration at constant volume: a rapid and precise technique.

Equilibrium binding isotherms have been measured as a function of temperature for the interaction of 2- and 8-aminoadenosine with poly(U) by continuous, constant-volume ultrafiltration. An analytical method is described for determining the entire isotherm in a single experiment on a preequilibrated ligand--polymer solution. The method is shown to produce the same results as classical equilibrium dialysis but to be much more rapid and convenient. Values of the enthalpy, equilibrium constant, and free energy of stacking as a function of temperature are derived from the binding data. Stoichiometry of interaction is obtained from the diafiltration profiles as well as from the binding isotherms. Over the temperature range studied, the cooperativity of binding is observed to increase with rising temperature.