A "release" protocol for isothermal titration calorimetry

Isothermal titration calorimetry (ITC) has become a standard method for inv estigating the binding of ligands to receptor molecules or the partitioning of solutes between water and lipid vesicles. Accordingly, solutes are mixe d with membranes (or ligands with receptors), and the subsequent heats of i ncorporation (or binding) are measured. In this paper we derive a general f ormula for modeling ITC titration heats in both binding and partitioning sy stems that allows for the modeling of the classic incorporation or binding protocols, as well as of new protocols assessing the release of solute from previously solute-loaded vesicles (or the dissociation of ligand/receptor complexes) upon dilution. One major advantage of a simultaneous application of the incorporation/binding and release protocols is that it allows for t he determination of whether a ligand is able to access the vesicle interior within the time scale of the ITC experiment. This information cannot be ob tained from a classical partitioning experiment, but it must be known to de termine the partition coefficient (or binding constant and stochiometry) an d the transfer enthalpy. The approach is presented using the partitioning o f the nonionic detergent C12EO7 to palmitoyloleoylphosphatidylcholine vesic les. The release protocol could also be advantageous in the case of recepto rs that are more stable in the ligand-saturated rather than the ligand-depl eted state.