Isothermal titration calorimetry (ITC) is a label-free technique that directly measures the heat change accompanying a binding interaction. It is the only method that provides a complete thermodynamic characterization of binding, including the equilibrium association constant (Ka), Gibbs free energy (ΔG), enthalpy change (ΔH), entropy change (ΔS), and binding stoichiometry (n) from a single experiment.
In an ITC experiment, a ligand solution is injected in small aliquots into a sample cell containing the macromolecule under constant temperature. Each injection produces a heat pulse as ligand molecules bind to the target. As the binding sites become saturated, the heat signal decreases until only the heat of dilution remains. The reference cell contains buffer only, and the instrument measures the differential power required to maintain identical temperatures between the two cells.
The resulting thermogram plots heat per injection versus time. Integration of each peak yields the cumulative heat released or absorbed as a function of the molar ratio of ligand to macromolecule. Nonlinear regression of the binding isotherm using a suitable binding model yields Ka, ΔH, and n. The ΔG is calculated from Ka, and ΔS is derived from ΔG = ΔH - TΔS.
ITC can measure association constants from 103 to 109 M−1. The detection limit depends on the magnitude of the binding enthalpy and the concentration of reactants. Typical experiments require 10–100 µM concentrations of macromolecule in the cell and 10–100 times higher ligand concentration in the syringe.
Experimental design considerations include optimizing the c-value (product of receptor concentration and Ka), selecting injection volumes, and determining the heat of dilution by injecting ligand into buffer alone. Buffer composition, pH, and temperature must be matched between sample and reference.
Applications include characterizing enzyme-inhibitor interactions, protein-protein binding, protein-DNA interactions, and antibody-antigen recognition. ITC also measures enzyme kinetics, membrane protein interactions, and nanoparticle-protein corona formation.
