Isothermal titration calorimetry (ITC) is a physical technique used to determine the thermodynamic parameters of interactions in solution. It is most often used to study the binding of small molecules (such as medicinal compounds) to larger macromolecules (proteins, DNA etc.).
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ITC is a quantitative technique that can directly measure the binding affinity (Ka), enthalpy changes (ΔH), and binding stoichiometry (n) of the interaction between two or more molecules in solution. From these initial measurements Gibbs energy changes (ΔG), and entropy changes (ΔS), can be determined using the relationship:
(where R is the gas constant and T is the absolute temperature).
An isothermal titration calorimeter is composed of two identical cells made of a highly efficient thermal conducting and chemically inert material such as Hastelloy alloy or gold, surrounded by an adiabatic jacket[1]. Sensitive thermopile/thermocouple circuits are used to detect temperature differences between the reference cell (filled with buffer or water) and the sample cell containing the macromolecule. Prior to addition of ligand, a constant power (<1 mW) is applied to the reference cell. This directs a feedback circuit, activating a heater located on the sample cell[2]. During the experiment, ligand is titrated into the sample cell in precisely known aliquots, causing heat to be either taken up or evolved (depending on the nature of the reaction). Measurements consist of the time-dependent input of power required to maintain equal temperatures between the sample and reference cells.
In an exothermic reaction, the temperature in the sample cell increases upon addition of ligand. This causes the feedback power to the sample cell to be decreased (remember: a reference power is applied to the reference cell) in order to maintain an equal temperature between the two cells. In an endothermic reaction, the opposite occurs; the feedback circuit increases the power in order to maintain a constant temperature (isothermic/isothermal operation).
Observations are plotted as the power needed to maintain the reference and the sample cell at an identical temperature against time. As a result, the experimental raw data consists of a series of spikes of heat flow (power), with every spike corresponding to one ligand injection. These heat flow spikes/pulses are integrated with respect to time, giving the total heat exchanged per injection. The pattern of these heat effects as a function of the molar ratio [ligand]/[macromolecule] can then be analysed to give the thermodynamic parameters of the interaction under study. It should be noted that degassing samples is often necessary in order to obtain good measurements as the presence of gas bubbles within the sample cell will lead to abnormal data plots in the recorded results. The entire experiment takes place under computer control.
Alternative ITC data analysis software: IC ITC