Principles
Isothermal titration calorimetry ( ITC) enables study of the thermodynamics of molecules binding to each other. This is a general method for studying the thermodynamics of any binding (association) process in solution. It detects and quantifies small heat changes associated with the binding and has the advantages of speed, accuracy and no requirement for either of the reacting species to be chemically modified or immobilised. The apparatus consists of a pair of matched cells (sample and reference) of approximately 2 cm 3 volume contained in a microcalorimeter (Figure 1a). One of the reactants (say the enzyme preparation) is added to the sample cell and the ligand (substrate, inhibitor or effector) is added via a stepper-motor-driven syringe. The mixture is stirred to ensure homogeneity. The reference cell contains an equal volume of reference liquid. A constant power of less than 1 mW is applied to the reference cell. This directs a feedback circuit activating a heater attached to the sample cell. The addition of the ligand solution causes a heat change due to the binding process and the dilution of both the enzyme and ligand preparations. If the reaction is exothermic, less energy is required to maintain the cell at constant temperature. If the reaction is endothermic, more energy is required. The power required to maintain a constant temperature is recorded as a series of spikes as a function of time (Figure 1b). Each spike is integrated to giveμcal s−1 and summed to give the total heat exchange per injection. The study is repeated with a series of increasing ligand concentrations and control experiments carried out replacing the ligand with buffer solution to allow the heat exchange (Δ H) associated solely with the addition of ligand to be calculated. A plot is then made of enthalpy change against the molar ratio of the ligand to enzyme. The plot is hyperbolic, from which it is possible to calculate enthalpy, free energy and entropy changes associated with the ligand binding and hence the dissociation constant, Kd , and stoichiometry of binding n.
Fig1. (a) Schematics of an isothermal titration calorimeter (ITC). (b) ITC data obtained for titrating calcium ions into a solution of a calcium-binding protein. The top panel shows the raw data. The area underneath each injection peak is equal to the total heat released for that injection. These data can be integrated to yield a plot of the integrated heat against the molar ratio of ligand added to protein (bottom panel). The dotted and dashed lines illustrate determination of stoichiometry at the point of inflection of the binding isotherm. In this example, the determination of x = 4.6 indicates a stoichiometry of five calcium ions per protein molecule.
Applications
Since ITC investigates thermodynamic parameters and kinetic information on molecular interaction processes in solution, a diverse range of interactions in biological systems can be studied, including proteins, peptides, DNA, carbohydrates, lipids, small molecules and cells. The technique allows label-free measurements of thermodynamics of binding reactions and results guide direct applications in many fi elds (life sciences, drug discovery, etc.). In particular, ITC has been successfully used for determination of rates of enzymatic reactions and their use in investigation of enzyme kinetics. A further important application of ITC has been in the study of the interconversion of protein conformations and the elucidation of the mechanism of allostery. An incremental increase of a component or an inhibitor addition allows the study of reaction mechanisms, but can also be used as an efficient tool to screen compound libraries of molecules. Combined with other techniques, for example chromatography, ITC may also be used to identify a target protein for a particular ligand within a biomolecular mixture.
