Isothermal Titration Calorimetry
Almost all reactions between molecules, whether involving the breaking and making of covalent bonds or non-covalent interactions, result in either the absorption or release of heat. Isothermal Titration Calorimetery (ITC) provides a direct measurement of these heats and is particularly well suited for the study of the noncovalent interactions involved in protein – protein and protein – ligand interactions. Data from a single experiment can often provide a complete thermodynamic characterization of the interaction, including the enthalpy (∆H) and equilibrium constant (Ka) as well as the stoichiometry of the interaction, and these in turn can be used to derive the Gibbs free energy (∆G) and the entropy (?S) for the reaction. If the reaction is measured at multiple temperatures, the change in constant pressure heat capacity (∆Cp) can also be derived. Because heat is the quantity measured in ITC, the method requires no spectroscopic or isotopic labeling of any of the molecular constituents in the reaction. Measurements using ITC and BIAcore SPR technology overlap to some extent; however, unlike the BIAcore approach, ITC does not require immobilization of any of the species involved in the interaction. The near universal production/absorption of heat during chemical transformations, make it possible to use the ITC to measure the kinetics of some reactions as has been applied to the determination of Michaelis – Menten kinetic constants in some enzymatic reactions.
Advances in instrument design and sensitivity accompanied by the development of user friendly software have made reliable and accurate ITC measurements by non-specialists possible, thus expanding the range of biological interactions studied by calorimetric methods. The VP-ITC has a temperature range of 2 to 80 °C. Affinity constants from 100 to ~109 M-1 can be directly measurable by ITC with even higher affinities possible through competition assays. In addition to the normal mode of operation, the current instrument is capable of the single-injection method (SIM), which can facilitate higher throughput. Software is available for the measurement and interpretation of kinetic studies as well as binding measurements.