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Pure Appl. Chem., 2008, Vol. 80, No. 9, pp. 2025-2040


Recommendations on measurement and analysis of results obtained on biological substances using isothermal titration calorimetry (IUPAC Technical Report)

Frederick P. Schwarz1, Timm Reinisch2, Hans-Jürgen Hinz2 and Avadhesha Surolia3

1 Center for Advanced Research in Biotechnology/National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA
2 Institut für Physikalische Chemie, Westfalische Wilhelms-Universität, Schlossplatz 4/7 D-48149, Münster, Germany
3 National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India-110067

Abstract: Isothermal titration calorimetry (ITC) is widely used to determine the thermodynamics of biological interactions including protein-protein, small molecule-protein, protein-DNA, small molecule-DNA, and antigen-antibody interactions. An ITC measurement consists of monitoring the transfer of heat between an analyte solution in a sample vessel and a reference solution in a reference vessel upon injection of a small aliquot of titrant solution into the sample vessel at a fixed ITC operating temperature. A binding isotherm is generated from the heat-transferred-per-injection data and values for the binding constants, the apparent binding enthalpies, and the apparent ratio of the amount of titrant to analyte for the binding reaction are then determined from fits of a binding model, whether it is a single site, identical multi-site, or an interacting multi-site binding model, to the binding isotherm. Prior to the fitting procedure, corrections should be made for contributions from extraneous heat of mixing determined separately from injections of the titrant into just the dialysate/buffer solution. Ultra-high binding constants, which cannot be directly determined from an ITC measurement, can be determined by a displacement ITC method where injections of the tight-binding titrant into a solution of a weaker-binding titrant-analyte complex displaces the weaker-binding titrant from the complex. The Michaelis and catalytic constants can be determined for an enzyme reaction from injections of a substrate or enzyme titrant into an enzyme or substrate analyte solution. Several binding reactions are suggested to check the operating performance of the ITC. The reporting of ITC results must be specific with regard to the composition of the titrant and the analyte solutions, the temperature, and the model used in the analysis.