Isothermal Titration Calorimetry
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Isothermal Titration Calorimetry (ITC) is a biophysical technique used to determine the thermodynamic parameters of (biochemical) interactions. 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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[edit] Thermodynamic Measurements
ITC is a quantitative technique that can directly measure the binding affinity (Ka), enthalpy (ΔH), and binding stoichiometry (n) of the interaction between two or more molecules in solution. From these initial measurements free energy (ΔG), and entropy (ΔS), can be determined using the relationship:
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- ΔG = -RTlnK = ΔH-TΔS
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(where R is the gas constant and T is the absolute temperature).
[edit] The Instrument
An isothermal titration calorimeter is composed of two identical cells made of a highly efficient thermal conducting material such as Hasteloy® alloy or gold, surrounded by an adiabatic jacket (reviewed by O’Brien et al, 2000). 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 (VP-ITC users manual, MicroCal Inc, Northampton, MA, USA. 2001). 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 in μcal/sec needed to maintain the reference and the sample cell at an indentical temperature. This power is given as a function of time in seconds. As a result, the raw data for an experiment consists of a series of spikes of heat flow (power), with every spike corresponding to a ligand injection. These heat flow spikes/pulses are integrated with respect to time, giving the total heat effect 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. The entire experiment takes place under computer control.
[edit] See also
[edit] References
O’Brien, R., Ladbury, J.E. and Chowdry B.Z. (2000) Isothermal titration calorimetry of biomolecules. Chapter 10 in Protein-Ligand interactions: hydrodynamics and calorimetry Ed. Harding, S.E. and Chowdry, B.Z, Oxford University Press. ISBN 0-19-9637466
VP-ITC Instruction Manual (2001). Microcal Inc., Northampton, MA. http://www.microcalorimetry.com
