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Concept
Titrage calorimétrique isotherme
Résumé
In chemical thermodynamics, 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.) in a label-free environment. It consists of two cells which are enclosed in an adiabatic jacket. The compounds to be studied are placed in the sample cell, while the other cell, the reference cell, is used as a control and contains the buffer in which the sample is dissolved.
The technique was developed by H. D. Johnston in 1968 as a part of his Ph.D. dissertation at Brigham Young University, and was considered niche until introduced commercially by MicroCal Inc. in 1988. Compared to other calorimeters, ITC has an advantage in not requiring any correctors since there was no heat exchange between the system and the environment.
ITC is a quantitative technique that can determine the binding affinity (), enthalpy changes (), and binding stoichiometry () of the interaction between two or more molecules in solution. This is achieved from integrating the area of the injection peaks and plotting the individual values by molar ratio of the binding event versus \Delta H (kcal/mol). From these initial measurements, Gibbs free energy changes () and entropy changes () can be determined using the relationship:
(where is the gas constant and is the absolute temperature).
For accurate measurements of binding affinity, the curve of the thermogram must be sigmoidal. The profile of the curve is determined by the c-value, which is calculated using the equation:
where is the stoichiometry of the binding, is the association constant and is the concentration of the molecule in the cell. The c-value must fall between 1 and 1000, ideally between 10 and 100. In terms of binding affinity, it would be approximately from ~ within the limit range.
Source officielle
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