Calorimétrie isotherme de titration

Caractérisation des interactions intermoléculaires grâce aux iTC200 et VP-ITC. Determination du profile thermodynamique complet : constante d'affinité, stoechiométrie, enthalpie et entropie.

Isothermal Titration Calorimetry (ITC) is a well-known and very powerful biophysical method to study intermolecular interaction between two compounds. It works by directly measuring the heat that is either released or absorbed during a biomolecular binding event. During typical ITC experiment small aliquots of titrant (compound one) are sequentially injected into a cell containing the second compound until its full saturation. The resulting heat produced by interacting molecules after each injection is measured at constant temperature, treated and analyzed using special software. ITC used in quantitative studies of a wide variety of biomolecular interactions.

ITC is the only technique that can simultaneously determine all binding parameters in a single experiment. Requiring no modification of binding partners, either with fluorescent tags or through immobilization, ITC measures the affinity of binding partners in their native states. To get all binding parameters experimental data should be fitted using the theoretical binding models to experimental curves by varying the free parameters of the model (such as stoichiometry, binding constant, enthalpy etc.). This is the key point of determining the thermodynamical parameters of intermolecular interaction using ITC. Thus, for proper data analysis, it is necessarily to choose theoretical binding model correctly. The main obstacle faced by researchers using ITC is the very limited set of binding models provided by manufacturers. Together with the fact that ITC has become widely used by non expert researchers, the lack of specialists in biology laboratories able to build and implement new mathematical models of interaction, frequently leads to significant slowdown of data analysis or even to misinterpretation of obtained data.

Measuring heat transfer during binding enables accurate determination of binding constants (KD), reaction stoichiometry (n), enthalpy (∆H) and entropy (ΔS). This provides a complete thermodynamic profile of the molecular interaction. ITC goes beyond binding affinities and can elucidate the mechanisms underlying molecular interactions. This deeper understanding of structure-function relationships enables more confident decision making in hit selection and lead optimization.