Isothermal Titration Calorimetry is a biophysical technique used to measure the heat change that occurs when two molecules interact with each other. It provides information on binding stoichiometry, affinity and thermodynamic parameters that drive the binding process. It can be used to characterize the interactions of small molecules, proteins, antibodies, nucleic acids, lipids etc. The PEAQ-ITC Automated system is equipped with an autosampler for walk-away operation.
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The ITC instrument consists of two cells (sample and reference cell) surrounded by an adiabatic jacket. As the binding partners are mixed within the sample cell, heat is either released or absorbed as a result of the redistribution of non-covalent bonds. ITC monitors the heat changes by measuring the differential power that is applied to the cell heaters in order to maintain zero temperature difference between sample and reference cell. The reference cell usually contains water, which has the same heat capacity as most of the sample buffers. The sample cell contains one of the binding partners (often, but not necessarily a macromolecule) and a stirring syringe which holds the other binding partner (often, but not necessarily the ligand). During the experiment, ligand is injected into the sample cell, typically in 0.5-3 µL aliquots. Each injection results in a heat pulse that is plotted against time and normalized for concentration to generate a titration curve of kcal/mol vs. molar ratio (ligand/sample). The resulting isotherm is fitted to a binding model and analysed to give thermodynamic parameters like affinity (KD), stoichiometry (N), entropy (∆S) and enthalpy of interaction (∆H). Binding affinity is a combined function of the binding enthalpy (∆H) and the binding entropy (∆S). Binding enthalpy reflects the strength of the interaction due to hydrogen bond formation and van der Waals interactions. Binding entropy is a combination of the change in entropy from desolvation and conformational changes upon complex formation.
The system has a sample capacity of 384 samples, which corresponds to four 96 well plates with a throughput of up to 42 samples per 42 h. As it is a label-free in solution technique, it ensures the analysis of unaltered biomolecules in their native state over a broad dynamic range. Dissociation constants can be measured down to the picomolar range using direct or competitive binding techniques.
Typical applications are the determination of binding affinities and based on that the appropriate choice of (drug) candidates for further optimization, characterization of mode of binding, determination of stoichiometry or measurement of enzyme kinetics.