Isothermal Titration Calorimetry | ITC
Every interaction between two molecules generates or absorbs heat, depending on the thermodynamic nature of the interaction. This heat change is measured in isothermal titration calorimentry (ITC) by titrating one interaction partner (usually present in a syringe) to the other interaction partner (usually present in a reaction cell). From the raw heat pulses binding isotherms are derived that deliver comprehensive information of the affinity, stoichiometry, and thermodynamics of molecular interactions.
ITC is widely used for quantifying binding affinity, for drug candidate selection, optimization, and validation, for measuring thermodynamics of molecular interactions, for confirmation of binding targets in small-molecule drug-discovery, for determination of binding specificity, or for validation of IC50 and EC50 values.
ITC directly measures the heat released or absorbed during molecular binding events and the concomitant formation of molecular complexes. The label-free, in-solution characteristic of ITC experiments allows for the direct and native-like determination of all important thermodynamic parameters that characterize a molecular interaction: the binding constant (KD), the reaction stoichiometry (n), the observed binding enthalpy (ΔHobs), the observed binding entropy (ΔSobs), the observed heat capacity of binding (∆Cobs) and finally the change in free enthalpy of binding (ΔG). Thus, ITC generates a complete thermodynamic profile of a molecular interaction and, for example, can help to differentiate between binding reactions that are driven by enthalpy (due to the formation of non-covalent interactions across the binding-interface) or by entropy (due to the release of water molecules from a binding pocket).
In order to determine the heat released or absorbed during molecular binding events an ITC apparatus comprises two coin-shaped cells: A reference cell, which is usually filled with water and a sample cell, which contains one of the two interaction partners. The two cells are kept at the same constant temperature so that the temperature difference (ΔT) is zero. Heat changes due to binding events in the sample cell are registered by the device by measuring the differential power (DP) that is required to maintain a temperature difference of zero between the sample and the reference cell. For example, in the case of an exothermic binding event, heat is released inside the sample cell and the heating power required to keep the sample cell at the predefined fixed temperature is lower than that for the reference cell. The other interaction partner (often called the ligand) is titrated into the sample cell via a rotating syringe, typically in aliquots of 0.5 to 2 µL. Each injection leads to a heat change in the sample cell, which is recorded as a heat pulse (see Figure 2). The heat pulses are integrated over time to generate a titration curve that related the heat (kcal/mol of injected ligand) to the molar ratio (amount of titrated interaction partner relative to amount of interaction partner in the sample cell). Fitting the resulting isotherm to different binding models (e.g. 1:1 binding) yields the thermodynamic parameters described above.
Here at 2bind, the state-of-the-art Malvern PEAQ-ITC platform is used. This system features exceptional sensitivity and excellent reproducibility. The low sample consumption enables the analysis of interactions in cases where only small amounts of proteins or other macromolecules are available. Typically, the sample cell is filled with 200 µL of one interaction partner at a concentration of 10 to 30 µM and the syringe is filled with 40 µL of the other interaction partner at a concentration of 100 to 300 µM. The PEAQ-ITC systems allows a throughput of about 6 to 10 measurements per working day. Interactions can be quantified in the temperature range from 25 to 80 °C.
- Determining enthalpic and entropic contribution to molecular interactions
- Determining changes in free energy upon molecular binding
- Determining stoichiometry of molecular interactions
- in various contexts:
protein-small molecule interactions
DNA-small molecule interactions
RNA-small molecule interactions
- No labeling required (close-to-native conditions)
- No immobilization required (measurements in solution)
- Low sample consumption for ITC standards (only 300 µl of a 20 µM protein solution required)
- Complete thermodynamic profiling (ΔH, ΔG, ΔS)
- Wide temperature range (from 10°C to 80°C)
- Tells binding characteristics (affinity and stoichiometry)
- Wide range of binding affinities (range from nM to mM affinities measurable)
- Wide molecule size range (small molecules to MDa)