The Science Behind ITC: Measuring the Heat of Interaction. Isothermal Titration Calorimetry (ITC) excels in directly measuring the heat released or absorbed during a controlled biomolecular interaction within a solution. Two identical cells are meticulously kept at a constant temperature. One cell holds your sample molecule, while the other serves as a reference. Precise amounts of a binding partner are gradually injected into the sample cell. Every injection triggers either an uptake or release of heat, which ITC's sensitive detectors quantify.
This analysis yields a wealth of thermodynamic data with unparalleled accuracy: Binding Affinity KD (quantifies how strongly molecules bind), Enthalpy Change ΔH (reveals the energy change associated with binding), Entropy Change ΔS (disentangles the forces driving the interaction), and Stoichiometry (the ratio of how molecules bind to each other).
Equilibrium dissociation constant. Can be obtained by kinetic or classical equilibrium binding analysis. Provides information about the strength but not the dynamics of an interaction.
Binding stoichiometry (ratio of ligand/target in bound state)
Change of binding enthalpy (heat released per mole of ligand bound). Derived directly from binding isotherm. Indication of changes in hydrogen bonding and van der Waals interactions upon target-ligand binding.
Change of free energy of binding. Derived from KD and temperature.
Change of binding entropy. Derived from ΔH and ΔG. Indication of changes in hydrophobic effects and conformational changes upon target-ligand binding.
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Biomolecular research: Probe protein folding, enzyme mechanisms, macromolecular interactions, and intricate biological processes with exceptional detail. 2bind’s ITC services mean optimized workflows, tailored experimental designs, and in-depth data analysis, all geared towards achieving your specific goals.
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 parameters that characterize the thermodynamics of 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.
With ITC it is possible to measure bi-molecular interactions between almost any kind of molecule, most importantly proteins, DNA, RNA, small molecule compounds, lipids, carbohydrates, as long as these molecules can be brought into solution.
No, ITC does neither require any form of labeling of the test molecules, nor an immobilization to any surface. All experiments are performed fully label-free and in solution.
It is necessary to accurately determine the concentration of the interaction partners in the cell and the syringe. Thus, it must be possible to precisely determine their concentration via spectroscopic means or color-based techniques. Also, the assay buffer has to be carefully selected in order to minimize any potential background heat. Furthermore, it has to be ensured that target and ligand molecules are fully in solution and do not form any precipitates or aggregates before or during the ITC experiment. For this, you can benefit from 2bind’s vast experience in protein and small molecule-targeted solution and solubility optimization.
The concentration of the interaction partner inside the sample cell should be around 30-times KD. If KD is unknown, a best guess will do as well. The concentration of the interaction partner inside the syringe should be around 10*n-times that of the partner in the sample cell, where n is the stoichiometry of the reaction. The following concentrations are frequently used to determine KD values between 10 µM and 10 nM: The interaction partner that is present in the sample cell should be in the concentration range of 10 µM to 30 µM. The other interaction partner, which is titrated, should typically be in a concentration range approximately 10-fold higher, thus between 100 µM and 300 µM (under the assumption of a 1:1 binding stoichiometry). However, the necessary concentration range depends on the heat released or absorbed during the specific interaction. If these requirements are a concern, it is best to consider alternative methods like TRIC, Spectral Shift, nanoDSF, or GCI that have much lower sample consumption.
Yes, of course. It is important for a successful and high-quality ITC experiment that both interaction partners are in identical buffer solutions. Other large heats of dilution can mask the actual heat generated or absorbed by the interaction. Usually, it is best practice to thoroughly dialyze both samples (interaction partners) against the same buffer prior to the ITC experiment or to use desalting columns for buffer exchange. 2bind performs all necessary dialysis or other buffer-exchange steps in-house.
In general, assay buffers or additives should not negatively affect the stability or homogeneity of the sample. Moreover, it is important to minimize artefactual heat by using a buffer with a low enthalpy of ionization (e.g. phosphate, citrate, or acetate buffers). Common biological buffers with amino groups such as TRIS or HEPES are not recommended for ITC due to their high heats of ionization. Also, avoid using DTT if possible, because DTT can cause high background heat. If a reducing agent is required nonetheless, use β-mercaptoethanol or TCEP at a maximum concentration of 1 mM.
No, DMSO is not problematic. If one interaction partner solution contains some DMSO (e.g. a small molecule dilution with 2% DMSO), DMSO must be added to the solution of the second interaction partner (e.g. protein in the sample cell) to the same concentration to avoid buffer mismatch and high dilution heats. Usually 2-10 % of DMSO can be added to protein solutions in the short term.
Isothermal Titration Calorimetry (ITC) is a truly label-free, in-solution method. It thus brings along several advantages:
