In chemical thermodynamics, isothermal titration calorimetry (ITC) is a physical technique used to determine the thermodynamic parameters of interactions in solution. ITC is the only technique capable of comprehensively characterizing thermodynamic and kinetic profiles of a molecular interaction by simultaneously determining binding constants (), reaction stoichiometry (), enthalpy (), Gibbs free energy () and entropy () within a single experiment. 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 quantifies the heat released or absorbed during the binding process by incrementally adding one reactant (via a syringe) to another (in the sample cell) while maintaining constant temperature and pressure. Heat-sensing devices within the ITC detect temperature variations between two cells, transmitting this information to heaters that adjust accordingly to restore thermal equilibrium between the cells. This energy is converted into binding enthalpy using the information about concentrations of the reactants and the cell volume. Compared to other calorimeters, ITC does not require any correctors since there is no heat exchange between the system and the environment. ITC is also highly sensitive with a fast response time and benefits from modest sample requirements. While differential scanning calorimetry (DSC) can also provide direct information about the thermodynamic of binding interactions, ITC offers the added capability of quantifying the thermodynamics of metal ion binding to proteins.
History of ITC The history of ITC can be traced back to the 1930s when isothermal calorimetry was used to study chemical reactions. In 1965 Christensen and Izatt introduced titration calorimetry as a method for simultaneously determining the equilibrium constant and enthalpy. The ITC technique was then 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. In 1978, Beaudette and Langerman conducted one of the earliest calorimetric binding studies using a small volume isoperibol titration calorimeter and a decade later, in 1989, Wiseman, Williston, Brandts, and Lin demonstrated its application in biological systems, marking the beginning of titration calorimetry as a valuable tool for studying biological equilibria. Originally, ITC was 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. Its application has now broadened, aided by modern improvements, making it possible to measure the heat effects as small as 0.1 μcal (0.4 μJ) and determine the binding constants (K) as high as 108–109 M−1.
Thermodynamic measurements
ITC is a quantitative technique that can determine the binding affinity (), reaction enthalpy (), and binding stoichiometry () of the interaction between two or more molecules in solution. This is achieved by measuring the enthalpies of a series of binding reactions caused by injections of a solution of one molecule to a reaction cell containing a solution of another molecule. The enthalpy values are plotted over the molar ratios resulting from the injections. From the plot, the molar reaction enthalpy , the affinity constant () and the stochiometry are determined by curve fitting. The reaction's Gibbs free energy change () and entropy change () 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. A steep sigmoidal curve signals a strong binding whereas a less steep sigmoidal curve points to a weaker binding. 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.Quick Start: Isothermal Titration Calorimetry (ITC) (2016). TA Instrument. New Castle, DE.VP-ITC Instruction Manual (2001). Microcal Inc., Northampton, MA. 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.Stevenson, M. J. (2016) Thermodynamic studies of Cu(I) and other d10 metal ions binding to proteins in the copper homeostasis pathway and the organomercurial detoxification pathway. Dartmouth College. If it does not fall within the above range, the thermodynamic values obtained from the experiment are less robust to interpretation.
Instrumental measurements
An isothermal titration calorimeter is composed of two identical cells made of a highly efficient thermally conducting and chemically inert material such as Hastelloy alloy or gold, surrounded by an adiabatic jacket. In this setup, one of the cells, the sample cell, holds the solution of one reactant, and the other cell, the reference cell, is filled with either a buffer or water.
The second reactant, which has a higher concentration than the first, is placed in a syringe and gradually injected into the sample cell in small aliquots throughout the titration process. This process is semi-automated and the sensitive thermopile/thermocouple circuits are used to detect temperature differences between the reference cell (filled with buffer or water) and the sample cell containing the macromolecule. Prior to addition of ligand, a constant power (<1 mW) is applied to the reference cell. This directs a feedback circuit, activating a heater located on the sample cell. During the experiment, ligand is titrated into the sample cell in precisely known aliquots, causing heat to be either taken up or evolved (depending on the nature of the reaction). Measurements consist of the time-dependent input of power required to maintain equal temperatures between the sample and reference cells.
In an exothermic reaction, the temperature in the sample cell increases upon addition of ligand. This causes the feedback power to the sample cell to be decreased (remember: a reference power is applied to the reference cell) in order to maintain an equal temperature between the two cells. In an endothermic reaction, the opposite occurs; the feedback circuit increases the power in order to maintain a constant temperature (isothermal operation).
Observations are plotted as the power needed to maintain the reference and the sample cell at an identical temperature against time. As a result, the experimental raw data consists of a series of spikes of heat flow (power), with every spike corresponding to one ligand injection. These heat flow spikes/pulses are integrated with respect to time, giving the total heat exchanged per injection. By integrating each peak from the baseline, the total heat associated with each injection is obtained, including both reaction-specific and non-reaction-related contributions. The pattern of these heat effects as a function of the molar ratio [ligand]/[macromolecule] can then be analyzed to give the thermodynamic parameters of the interaction under study.
Experimental Procedures
In order to accurately and precisely measure the thermodynamic parameters using ITC, certain procedures must be followed, involving instrument set up, parameter configuration, sample loading, buffer selection, and instrument cleaning. To obtain an optimum result, each injection should be given enough time for a reaction equilibrium to reach. Degassing samples is often necessary in order to obtain good measurements as the presence of gas bubbles within the sample cell will lead to abnormal data plots in the recorded results. The entire experiment takes place under computer control.
Direct titration is performed most commonly with ITC to obtain the thermodynamic data, by binding two components of the reaction directly to each other. However, many of the chemical reactions and binding interactions may have higher binding affinity above what is desirable with the c-window. To troubleshoot the limitation of c-window and conditions for certain binding interactions, various different methods of titration can be performed. In some cases, simply doing a reverse titration of changing the samples between the injection syringe and sample cell can solve the issue, depending on the binding mechanism. However, the process of introducing a ligand to a macromolecule is distinct from the process of adding a macromolecule to a ligand. While the binding equilibrium remains unchanged in both direct and reverse titrations, the route to equilibrium and the accessible binding states varies, particularly when one molecule possesses multiple binding sites for the other. Most of the high or low affinity bindings require chelation or competitive titration. This method is done by loading pre-bound complex solution in the sample cell and chelating one of the components out with a reagent of higher observed binding affinity within the desirable c-window.
In order to ensure optimum instrument stability, the ITC instrument should be powered on at least one day before use. Samples should ideally be pre-equilibrated to approximately 2 °C below the target experimental temperature to reduce stabilization time after loading, although starting at the exact experimental temperature is also an option. For instrument cleaning, sample cell should be rinsed with the experimental buffer and dried under vacuum, and any remaining rinsed solution should be discarded manually with a syringe. Then, the sample cell is filled with the experimental solution and the reference cell with either high-purity water or the same buffer. To prevent air contamination, gas-tight Hamilton syringes are used, ensuring the needle is positioned near the bottom of the sample cell before dispensing the liquid slowly. Experimental parameters such as the number of injections, initial injection volume, subsequent injection volumes, temperature, reference power, stirring speed, spacing, initial delay, and filter period should be adjusted according to the specific study.
If the experiment is to be repeated, the syringe should be emptied, with the solution either discarded or saved for further analysis. Before refilling with the same solution, the syringe needle should be externally rinsed and dried with lint-free paper, without the need for an internal wash. However, when switching to a different reagent or concentration, the syringe must be thoroughly washed, flushed with ethanol or methanol, and carefully dried under vacuum-using a ThermoVac for VP-ITC or the integrated vacuum systems in other models. The sample cell should be emptied using the loading syringe and rinsed multiple times with water.
Analysis and interpretation
Post-hoc analysis and proton inventory It is necessary to conduct a post-hoc analysis to determine the buffer or solvent-independent enthalpy from the experimental thermodynamics. The collected experimental data reflects not only the binding thermodynamics of the interaction of interest, but any contributing competing equilibria associated to it. A post-hoc analysis can be performed to determine the buffer or solvent-independent enthalpy from the experimental thermodynamics, by simply going through the process of Hess' law. Below example shows a simple interaction between a metal ion (M) and a ligand (L). B represents the buffer used for this interaction and H+ represents protons.M - B <=> M + B -\Delta H_{MB}
L - H <=> L + H+
H+ + B <=> H - B
M + L <=> M - L