Isothermal Titration Calorimetry (ITC) Service
ITC is a technique that measures the heat released or absorbed during a biochemical reaction. The technique allows for the measurement of thermodynamic parameters such as enthalpy (∆H), entropy (∆S), and binding affinity (KD). In ITC, a small amount of one molecule (usually the ligand) is added stepwise to a solution containing the other molecule (usually the protein) while monitoring the heat changes using a highly sensitive calorimeter. The heat changes are then plotted against the molar ratio of the two molecules, giving a binding isotherm, which can be used to determine the thermodynamic parameters.
Competition ITC is a variant of the technique where a competing ligand is added in excess to the protein, and then the primary ligand is titrated into the mixture. This technique is used to determine the affinity of the primary ligand in the presence of the competitor. Competition ITC is particularly useful in drug discovery, where it can be used to determine the relative binding affinities of potential drugs to a target protein.
ITC measures the heat released or absorbed during a reaction, which can be related to the thermodynamic parameters of the reaction. The instrument typically consists of a sample cell containing the protein or macromolecule of interest and a reference cell containing the buffer or solvent used in the reaction. The sample cell is then filled with a solution containing the ligand, and the reaction is initiated by injecting small aliquots of the ligand solution into the sample cell while monitoring the heat changes.
Basic principle of isothermal titration calorimetry (Song et al., 2015).
One of the primary advantages of ITC is that it provides direct measurement of the thermodynamic parameters of a reaction. This allows for a more complete understanding of the reaction than other techniques such as fluorescence or absorbance spectroscopy, which only measure changes in the concentration of the reacting species. ITC is also a label-free technique, meaning that the interacting molecules do not need to be modified with a fluorophore or other label, which can be costly and time-consuming.
However, ITC also has some disadvantages. One of the main limitations of the technique is that it requires relatively high sample concentrations, typically in the micromolar to millimolar range. This means that the technique may not be suitable for weakly binding molecules, which can require concentrations that are difficult or impossible to achieve. ITC also requires a relatively large amount of sample, typically in the milligram range, which may not be feasible for some protein systems.
The data obtained from ITC experiments are typically analyzed using software that fits the data to a binding model to extract the thermodynamic parameters. The most commonly used binding models include the one-site, two-site, and independent binding models. These models assume that the binding is a simple interaction between two molecules and that there are no cooperativity or allosteric effects. However, in some cases, the binding may be more complex, and more sophisticated models may be required to extract the thermodynamic parameters accurately.
ITC has a wide range of applications in the field of biophysics, particularly in the study of protein-ligand and protein-protein interactions. One of the primary applications of ITC is in drug discovery, where it is used to determine the binding affinity of potential drug candidates to target proteins. ITC can also be used to study enzyme kinetics, protein folding, and protein stability. Another application of ITC is in the study of protein-protein interactions, where it can be used to determine the stoichiometry and thermodynamics of complex formation between two or more proteins.
Typical data obtained from isothermal titration calorimetry measurements of polystyrene nanoparticles (PS-NPs) titrated with human serum albumin (HSA) (Prozeller et al., 2019).
ITC is particularly useful for studying protein-ligand interactions because it provides direct measurement of the thermodynamic parameters of the interaction. This allows for a more complete understanding of the interaction, including the strength of the interaction, the enthalpy and entropy changes, and the stoichiometry of the interaction. ITC can also be used to determine the binding kinetics of the interaction by measuring the rate of heat exchange during the reaction.
ITC can also be used to study protein-protein interactions, which are often more complex than protein-ligand interactions. ITC can determine the stoichiometry and thermodynamics of the interaction, including whether the interaction is cooperative or allosteric. One of the challenges of studying protein-protein interactions using ITC is the requirement for large amounts of protein, typically in the milligram range. However, advances in protein expression and purification techniques have made this more feasible in recent years.