Isothermal Titration Calorimetry (ITC) is a technique for understanding molecular interactions. This method measures the heat changes that occur when molecules bind together. It provides a comprehensive thermodynamic profile of the interaction without the need for labels or modifications to the molecules. It studies interactions directly in solution, mimicking physiological conditions. The technique is versatile, applicable across various molecular systems, from small drug candidates binding to large proteins to protein-protein interactions. Its ability to quantify binding events based on heat makes it a valuable tool in molecular science.
How ITC Works
An ITC instrument uses two identical cells: a reference cell and a sample cell, both maintained at a constant temperature within an insulated chamber. The reference cell contains buffer, while the sample cell holds a solution of the macromolecule, such as a protein. This setup ensures any temperature differences observed are due to binding.
A precise amount of a second molecule, the titrant, is incrementally injected into the sample cell. As the titrant binds to the macromolecule, heat is either absorbed (an endothermic reaction) or released (an exothermic reaction). This heat change causes a slight temperature difference between the sample and reference cells.
The calorimeter then measures the power required to return the sample cell to the same temperature as the reference cell. This power adjustment maintains the isothermal condition. The heat measured during each injection is directly proportional to the binding occurring between the two molecules.
As more titrant is added and the macromolecule becomes saturated, less heat is generated or absorbed with subsequent injections. Plotting the heat change per injection against the cumulative amount of titrant added generates a binding isotherm. This curve, representing heat flow, serves as raw data for extracting quantitative information about the molecular interaction.
What ITC Reveals
ITC experiments yield several quantitative parameters that offer insights into molecular interactions. One primary output is the binding affinity, often expressed as the dissociation constant (Kd). A lower Kd value signifies a stronger interaction, meaning they bind tightly and dissociate less readily.
The technique also determines the stoichiometry (n) of the binding reaction. This value indicates the ratio at which molecules interact, for example, one drug molecule binding to one protein active site. This ratio is important for characterizing the molecular complex.
Beyond affinity and stoichiometry, ITC provides a thermodynamic profile of the binding event, including the enthalpy change (ΔH) and entropy change (ΔS). The enthalpy change reflects the heat absorbed or released during binding, revealing types of bonds formed or broken, like hydrogen bonds or van der Waals interactions. A negative ΔH indicates an exothermic reaction (heat released), while a positive ΔH indicates an endothermic reaction (heat absorbed).
The entropy change (ΔS) describes the change in disorder upon binding. An increase in entropy (positive ΔS) can indicate the release of ordered water molecules or conformational changes that increase molecular flexibility. Together, these thermodynamic parameters reveal the underlying forces driving the molecular interaction, providing a picture beyond just binding strength.
Applications of ITC
Isothermal Titration Calorimetry applies across scientific disciplines, especially for molecular recognition. In drug discovery, ITC identifies drug candidates by quantifying their binding to disease-related proteins. It also optimizes lead compounds, refining chemical structures for improved binding.
The technique studies protein-ligand interactions, including how proteins interact with small molecules, ions, or other proteins. This elucidates biological pathways and mechanisms. ITC also investigates enzyme kinetics, providing binding information for substrates or inhibitors, valuable for understanding catalytic mechanisms and designing modulators.
ITC studies nucleic acid interactions, such as DNA or RNA with proteins, small molecules, or other strands. This aids understanding gene regulation and genetic material function. The method also extends to materials science and biophysics, characterizing interactions relevant to biomaterials, nanoparticle binding, or other biophysical systems.