Theory and Practice of Isothermal Titration Calorimetry
contributed by Malvern Panalytical |
This white paper takes you through ITC principles and gives details and tips on a broad application range from studies of a simple 1:1 interactions to complex binding mechanisms and linked equilibria.
Isothermal titration calorimetry (ITC) is an analytical technique that has become the “gold standard” for studying intermolecular interactions. As its name indicates, it is a titrimetric technique, that is, a volumetric laboratory method for quantitative chemical analysis (traditionally intended to determine the unknown concentration of an identified analyte) where a reagent solution, the titrant, is made to react with a solution of analyte or titrand. The ITC instrument measures the heat produced or consumed by the interaction reaction along the titration to determine the endpoint (related to the interaction stoichiometry). By performing the titration at constant temperature and pressure, a single ITC experiment provides information on the equilibrium association constant, the binding enthalpy and the stoichiometry, from which the Gibbs energy and the entropy of binding can be readily calculated. Thus, a single ITC experiment gives direct access to the main thermodynamic potentials associated with the interaction process: Gibbs energy, enthalpy and entropy.
The high sensitivity of modern ITC instruments allows monitoring the formation of non-covalent complexes where molecules interact through a combination of multiple weak interactions: hydrogen bonds, electrostatic interactions, van der Waals interactions, and hydrophobic interactions, mainly. Then, it is possible to study the interaction between any type of molecules from ions and polymers to nanoparticles and biomolecules. Much of the ITC applications are focused on biomolecular interactions, such as the formation of well-defined protein-ligand, protein-protein, protein-DNA, protein-membrane, or DNA-ligand binding complexes (1, 2).
ITC allows a comprehensive quantitative description of a given interaction by providing valuable information at different levels: 1) whether or not two given molecules interact; 2) the binding stoichiometry; 3) the binding affinity or strength of the complex formation through the association/dissociation constant and the binding Gibbs energy; and 4) the partition of the Gibbs energy of binding into enthalpic and entropic contributions. For a biomolecular interaction careful analysis of the experimental results, together with a well-planned set of experiments under different experimental conditions, provides additional information for further characterizing the interaction and addressing: 1) possible conformational changes coupled to the binding process; 2) additional concomitant association/dissociation processes (e.g. net exchange of protons and other ions, as well as water molecules) coupled to and modulating the binding process; and 3) cooperativity phenomena involving homotropic (same ligand) or heterotropic interactions (a different ligand). Thus, ITC gives access to direct information on the interaction, and also on the regulation and modulation of the biomolecular interaction.
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