Isothermal Titration Calorimetry (ITC) is a label-free quantification technique used in studies of a wide variety of biomolecular interactions. Isothermal Titration Calorimetry (ITC) works directly by measuring the heat released or absorbed during a biomolecular binding event. Isothermal Titration Calorimetry (ITC) is used to measure reactions between biomolecules. The methodology enables the determination of the binding affinity, stoichiometry, entropy, and enthalpy of the binding reaction in solution, without the need for labels. The technique was developed by H. D. Johnston in 1968 as part of his Ph.D. dissertation at Brigham Young University. It was considered niche until it was introduced commercially by MicroCal Inc. in 1988.
Isothermal Titration Calorimetry (ITC) Principle
Isothermal Titration Calorimetry (ITC) is an analytical technique often regarded as the gold standard for analyzing intermolecular interactions. Isothermal Titration Calorimetry (ITC) is a titrimetric method, a volumetric technique for quantitative chemical analysis, wherein a reagent solution, the titrant, reacts with an analyte or titrant solution.
Instrumentation
Isothermal Titration Calorimetry (ITC) 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. 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. 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. During the experiment, the 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.
Isothermal Titration Calorimetry (ITC) Applications
Isothermal Titration Calorimetry (ITC) can be used to measure the thermodynamic parameters of biomolecular interactions, including affinity (KA), enthalpy (ΔH), entropy (ΔS), and stoichiometry (n). Isothermal titration calorimetry (ITC) is an analytical technique used to measure the thermodynamics of interactions between molecules in solution. Isothermal Titration Calorimetry (ITC) is widely used in pharmaceutical, biochemical, and biophysical research for understanding the fundamentals of molecular binding events.
Isothermal Titration Calorimetry (ITC) Limitations
Isothermal Titration Calorimetry (ITC) requires a large number of samples, and only one ligand can be tested at a time. Isothermal Titration Calorimetry (ITC) has an intrinsic sensitivity. Typical heats (∆H) of complex reactions are 10 kcal/mol, which limits the measurements of Kd’s to interactions that are 10-7 M and greater. Isothermal Titration Calorimetry (ITC) requires a significant amount of material and the washing of instrument sample cells, which is a tedious task.
Conclusion
Isothermal Titration Calorimetry (ITC) is a powerful tool for determining the enthalpies of binding in various reactions, including protonation, coprecipitation, and protein-ligand binding. However, there is a need for more careful validation of all calorimeters, more meticulous experimental planning regarding the buffers, pHs, and temperatures used, and more precise interpretation of calorimetric data before reporting the thermodynamics of the binding reactions studied. Our recommendation is to select several reactions, such as 1) Tris base with nitric acid, 2) silver nitrate with sodium iodide or bromide, and 3) bovine carbonic anhydrase II with CBS, and to validate calorimeters by carrying out these reactions. Validation should be reported in the experimental section of every ITC manuscript.
