Isothermal titration calorimetry
[1][2] It is most often used to study the binding of small molecules (such as medicinal compounds) to larger macromolecules (proteins, DNA etc.)
The technique was developed by H. D. Johnston in 1968 as a part of his Ph.D. dissertation at Brigham Young University,[5] and was considered niche until introduced commercially by MicroCal Inc. in 1988.
Compared to other calorimeters, ITC has an advantage in not requiring any correctors since there was no heat exchange between the system and the environment.
[8] 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.
Prior to addition of ligand, a constant power (<1 mW) is applied to the reference cell.
[6] 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.
[5] In an exothermic reaction, the temperature in the sample cell increases upon addition of ligand.
In an endothermic reaction, the opposite occurs; the feedback circuit increases the power in order to maintain a constant temperature (isothermal operation).
[1] Observations are plotted as the power needed to maintain the reference and the sample cell at an identical temperature against time.
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.
To obtain an optimum result, each injection should be given enough time for a reaction equilibrium to reach.
To troubleshoot the limitation of c-window and conditions for certain binding interactions, various different methods of titration can be performed.
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.
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.
As a part of the analysis, a number of protons are required to calculate the solvent-independent thermodynamics.
Competition would include buffer interactions and other pH-dependent reactions depending on the experimental conditions.
For the past 30 years, isothermal titration calorimetry has been used in a wide array of fields.
In the old days, this technique was used to determine fundamental thermodynamic values for basic small molecular interactions.
[12] In recent years, ITC has been used in more industrially applicable areas, such as drug discovery and testing synthetic materials.
Although it is still heavily used in fundamental chemistry, the trend has shifted over to the biological side, where label-free and buffer independent values are relatively harder to achieve.
[15][16] ITC collects data over time that is useful for any kinetic experiments, but especially with the proteins due to constant aliquots of injections.
In terms of calculation, equilibrium constant and the slopes of binding can be directly utilized to determine the allostery and charge transfer, by comparing experimental data of different conditions (pH, use of mutated peptide chain and binding sites, etc.)
Membrane proteins are known to have difficulties with selection of proper solubilization and purification protocols.
[17] This feature also applies in studies of self-assembling proteins, especially in use of measuring thermodynamics of their structural transformation.
However, determining enthalpy changes and optimization of thermodynamic parameters are hugely difficult when designing drugs.
Binding metal ions to protein and other components of biological material is one of the most popular uses of ITC, since ovotransferrin to ferric iron binding study published by Lin et al. from MicroCal Inc.[21] This is due to some of the metal ions utilized in biological systems having d10 electron configuration which cannot be studied with other common techniques such as UV-vis spectrophotometry or electron paramagnetic resonance.
[8][10] It is also closely related to biochemical and medicinal studies due to the large abundance of metal binding enzymes in biological systems.
PVA assembly process can be measured thermodynamically as mixing of the two ingredients is an exothermic reaction, and its binding trend can be easily observed by ITC.
