Isothermal Titration Calorimetry
Isothermal Titration Calorimetry (ITC) works by direct measurement of heat that is released or absorbed during a biomolecular binding event.
ITC is one of the rare techniques that are fully label and immobilization free. The methodology allows determination of the binding affinity (K D, stoichiometry (n), enthalpy (δH) and entropy (δS) between two or more molecules in solution.
A typical microcalorimeter consists of two cells, one of which contains a reference solution and the other contains the sample. The microcalorimeter has to keep the temperature of the two cells exactly the same. The energy used to keep the temperature in the cells the same is proportional to the heat used in the binding reaction.
An ITC experiment consists of successive additions of small volumes of a solution A (ligand) to a volume of solution B in the reaction cell. For both solutions the exact molar concentration are known. The interaction between the molecules either gives heat uptake or release and this is closely monitored. The figure depicts an exothermic reaction, which means that the sample cell becomes warmer compared to the reference cell. This causes a downward peak in the recorded signal. Compensating the heat change returns the signal to the base line. During the next injections the protein in the sample cell gets more saturated, is binding less ligand and the signal change decreases. At full saturation there is no heat change anymore and the experiment ends.
The area of each peak is integrated and plotted to the time of injection. A fitting to the isotherm gives the total enthalpy change (H), the dissociation constant (KD) and the stoichiometry (n).
Unlike SPR, ITC is not affected by high DMSO or glycerol concentrations. In addition, optical clarity is not important and there are no molecular weight limitations. On the other hand, ITC cannot be used for very low or very high affinity interactions since the change in heat capacity is not properly recorded.
ITC is a quantitative technique that can determine the thermodynamics of an interaction in solution. From these measurements, Gibbs energy (ΔG) and entropy (ΔS) can be determined using the relationchip ( R: gas constant; T: absolute temperature).
ΔG (Gibbs energy) is a measure of the change in free energy. ΔG is negative for spontaneous processes and the more negative the higher the affinity is, between the binding partners. ΔH (enthalpy change) is a measure of the energy content of the bonds broken and created. Hydrogen bonds contribute the most to the enthalpy change; therefore it is also dependent on the solvent in which the experiment is done. A negative value for ΔH indicates favouring binding. ΔS (entropy change) is a measure of how the motional energy is distributed.
ITC can be used to study the interactions between:
- Protein – small molecules
- Enzyme – inhibitor
- Protein – protein
- Protein – DNA
- Protein – lipid
- Protein – carbohydrate
|MicroCal TM ITC systems||GE Healthcare|
|MMC 274 Nexus||Netzsch|
|(3)||Kastritis, P. L. and A. M. J. J. Bonvin On the binding affinity of macromolecular interactions: daring to ask why proteins interact. Journal of the Royal Society Interface 10: 20120835; (2013). Goto reference|
|(4)||Day, Y. S., C. L. Baird, R. L. Rich, et al. Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods. Protein Science 11: 1017-1025; (2002).|
|(5)||Freyer, M. W. and E. A. Lewis Isothermal Titration Calorimetry: Experimental Design, Data Analysis, and Probing Macromolecule/Ligand Binding and Kinetic Interactions. (2008). Goto reference|