Isothermal Titration Calorimetry
Isothermal Titration Calorimetry (ITC) is an analysis technique used to understand the chemical dynamics of biomolecule interactions, usually between small and large molecules. The ITC technique has applications across the biochemical, medical, genetic, and pharmacological fields.
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What is ITC analysis used for?
Isothermal titration calorimetry is most commonly used in biochemical studies to analyze the interactions between larger molecules such as proteins, enzymes, and DNA, with small ligand molecules. The technique can measure factors such as the stoichiometry of reactions, as well as the binding constant, enthalpy, and entropy changes associated with these biochemical interactions. ITC is also used in research and industry to characterize biological processes, determine how enzymes operate, and develop drugs for specific bimolecular targets.
How does isothermal titration calorimetry work?
Whenever a chemical interaction between two species occurs, there will be a very small change in the temperature of the surrounding solution as energy is either absorbed or released. By measuring the temperature changes it is possible to determine the exact nature of the interaction that occurred.
ITC works off of this principle by measuring the power it takes to keep the temperatures of two cells constant, one as a reference and one containing the biomolecules of interest. A solution containing the ligand molecule to be tested is injected into the test cell and begins to interact with the main biomolecule inside. The calorimeter monitors the power needed to reset the minuscule changes in the temperature of the test cell. By repeating this process and logging temperature changes over time, information is obtained on the thermodynamic properties of the interaction
Sample requirements and preparation
ITC is performed in solution, so both the larger biomolecule and the test ligand will need to be in a suitable concentration for the test to be performed. Due to the nature of the types of molecules that are usually tested, a buffer solution may be implemented to ensure that pH is kept within a certain range throughout testing. In this case, both the biomolecule and ligand samples must be incorporated into the same buffer to minimize interference.
Advantages and limitations of ITC analysis
The key advantage of ITC is that it can be used to monitor biochemical interactions in a non-destructive way. It does not require the use of chemical markers or other preparation that could adversely affect the samples. It can monitor chemical interactions in solution, providing a suitable model for how interactions occur inside the body. Finally, ITC is non-specific, meaning that it can be used to monitor a very wide range of biological interactions, regardless of the exact chemicals involved in them.
The downside to ITC is that the signal provided during chemical interactions can be minuscule, to the point that in some cases it is difficult to detect. Therefore, a relatively large sample is often required to obtain substantial results; more so than in most other biochemical assays. To achieve the best results, each sample must be tested over a relatively long period, leading to a low general throughput of samples. Furthermore, some samples can be susceptible to contamination or changes in pH, so careful sample preparation is required.
ITC vs. DSC – what are the differences?
Differential Scanning Calorimetry (DSC) is a similar thermal analysis technique that is used to measure changes in a sample across a temperature range. Here, the sample is exposed to a temperature ramp. The energy required to change the temperature of the sample is recorded, helping to reveal how the sample absorbs energy compared to the reference.
The key difference is that in DSC the sample is exposed to a range of external temperatures, making it more suitable for measuring how processes will change under these conditions. This makes it ideal for studying the stability of proteins and other molecules under adverse conditions. ITC, on the other hand, is used at a set temperature and is more focused on monitoring the chemical processes themselves, rather than the effect of external conditions.
Suitable sample matrices
- Drug molecules
- Biological ligands
Typical applications of ITC analysis
- Determining the stoichiometry of biochemical reactions
- Calculating the binding constant for a ligand-macromolecule interaction
- Monitoring enthalpy changes during chemical processes
- Calculating entropy changes from binding reactions
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Frequently asked questions
ITC is most often used in the pharmacological industry to study the binding affinity of new drug candidates. The method has additional applications in biochemistry and biology.
ITC has a relatively low throughput and usually requires repetition with several protein and ligand samples before significant results are obtained.
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