Isothermal Titration Calorimetry | ITC

Overview

Interactions between biomolecules or macromolecules like proteins, nucleic acids, or lipids are essential for all cellular processes, ranging from replication, transcription, translation over protein biosynthesis and metabolism to signal transduction and cell-cell communication. The characterization of interactions between biomolecules is thus an important task in basic and applied life science, as well as in the pharmaceutical and biotech industry.

Every interaction between two molecules generates or absorbs heat, depending on the thermodynamic nature of the interaction. This heat change is measured in isothermal titration calorimentry (ITC) by titrating one interaction partner (usually present in a syringe) to the other interaction partner (usually present in a reaction cell). From the raw heat pulses binding isotherms are derived that deliver comprehensive information of the affinity, stoichiometry, and thermodynamics of molecular interactions.

ITC is widely used for quantifying binding affinity, for drug candidate selection, optimization, and validation, for measuring thermodynamics of molecular interactions, for confirmation of binding targets in small-molecule drug-discovery, for determination of binding specificity, or for validation of IC50 and EC50 values.

Technology

Isothermal titration calorimetry (ITC) is an essential tool for the study and analysis of molecular and macromolecular interactions between proteins, nucleic acids, lipids, and small molecule inhibitors or effectors. ITC is also widely used in drug discovery for hit validation, lead optimization, and elucidating mechanisms of action.

ITC directly measures the heat released or absorbed during molecular binding events and the concomitant formation of molecular complexes. The label-free, in-solution characteristic of ITC experiments allows for the direct and native-like determination of all important thermodynamic parameters that characterize a molecular interaction: the binding constant (KD), the reaction stoichiometry (n), the observed binding enthalpy (ΔHobs), the observed binding entropy (ΔSobs), the observed heat capacity of binding (∆Cobs) and finally the change in free enthalpy of binding (ΔG). Thus, ITC generates a complete thermodynamic profile of a molecular interaction and, for example, can help to differentiate between binding reactions that are driven by enthalpy (due to the formation of non-covalent interactions across the binding-interface) or by entropy (due to the release of water molecules from a binding pocket).

In order to determine the heat released or absorbed during molecular binding events an ITC apparatus comprises two coin-shaped cells: A reference cell, which is usually filled with water and a sample cell, which contains one of the two interaction partners. The two cells are kept at the same constant temperature so that the temperature difference (ΔT) is zero. Heat changes due to binding events in the sample cell are registered by the device by measuring the differential power (DP) that is required to maintain a temperature difference of zero between the sample and the reference cell. For example, in the case of an exothermic binding event, heat is released inside the sample cell and the heating power required to keep the sample cell at the predefined fixed temperature is lower than that for the reference cell. The other interaction partner (often called the ligand) is titrated into the sample cell via a rotating syringe, typically in aliquots of 0.5 to 2 µL. Each injection leads to a heat change in the sample cell, which is recorded as a heat pulse (see Figure 2). The heat pulses are integrated over time to generate a titration curve that related the heat (kcal/mol of injected ligand) to the molar ratio (amount of titrated interaction partner relative to amount of interaction partner in the sample cell). Fitting the resulting isotherm to different binding models (e.g. 1:1 binding) yields the thermodynamic parameters described above.

Here at 2bind, the state-of-the-art Malvern PEAQ-ITC platform is used. This system features exceptional sensitivity and excellent reproducibility. The low sample consumption enables the analysis of interactions in cases where only small amounts of proteins or other macromolecules are available. Typically, the sample cell is filled with 200 µL of one interaction partner at a concentration of 10 to 30 µM and the syringe is filled with 40 µL of the other interaction partner at a concentration of 100 to 300 µM. The PEAQ-ITC systems allows a throughput of about 6 to 10 measurements per working day. Interactions can be quantified in the temperature range from 25 to 80 °C.

Typical applications

  • Determining enthalpic and entropic contribution to molecular interactions
  • Determining changes in free energy upon molecular binding
  • Determining stoichiometry of molecular interactions
  • in various contexts:
    protein-protein interactions
    protein-DNA interactions
    protein-RNA interactions
    protein-small molecule interactions
    DNA-small molecule interactions
    RNA-small molecule interactions

Advantages

  • No labeling required (close-to-native conditions)
  • No immobilization required (measurements in solution)
  • Low sample consumption for ITC standards (only 300 µl of a 20 µM protein solution required)
  • Complete thermodynamic profiling (ΔH, ΔG, ΔS)
  • Wide temperature range (from 10°C to 80°C)
  • Tells binding characteristics (affinity and stoichiometry)
  • Wide range of binding affinities (range from nM to mM affinities measurable)
  • Wide molecule size range (small molecules to MDa)

FAQ – General

What kind of molecular interaction can I measure?

You can measure bi-molecular interactions between any kind of molecule: protein, DNA, RNA, small molecule compounds, lipids, carbohydrates.

Can I measure without labeling molecules?

Yes, ITC does not require any labeling prior to the measurement.

Can I measure without immobilizing molecules?

Yes, ITC does not require any immobilization on a surface prior to the measurement.

What type of information do I get?

The ITC technology is not only able to determine binding affinity and interaction stoichiometry, but also to generate a complete thermodynamic profile of the interaction including the binding enthalpy ΔH, the binding entropy ΔS, as well as the free enthalpy of binding ΔG.

Which range of affinities can be quantified?

ITC can determine binding affinities in a wide range from nanomolar (10-9) to millimolar (10-3) affinity.

Can I measure binding kinetics with ITC?

ITC measures equilibrium binding constants and it is this not possible to determine association and dissociation rates.

What is necessary for a successful determination of the thermodynamic parameters of an interaction?

It is necessary to accurately determine the concentration of the interaction partner in the syringe. Thus, it must be possible to precisely determine its concentration via spectroscopic means or color-based techniques.

FAQ – Samples

What are the required compound concentrations?

In general, the concentration of the ligand in the syringe should be 10-fold that of the interaction partner in the sample cell. In general, the concentration of the interaction partner inside the sample cell should be around 30-times KD. If KD is unknown, a best guess will do as well. The concentration of the interaction partner inside the syringe should be around 10*n-times that of the partner in the sample cell, where n is the stoichiometry of the reaction. The following concentrations are frequently used to determine KD values between 10 µM and 10 nM: The interaction partner that is present in the sample cell should be in the concentration range of 10 µM to 30 µM. The other interaction partner, which is titrated, should typically be in a concentration range approximately 10-fold higher, thus between 100 µM and 300 µM (under the assumption of a 1:1 binding stoichiometry). However, the necessary concentration range depends on the heat released or absorbed during the specific interaction.

How much sample do I need?

To fill the sample cell, 280 µL are needed. The minimum volume to fill the syringe is 40 µL, although we recommend that 60 µL be used to avoid bubbles or foaming during automatic loading of the syringe.

Can I measure the binding of small molecules?

Yes, ITC is not only able to characterize the interaction between macromolecules like proteins or nucleic acids, but also between macromolecules and small molecular inhibitors, effectors, or the like.

Do I need to prepare my samples in a specific way?

It is important for a successful and high-quality ITC experiment that both interaction partners are in identical buffer solutions. Other large heats of dilution can mask the actual heat generated or absorbed by the interaction. Usually it is best practice to thoroughly dialyze both samples (interaction partners) against the same buffer prior to the ITC experiment or to use desalting columns for buffer exchange.

FAQ – Assay Conditions

How high is the throughput?

Establishment of specific experimental conditions usually requires one working day. After that, depending on the number of injections and time spacing between injections, around 6-10 experiments are possible over a typical working day.

What is the temperature range?

10°C to 80°C.

Are there limitations to the buffers or additives I can use?

In general, the buffer or additive should not negatively affect the stability or homogeneity of the sample. Moreover, it is important to minimize artefactual heat by using a buffer with a low enthalpy of ionization (e.g. phosphate, citrate, or acetate buffers). Common biological buffers with amino groups such as TRIS or HEPES are not recommended for ITC due to their high heats of ionization. Also, avoid using DTT if possible, because DTT can cause high background heat. If a reducing agent is required nonetheless, use β-mercaptoethanol or TCEP at a maximum concentration of 1 mM.

Can I measure samples that contain DMSO?

Yes, this is possible. If one interaction partner (e.g. a small peptide dissolved in DMSO) contains DMSO, it has to be added to the solution of the second interaction partner (e.g. protein in the sample cell) to the same concentration in order to avoid buffer mismatch and high dilution heats. Usually 2-5 % of DMSO can be added to protein solutions in the short term.
+49 941 2000 0890
Customer Service
Am Biopark 11
93053 Regensburg
Germany

Get In Touch

2bind Work with the experts in biophysics!
 

info@2bind.com
© 2018 2bind GmbH. All Rights Reserved.

Contact Specialist