Ask an Expert: Microcalorimetry

por Peter Gimeson, Wednesday, 15th September 2021

A versatile tool for the characterization of biomolecular interactions

Isothermal titration microcalorimetry (ITC) is widely used in the life sciences and is an essential tool in drug discovery and the study and regulation of protein interactions. It measures the binding affinity and thermodynamics of biomolecular interactions, helping to understand why interactions occur. The technology is based on the measurement of heat evolved or absorbed when complexes form between molecules.

With ITC one can measure all binding parameters in a single label-free, in-solution experiment. The parameters measured, include binding affinity (KD), reaction stoichiometry (n), enthalpy (ΔH), and entropy (ΔS). This reveals thermodynamic data. In turn, thermodynamic data reveal the forces that drive complex formation to describe function and mechanism at a molecular level.

The power of ITC

As shown in figure A, ITC determines thermodynamic properties including the stoichiometry of the interaction (n), the affinity constant (KD), change in enthalpy (ΔH), and change in entropy (ΔS).

Figure B shows thermodynamic signatures of three interactions that have the same binding energy (ΔG). The binding energy is related to the affinity. The binding affinity is a combined function of the binding enthalpy (ΔH) and the binding entropy (ΔS). Binding enthalpy reflects the strength of the interaction due to hydrogen bond formation and van der Waals interactions. Binding entropy is a combination of the change in entropy from desolvation and conformational charges upon complex formation.

ITC systems

Let´s dive a bit deeper into the theory. Isothermal titration microcalorimeters measure the heat change that occurs when two molecules interact. Heat is released or absorbed as a result of the redistribution and formation of non-covalent bonds when the interacting molecules go from the free to the bound state. ITC monitors these heat changes by measuring the differential power, applied to the cell heaters, required to maintain zero temperature difference between the reference and sample cells as the binding partners are mixed.

The reference cell usually contains water, while the sample cell contains one of the binding partners (the sample, often but not necessarily a macromolecule) and a stirring syringe that holds the other binding partner (the ligand).

The ligand is injected into the sample cell, typically in 0.5 to 2 μL aliquots until the ligand concentration is two- to three-fold greater than the sample. Each injection of ligand results in a heat pulse that is integrated with respect to time and normalized for concentration to generate a titration curve of kcal/mol vs molar ratio (ligand/sample). The resulting isotherm is fitted to a binding model to generate the affinity (KD), stoichiometry (n), and enthalpy of interaction (ΔH).

MicroCal ITC calorimeters

Malvern Panalytical developed their MicroCal ITC calorimeters specifically to meet the needs of life scientists. They directly measure the heat released or absorbed during a biomolecular binding event. The result is a direct, label-free measurement of binding affinity and thermodynamics in a single experiment. With a complete thermodynamic profile of the molecular interaction, ITC goes beyond binding affinities and can elucidate the mechanisms underlying molecular interactions.

Ask an Expert!

Microcal ITC systems are the ideal instruments for the measurement of a wide range of biomolecular interactions but, how is it working for you? During our next ´Ask an Expert!´ webinar, we will give a special focus to the benefits of PEAQ-ITC and PEAQ-DSC, the latest generation of MicroCal instruments, and the solutions they offer for addressing current bottlenecks. We will also discuss experimental practicalities and how to obtain quality data. Join us on October 5th and put your bottlenecks to the expert.

You can send in your questions and/or data prior to the webinar by emailing askanexpert@malvernpanalytical.com or using the #MPexpert hashtag on Twitter. Our in-house experts will then handle a selection of the incoming questions during the live webinar. In other words, it´s the ultimate way of improving your research.

There’s more

Malvern Panalytical is also planning a virtual DSC user meeting on October 27th and a virtual ITC user meeting in November. Do not miss this unique opportunity to gain knowledgeable user insight and share your experiences with both our expert panel and other users. Keep an eye on the event calendar.

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