Isothermal Titration Calorimetry: An essential tool for binding affinity measurements in life sciences research
What is Isothermal Titration Calorimetry?
Isothermal titration calorimetry (ITC) is established in biophysical characterization circles as the “gold standard” for measuring binding affinity interactions involving proteins, lipids, nucleic acids and other biopolymers.
Since there are so many ways to measure binding affinity, why has Isothermal Titration Calorimetry (ITC ) become the gold standard?
ITC (Isothermal Titration Calorimetry) is a direct, label-free binding assay that measures the heat change due to a binding interaction. Every binding event and chemical process gives off heat (exothermic) or absorbs heat (endothermic). This heat change is used to determine the binding affinity, as well as the enthalpy and entropy of binding.
Since heat change is being measured, there is no need to use a label, dye, probe or tag. Binding can be explored in essentially all biological buffers with additives like surfactants, salts, reducing agents, cofactors, stabilizers like glycerol, etc present.
However ITC (Isothermal Titration Calorimetry) was not always the “gold standard” for binding assays. When I was a graduate student of biochemistry in the early 1980s, ITC was only being performed in a handful of labs, using homemade instruments designed by calorimetrists. These ITCs required large sample volumes, and took a long time to perform an experiment. I did not even know this method existed during my graduate student days! Looking in Google Scholar I can find only 15 results for “isothermal titration calorimetry” prior to 1985.
ITC in the 1970s and 1980s was a “niche” instrument used by thermodynamics experts. Today ITC (Isothermal Titration Calorimetry) has become a core technology found in academic, industrial, and government labs around the world, used by life scientists to understand how and why biomolecules bind to each other. This chart shows the dramatic growth of publications citing MicroCal ITC systems from 1986 to today. When I joined MicroCal in 2001 as an applications scientist, there were about 150 articles that same year which cited use of a MicroCal ITC. In 2015 there were over 1,100!
There are many researchers who use ITC (Isothermal Titration Calorimetry) as their primary binding assay. Many other researchers use ITC to validate and confirm the binding affinity reported by another assay. It is often necessary to compare values from “orthogonal” assays which measure the same parameter by means of a different method, and having at least 2 different assays reporting the same binding affinity is important in a high quality publication.
ITC is also the only direct assay which can determine binding thermodynamics (enthalpy and entropy). Many researchers are interested in studying the thermodynamics of their interaction to be used in structural biology, structure-activity relationships, protein engineering and drug design. Most biophysical tools which provide thermodynamic data calculate van’t Hoff enthalpy; ITC (Isothermal Titration Calorimetry) is a direct measure of the calorimetric enthalpy.
ITC is primarily used to look at biomolecular interactions. However, ITC can also be used whenever there is a process/reaction involving a detectable heat change, and with samples of any molecular weight, from ions to large polymers. Below are some common uses of ITC:
- Guest-host binding involving cyclodextrin; for example, the study of surfactant-polymer interactions involved in paint formulation, drug delivery, skin care products, detergents, etc.
- Determination of critical micelle concentrations, and heats of dissociation of oligomeric molecules
- Characterization of nanoparticle formation and interactions with drugs and proteins. Nanoparticle characterization is important in drug delivery, diagnostics like quantum dots, and developing consumer products such as creams and lotions
- In enzyme kinetics assays, by measuring the enzymatic conversion of substrate to product by the power change of the ITC instrument due to product formation. Since ITC is label-free, it can replace more cumbersome and time-consuming enzyme kinetics assays that require labels, chromatography, and/or multiple steps. ITC enzyme assays are used in academic research, as well as the R&D labs of biofuel companies to look at biomass conversion
And ITC applications continue to be developed in biomedical research, food science, and industrial applications.
John Brandts and his colleagues at MicroCal Inc. in Northampton Massachusetts (now part of Malvern Instruments) designed the OMEGA ITC, the first commercially available isothermal titration microcalorimeter, in 1988. OMEGA ITC launched the modern era of ITC, and scientists started to incorporate ITC experiments in their biophysical characterization studies. There are still some OMEGA ITCs functioning in the field today, and researchers still publish data from their thirty-year-old instruments.
The new MicroCal PEAQ-ITC (launched in 2015), the 5th generation ITC from MicroCal/Malvern, continues the tradition of high quality, user friendly ITCs with innovations in instrument design, throughput and data analysis.
If you would like more information on ITC in life sciences research, below are links to three recent publications which contain review articles and practical guides on experimental design and data analysis:
- Biocalorimetry, edited by JB Chaires, LD Hansen, S Keller, CA Brautigam, H Zhao and P Schuck, Methods Vol 76, 2015 (published by Elsevier)
- Calorimetry, edited by A. Feig, Methods in Enzymology Vol 567, 2016 (published by Elsevier)
- A special edition of Biochim Biophys Acta, entitled Microcalorimetry in the BioSciences – Principles and Applications, edited by F. Bou-Abdallah, (published in 2015, to be printed March 2016)
There are also many ITC application notes available on the Malvern web site:
- Analysis of demicellization data from isothermal titration calorimetry
- Microcalorimetry as a tool for structural biology
- Application of microcalorimetry in drug discovery at Exelixis
- Targeting protein/protein interactions in drug design
- Applications of Isothermal Titration Calorimetry and Differential Scanning Calorimetry in biopharmaceutical formulation development
- Revealing kinase inhibitor mechanisms: ITC leads the way
- Multiple applications for the Malvern MicroCal Auto-iTC200 in a fragment-based drug discovery campaign
- From assay development to lead optimization using Isothermal Titration Calorimetry