Experimental Techniques Explained: Isothermal Titration Calorimetry (ITC)

ITC graphs
You’re sat at your desk, reading a journal article. You see a graph that looks like this:
the first thing that comes to mind? WHAT ON EARTH DOES IT MEAN?!
At this point many people will skip the methods section of their article and head straight to the discussion- But these graphs and techniques aren’t so scary once you understand the science behind them.

What is ITC used for?
Isothermal Titration Calorimetry, or ITC, measures a very basic interaction- how does one thing bind to another? Imagine you have a protein, and want to measure how well it binds to its substrate. ITC allows you in a single experiment to gain a huge amount of information. Here are a few of the main things you can find out:

Enthalpy change (Termed ΔH): The total amount of energy that is released or gained during a reaction, in this case as heat energy
Entropy change (Termed ΔS): A measure of “disorder”. When your protein and substrate are separate they have high entropy- they can exist in many different ways, they can move around and rotate. When the protein and substrate bind their entropy is low, because there are few ways in which they can bind one another and they aren’t flexible to move around
Binding affinity: How strongly your protein and substrate will bind. This is defined numerically as Kb (Association/Binding rate) or Kd (Dissociation rate. Kd= 1/Kb). Low Kd, for example, means that very little of the protein-substrate complex will dissociate, so the binding must be strong
Stoichiometry (Termed n): This tells you how much of each reactant you need to make a certain amount of product.  Essentially, if you drew a reaction as an equation, it’s the numbers you put in front of each reactant and product to balance the equation.  With our protein/substrate, this allows us to quantify the number of places which the substrate can bind onto the protein (If 3 molecules of substrate + 1 molecule of sample become 1 complex you know that 1 molecule of protein in the sample has 3 binding sites).

How does it work?ITC pic simple
When two molecules interact, there is an enthalpy change. Either heat is given off by the reaction (This is called an exothermic reaction), or absorbed (This is called an endothermic reaction). The main principle of ITC is that two substances are kept under the same conditions- a sample and a reference. When you add a set amount of ligand (something you want to interact with the sample-this is in a high concentration, but a small volume) there will be a temperature change. The amount of power it takes to keep the reference chamber and sample chamber at the same temperature is what is recorded and converted into a “heat of interaction”.

When you add your first amount of ligand, there will be lots of binding sites available in the sample, so you will get your largest temperature change here (this is your ΔH value). As you keep adding set volumes of sample, the temperature “spikes” will decrease in size as there is less and less binding occurring with each addition (the previous molecules are still bound, so there are less binding sites available). When no more spikes appear, you know that your protein sample has been “saturated”.


Rajarathnam et al, 2014

These spikes are then used to plot a curve:

graph itc

Adapted from Rajarathnam et al, 2014

This information can be used to calculate a number of thermodynamic properties (some defined earlier) using some clever maths, the equations for which are shown below:

ITC equations gibbs

Click picture for more info on Gibbs Free Energy

You can also repeat your experiment at a number of different temperatures to calculate the heat capacity of binding, or ΔCp.  This value can tell you a lot about the structure of your protein and how it interacts with its surroundings (mainly water).  To do this you plot the ΔH values observed at each temperature on a graph.  Proteins are quite fragile molecules and they only work within certain temperature ranges, so when your line of best fit starts to curve this is the temperature at which your protein is unfolding.  This information can come in very handy.

So that, in a nutshell, is Isothermal Titration Calorimetry. I hope next time you see it mentioned in a journal article it seems a little less scary!



Rajarathnam K, & Rösgen J (2014). Isothermal titration calorimetry of membrane proteins – Progress and challenges. Biochimica et biophysica acta, 1838 (1), 69-77 PMID: 23747362

Ghai R, Falconer RJ, & Collins BM (2012). Applications of isothermal titration calorimetry in pure and applied research–survey of the literature from 2010. Journal of molecular recognition : JMR, 25 (1), 32-52 PMID: 22213449

Jing M, & Bowser MT (2011). Methods for measuring aptamer-protein equilibria: a review. Analytica chimica acta, 686 (1-2), 9-18 PMID: 21237304

O’Brien, R., Ladbury, J., Chowdhry, B. (2001). Isothermal Titration Calorimetry of Biomolecules. In: Harding, S., Chowdhry, B Protein-Ligand Interactions: Hydrodynamics and Calorimetry. Oxford: Oxford University Press. 263-286.

Image 1 from:

Alan Cooper (1998). Microcalorimetry of Protein-Protein Interactions Methods in Molecular Biology, 88, 11-22 DOI: 10.1385/0-89603-487-9:11


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