Isothermal Titration Calorimetry (ITC) is a powerful analytical tool that allows the measurement of binding affinities and of their related thermodynamic parameters. ITC directly measures the heat released or absorbed during molecular binding. Measurement of this heat enables direct accurate determination of a number of thermodynamic parameters in a single experiment: binding affinity (KD), stoichiometry (n), enthalpy (ΔH). From the former and the latter of these parameters we can derive as well the entropy (ΔS) of the reaction.
This technology presents a wide range of applications including the characterization of molecular interactions involving small molecules, proteins, antibodies, nucleic acids, lipids, metal ions, etc. ITC is as well useful in enzyme kinetics and inhibition/competition studies. No labelling or immobilisation are required and there are almost no buffer restrictions.
We use an iTC200 system from Microcal (GE Healthcare), one of the most sensitive isothermal titration calorimeters available. The sample cell is only 200 µL. It can directly measure sub-millimolar to nanomolar binding constants (10-7 to 10-9 M-1). Measure of nanomolar to picomolar binding constants (10-9 to 10-12 M-1) is also possible by using the competitive binding technique.
How much sample do I need?
You will need 300 μl of sample for the cell and 60 μl of ligand for the syringe. The sample and the ligand must be in the same buffer. Ideally, you should dialyse both against the same buffer. It is important to perform a control titration of ligand into buffer allowing subtraction of the observed heat of dilution from the observed binding data.
At which concentration ?
The appropriate protein and ligand concentrations to be used in the ITC cell can be estimated from the so-called c value:
Accurate determination of K requires C values to be between 1 and 1000. The concentration of ligand used in the seringue must be sufficiently high to ensure that by the end of the titration the protein in the cell is saturated.
A good start could be :
If you do not know the value of the K, assume a value of 1μM and start doing an experiment with 10 μM in the cell and 100 μM in the syringe.
Highly purified proteins with accurately determined concentrations are required for the correct determination of binding parameters.
At what working temperature?
Enthalpy (ΔH) is temperature dependent, the plot of ΔH versus temperature is linear with a slope corresponding to the change in heat capacity ΔCp. For a certain temperature ΔH will be zero. If the ITC experiment is done at such temperature almost no signal will be detected. In conclusion, temperature is a parameter to explore when doing an ITC experiment