Isothermal Titration Calorimetry (ITC)

Isothermal Titration Calorimetry (ITC) is a label-free method for measuring binding of any two molecules that release or absorb heat upon binding. ITC can be used to measure the thermodynamic parameters of biomolecular interactions, including affinity (Ka), enthalpy (ΔH), entropy (ΔS), and stoichiometry (n).

 

The CMI has a Microcal ITC200 from Malvern.

For information on access fees, policies and getting started at the CMI, see the CMI Access Page.

ITC Theory

Energetically favorable binding reactions have negative free energy values, ΔG = RTlnKD.  ΔG, has two energetic components, enthalpy (ΔH) and entropy (ΔS) and their contributions are expressed as:

ΔG = ΔH -TΔS.  

In an ITC experiment, ΔH of binding is measured directly.  The ITC200 microcalorimeter has two cell: one contains water and acts as a reference cell, the other contains the sample. The microcalorimeter needs to keep these two cells at exactly the same temperature during the course of an experiment. Heat sensing devices detect temperature difference between the cells when binding occurs in the sample cell and give feedback to the heaters, which compensate for this difference and return the cells to equal temperature.  

Data collection

To perform an experiment, the calorimeter is set to the experimental temperature. The sample cell is filled and the injection syringe is loaded with ligand. The syringe is inserted into the sample cell and and a series of small aliquots of ligand are injected into the sample solution, while stirring. If there is a binding of the ligand to the sample, heat changes of a few millionths of a degree Celsius are detected and measured.  The microcalorimeter measures all heat released until the binding reaction has reached equilibrium.  The instrument records the differential power (in µcal/sec) applied to the sample cell vs. the reference cell to keep the two cells at the same temperature.  

Results 

The example below shows an exothermic reaction, which means the sample cell becomes warmer than the reference cell and causes a downward peak in the signal. As the temperature of the two cells equilibrate, the signal returns to its starting position. As the molar ratio between the ligand and sample increases, the sample becomes saturated and fewer injected ligand molecules bind and the heat change decreases.  By integrating the area of the injection peaks and plotting molar ratio vs. ∆H (kcal/mol), an ITC binding curve can be fit for binding affinity (KA = 1/KD), reaction stoichiometry (n), enthalpy (∆H) and entropy (ΔS).  The relative contribution of enthalpy and entropy to the binding energetics can provide insights into molecular mechanism:  ∆H is an indication of changes in hydrogen bonding and van der Waals interactions, and ∆S is an indication of changes in hydrophobic effects and conformational changes.

 

isothermal titration calorimetry

Image from Malvern Instruments.

 

 

Supplies provided by the CMI:

  • tubes for syringe filling
  • water and methanol for instrument cleaning

Samples

  • Molar concentrations must be known accurately. 
    • Errors in cell concentration affect the stoichiometry (n). 
    • Errors in the syringe concentration affect n, Ka and ΔH.
  • Filter or spin down your samples to remove precipitates and debris.
  • Don't use samples that are not soluble and well-behaved.
  • Sample in the cell
    • ≥ 300 µl  (202 µl + ~ 100 µl for filling)
    • ≥ 5-10 µM (need high enough concentration to measure heat) AND ≥ 10X Kd 
  • Sample in the syringe
    • ≥ 120 µl (60 µl for each of two injections: sample injection and control injection into buffer)
    • ≥10x concentration in cell for 1:1 stoichiometry)

Reaction Buffers

Protein and ligand must be in identical buffers, to minimize heats of dilution which can mask heats of binding. Reducing agents can cause erratic baseline drift and artifacts.  TCEP is recommended over βMe and DTT.  Avoid or keep ≤ 1 mM, especially if ΔH is small.

  • It is essential to bring matched buffer for rinsing, baselines and dilutions.