How can I get kinetic information from an ITC experiment?

The elucidation of kinetic aspects of molecular interactions has been gaining interest in many research areas. For instance, the quantitative analysis of binding kinetics helps to a better understanding of the biological function of molecular interactions; it also serves to identify and characterize lead compounds in drugs discovery programs. Getting kinetic information of a binding event requires the use of real-time techniques in which, an observable is monitored as a function of time during the course of the titration.

Isothermal Titration Calorimetry (ITC) has been formally considered a technique to get steady-state binding information. However the primary data of an ITC experiment (ITC raw data), the power vs plot time, is the result of monitoring the heat flow as a function of time. Therefore, the power vs plot time could deliver kinetic information as well.

KinITC (1) is a new analytical tool implemented in AFFINImeter and developed to obtain kinetic information from ITC data of 1:1 interactions. The method consists in determining the Equilibration Time for every peak of the power vs time plot (that is, the time needed to return to baseline after injection) and plot it against the titrant to titrate molar ratio to obtain the so called Equilibration Time Curve (ETC). Noteworthy, a clear sign that the ITC raw data plot contains kinetic information is the increase of the Equilibration Time of the peaks close to mid-titration. Under these conditions, fitting of the ETC yields the dissociation rate constant, koff. Fitting of the corresponding isotherm yields the association constant, KA; ultimately, the association rate constant, kon, is calculated as the product of koff * KA.



Use of precise standard reactions for Isothermal Titration Calorimeter validation

Use of  precise Standard Reactions for Isothermal Titration  Calorimeter Validation

Many published papers report inconsistent thermodynamic values of the same interactions between chemical reactants or macromolecular binding. One of the reasons for this discrepancies is the difficulty of repeating the same conditions in the ITC experiments (buffer, pH, concentrations, ionic strength, source of the materials…). But users start to be more aware that some systematic errors of the calorimeters may also have an important effect in the reported values.

For instance, the interaction between 4-carboxybenzenesulfonamide and bovine carbonic anhydrase II is considered a standard reaction to be measured by ITC and its enthalpy has been measured by 14 operator using different calorimeters (1). The resulting value considering all these independent measurements is -10.4±2.5 kcal·mol-1. The error of the enthalpy is surprisingly high and significantly higher than those typically reported for ITC measurements.

Baranauskiené and co-workers (1) suggest the use of precise standard reactions for  Isothermal Titration calorimeter validation after the calibration. The table below shows the series of chemical reaction they propose as standards where the enthalpy of binding has been determined to high precision and the reagents are readily available from commercial sources.



They also used these standards reactions to compare the results obtained with different micro calorimeter. Their study concluded that Microcal calorimeters are more reliable than TA Calorimeters; and the most recent Microcal ITC200 is less accurate than Microcal VP-ITC. Nano ITC-III calorimeter results were very reproducible, but enthalpy values were systematically underestimated. To learn more about Isothermal Titration Calorimeter validation,, visit the references from where this article was taken.

Enthalpy of the interaction between Tris base and HNO3 as a function of temperature and measured in different ITC calorimeter. Literature values were taken from (2).


(1) Int. J. Mol. Sci 2009, 10, 2752-2762.

(2) Handbook of proton Ionization Heats Wiley-Interscience: Hoboken, NJ, USA, 1979