Extending ITC to Kinetics with kinITC


Title: Extending ITC to Kinetics with kinITC

Authors: Philippe Dumas, Eric Ennifar, Cyrielle Da Veiga, Guillaume Bec, William Palau, Carmelo Di Primo, Angel Piñeiro, Juan Sabín, Eva Muñoz, Javier Rial.


Isothermal titration calorimetry (ITC) has long been used for kinetic studies in chemistry, but this remained confined to enzymatic studies in the biological field. In fact, the biological community has long had the tendency of ignoring the kinetic possibilities of ITC considering it solely as a thermodynamic technique, whereas surface plasmon resonance is seen as the kinetic technique par excellence. However, the primary signal recorded by ITC is a heat power which is directly related to the kinetics of the reaction. Here, it is shown how this kinetic signal can be recovered by using kinITC, the kinetic extension of ITC. The theoretical basis of kinITC is detailed for the most common situation of a second-order reaction A + B Ω C characterized by kinetic parameters kon,koff. A simplified kinITCETC method based upon the determination of an “Equilibration Time Curve” (ETC) is presented. The ETC is obtained by automatic determination of the “effective end” of each injection. The method is illustrated with experimental results with a comparison to Surface Plasmon Resonance (SPR) data. kon values were obtained in a wide range, from 103 to 0.5 × 106 M− 1 s− 1. All procedures were implemented in the program AFFINImeter (https://www.affinimeter.com/).

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Global fitting: the key for a robust analysis

The Indian parable of “the six blind men and the elephant” tells the story of six blind men who touch an elephant in the hope of learning what it is like. As each one can only feel a different part of the animal the individual conclusions obtained are in disagreement and none of them provides a real view of the full elephant. “only by sharing what each of you knows can you possibly reach a true understanding”; that´s the moral behind this nice story.

The six blind men and the elephant: only a global analysis of the overall data provides a true understanding

Sometimes, when we perform two or more ITC titration experiments of a complex interacting system under different experimental conditions (i.e. different concentrations and/or experimental setup) we find out that the individual analysis of the corresponding isotherms yields different values of the thermodynamic parameters. This result can be very confusing, especially for newcomers in the field of molecular recognition, because all these experiments are a representation of the same interaction and should converge to provide the same information. Frequently, the explanation for this behaviour is that each individual ITC experiment lacks of sufficient information to unequivocally determine the thermodynamic parameters of the binding event. “It´s like feeling only a separate part of the elephant”.
Analogous to the parable of the six men and the elephant, the way to get a true understanding of the binding event consist of the global analysis of the different isotherms.
Being aware of the relevance of global analysis, in AFFINImeter we count with the possibility to perform Global fitting of multiple dataseries (isotherms) to tailored binding models where one or more fitting parameters are shared between isotherms. The number and identity of the parameters shared are selected by the user. Moreover, two or more parameters can be related trough mathematical relationships designed by the user. All these features make our global fitting tool the most potent among others to perform a robust analysis of ITC data of complex interactions.

Learn how to perform a global analysis with AFFINImeter:

Thermodynamic and kinetic aspects of Molecular Recognition Processes

The understanding of molecular recognition processes requires a thorough examination from different perspectives including thermodynamic and kinetic aspects of the binding interaction and structural aspects of the interactants and the complex.

The determination of the binding affinity of two (or more) interactants (i.e. a protein/ligand system), through a steady state analysis provides information on how strong is the complex formed, and it is typically expressed in terms of equilibrium binding constants (association, KA , or dissociation, KD constants). The kinetic analysis of the interaction offers information on how fast the complex is formed and how fast it dissociates, expressed in terms of association and dissociation rate constants, respectively (kon and koff).


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