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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KinITC: Obtain Thermodynamic and Kinetic Data from your ITC Measurements in just five clicks

In AFFINImeter we have implemented KinITC, this is a new method to obtain kinetic information from Isothermal Titration Calorimetry Data. With one single titration experiment it calculates the kinetic constants (kon and koff) and the thermodynamic data (KD and ΔH) of 1:1 binding interactions.

Blow the dust off your old ITC binding experiments, you still can get kinetic information out of them

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How many experiments have you carried out in your Isothermal Titration Calorimeter during the last years? Do you know that you can reuse them to obtain kinetic information?




KinITC is a methodology recently developed by Philippe Dumas (CNRS, France) to simultaneously get kinetic and thermodynamic information from a standard ITC experiment. The current implementation of kinITC in AFFINImeter is valid only for 1:1 interactions but we intend to extend this for more complex systems in the near future.




If you have performed experiments of 1:1 interactions in the past, bring them back and use KinITC to get kinetic constants (kon and koff). Under ideal conditions (perfectly clean sample cell, accurate characterization of the response time of your instrument and sufficiently slow kinetics) the resulting values are expected to be equivalent to those obtained by surface plasmon resonance (SPR). The whole process takes just a few seconds and you can analyse multiple files simultaneously.

Blow the dust off your old ITC binding experiments! Go to AFFINImeter!



(1) Burnouf D1, Ennifar E, Guedich S, Puffer B, Hoffmann G, Bec G, Disdier F, Baltzinger M, Dumas P. kinITC: a new method for obtaining joint thermodynamic and kinetic data by isothermal titration calorimetry. J. Am. Chem. Soc. 2012 Jan 11; 134(1):559-65. doi: 10.1021/ja209057d. Epub 2011 Dec 16.



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.



New version with AFFINImeter KinITC Included

AFFINImeter is the most complete software for Isothermal Titration Calorimetry data Analysis. You can easily  build your own Binding Models or Perform Global Simultaneous Analysis of several Isotherms, among other features.

In this new AFFINImeter  version we have implemented KinITC, this is a new method to obtain kinetic information from Isothermal Titration Calorimetry Data. With one single titration experiment it calculates the kinetic constants (kon and koff) and the thermodynamic data (KD and ΔH) of 1:1 binding interactions.

AFFINImeter KinITC (soon to be released) offers a general solution for the smart and efficient analysis of the primary signal obtained from ITC experiments. This solution will include an automatic detection of outliers to remove the noise, the detection of the end injection time, the integration of the injection peaks and a preliminary analysis of the peak shape during the titration. The latter feature will allow to provide an estimation of the kinetic constants in the most typical cases where the experiment involves only the formation of 1:1 complexes.

Simulation, advanced ITC data fitting and Kinetics Analysis
Simulation, advanced ITC data fitting and Kinetics Analysis


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).