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.



Stoichiometric and site constants: two approaches to analyze data with AFFINImeter.

The interaction between two species, i.e. a protein and its ligand, is defined by means of the equilibria existing between free and bound species and the binding constant(s) associated to each equilibrium. This scenario can be described in terms or reaction schemes following two approaches:

a) Based on equilibria between existing stoichiometric species, to obtain stoichiometric binding constants and

b) Based on equilibria between the ligand and specific interaction site(s) of the protein, to obtain site binding constants.


The understanding of both approaches/type of binding constants is key for a correct interpretation of the results after data analysis, in order to get key structural and mechanistic information of the binding event; i.e. the presence or absence of cooperative interactions when a ligand binds to a multivalent receptor.
The design of binding models for ITC curve fitting with AFFINImeter can be done following these two approaches, to perform analysis based on stoichiometric and/or site binding constants.

The scientific team of AFFINImeter has just released three NOTES regarding this subject to guide users into the right selection of binding model approach and a better understanding of stoichiometric vs site binding constants.

Comparative table of the two approaches for binding model design available in AFFINImeter
Characteristics of the two approaches for binding model design available in AFFINImeter



Or visit the RESOURCES section of AFFINImeter web page where you find tutorials, webinars, cases of use, among others.

The importance of the treatment of ITC raw data in calorimetry experiments

Isothermal titration calorimetry (ITC) is an extremely sensitive technique to assess for the formation/disruption of complex chemical/biological species in solution. During the last years, the increase in instrument sensitivity as well as the reduction of the sample concentration required to perform experiments, have made possible to expand the application range of ITC, which is expected to continue growing.

Quality of the ITC Raw Data?

The amount and the quality of useful information that can be obtained from an ITC experiment depend on several factors including the purity of the samples, the concentration of the solutions prepared, the choice of injection volume and its length in time. The researcher handling the instrument is responsible for the appropiate selection of these variables as part of the experimental setup. They can be optimized on the basis of previous experience and also taking advantage of computational simulations. A key factor for this is that ITC is an incremental technique and so the results depend strongly on the injection volume employed to perform the experiment.

Kinetic information from ITC Experiments


How to perform a Global Fitting Analysis?

The Global fitting of multiple isotherms is one of the advanced tools that AFFINImeter offers to facilitate the analysis and interpretation of isothermal titration experiments and to expand the range of applications of this technique.

The following video tutorial describes the global fitting of three isotherms of a displacement assay describing, a receptor interacting with a tight ligand, with a weak ligand, or with both ligands simultaneously, in a competitive experiment where the ligands are mixed in the syringe of the ITC equipment.


If you want to know more about global fittings with AFFINImeter you can also download the case of use “Global Analysis in ITC Displacement Titrations with AFFINImeter” that describes a Displacement Titration Assay to determine the thermodynamics of HIV-protease with indinavir, a high-affinity binder, and with acetyl-pepstatin, a weaker ligand.

ITC displacement titrations offer an attractive alternative to standard assays when working with ultra-high or ultra-low- affinity interacting systems. The method requires the fitting of at least two isotherms that share various adjustable parameters. The case study exemplifies the potential advantages of using AFFINImeter in ITC displacement assays. The software offers unique advanced tools that enhance the robustness of the method and makes it more versatile, facilitating the acquisition of reliable thermodynamic data from ultra-high of ultra-low affinity systems. Thus, it opens a door for new applications of the displacement assay.




Multiple Independent Sites: Advanced tools for a successful analysis.

Isothermal titration Calorimetry experiments of a ligand binding to a macromolecule with multiple independent sites

A successful Isothermal Titration Calorimetry (ITC) experiment requires the acquisition of high quality experimental data together with a careful analysis. Choosing the right binding model to fit the ITC isotherm is critical in order to get the true thermodynamic profile of the interaction. Often, the main limitation to achieve good results arises when the evaluation software lacks of the mathematical model that best describes our binding experiment. A good example is the case of a ligand binding to a macromolecule with multiple independent sites, i.e ligand – DNA interactions (1). Until now the readily available mathematical models to fit such experiments was limited to one or two sets of “n” independent identical sites; frequently, these models offer a poor description of the interaction due to the inherent higher complexity of the system, where many distinct binding equilibria coexist.


Multiple Sets of Independent Sites

AFFINImeter ITC offers an unlimited number of user-defined binding models. Particularly, it counts with a feature to easily design models based on multiple independent binding sites. Here, a model with a number of sets of independent sites can be created with no limitation in the number of sets or sites. Noteworthy, the number of sites in each set can be considered as a fitting parameter throughout the data analysis. As an illustration, the following figure shows the reaction parameters of a model generated with AFFINImeter that describes a ligand binding to a receptor having 3 sets of sites, each set having an unknown number of sites. Fitting the experimental data to such model yields the microscopic association constant (K) and the change in enthalpy (ΔH) of the ligand binding to each site type, and the number of sites in each set (n).

Scheme of the interaction of a ligand with a multisite receptor
Reaction parameters table of a model that describes the interaction of a ligand (in syringe) with a multi-site receptor (in cell) having 3 sets of independent sites. Note that the option “Fit” was checked to consider the number of sites (n) as fitting parameters.

These binding models, described by numerous variable parameters, may end up in an over-parameterized fitting function. Thus, the best strategy to achieve a robust and consistent analysis involves the global fitting of several ITC curves acquired under different experimental conditions. In this sense, AFFINImeter also supports global fitting of multiple isotherms wherein parameter linkage between curves is used to decrease the relative number of estimated parameters per experiment.

(1) Methods 2007, 42, 162–172.

How to design an Isothermal Titration Calorimetry experiment?

Isothermal Titration Calorimetry Experiment Simulation



The Simulator tool available in AFFINImeter is completely free under registration. This is currently the only alternative to design complex Isothermal Titration Calorimetry (ITC) experiments. The Simulator allows plotting ITC curves (evolved heat as a function of the system concentration) together with a phase diagram of the different chemical species that are present in the solution regardless the complexity of the interaction mechanism between the involved molecules.

Scheme of a ITC Experiment
Isothermal Titration Calorimetry Experiment


Avoid Trial and Error Assays

Using the AFFINImeter Simulator you will be able to pre-visualize the results of an experiment, provided that you have an approach for the interaction mechanism of your molecules and of the corresponding thermodynamical parameters. This tool will guide you in the optimization of the most advantageous combination of experimental parameters: the concentration and location (in the sample cell or in the syringe) of your compounds, the injection volume and the number of titrations; thus avoiding trial-and-error assays and saving time, reactants and money.

This tool is also useful to set the conditions under which the distribution of chemical species meet some special requirement (for instance, the solution dominated by a given chemical species). It can also be used for didactic purposes since it helps to illustrate how a chemical species can be displaced by another, to explain the difference between cooperative and non-cooperative processes or to explain the effect of endothermic and exothermic processes.


Applications in Drug Discovery

Isothermal Titration Calorimetry is a key technique in the development of drugs since it assess the affinity between molecules. The most typical application is to determine the free energy of interaction between proteins and inhibitors. The AFFINImeter simulator tool allows simulating the displacement of a weak ligand by a strong ligand as a function of the concentration of the compounds involved in the experiment.

Advantages of the Simulator

Introduce your personalized thermodynamic model directly in chemical language (reaction scheme) and an estimation for the corresponding thermodynamic parameters. Even the most advanced models are easy to implement. Through the model builder AFFINImeter offers an unlimited amount of thermodynamic models for Isothermal Titration Calorimetry data analysis. If the model required for your system is not available, please, do not hesitate to contact us and we will try to implement it.

Sequential Binding Sites Model
This is a Sequential Binding Model that considers the free species of both solutes M and A, plus the hybrid complexes with stoichiometries (1:1, 1:2, 2:1, 2:2).


Start Using the Simulator

The AFFINImeter Simulator is free under registration. To learn how to use it, please read this tutorial.