AFFINImeter binding models for Nuclear Magnetic Resonance

AFFINImeter is already well known in ITC binding data analysis for providing the possibility to use tailored binding models created by the user. The models are generated with the tool “model builder” that includes a letter code “M-A-B” to describe titrate (M), titrant (A) and if necessary, the presence of a third species (B) (Figure 1).


Fig.1 Example of a competitive binding model created in AFFINImeter where the titrant in syringe “A” binds to the titrate in cell “M” to form a 1:1 complex “MA” and a second ligand “B” mixed in the cell with “M” forms the complex “MB” and thus competes with “A”.


Following the same approach, the binding models available for the software AFFINImeter for Nuclear Magnetic Resonance are generated with the model builder and based on the “MAB” code. But there are significant differences between ITC and NMR data analysis when the time comes to select a binding model from AFFINImeter, which have an origin in the inherent characteristics of each technique and in the different experimental design. In chemical shift perturbation (CSP) NMR titration experiments, the observed parameter used to monitor the progress of the binding event is the chemical shift of titrate resonance signals. Hence, the models used for NMR data analysis require the presence of compound “M” (titrate) as it is the species from which changes associated with the binding process are monitored. Conversely, in ITC the observed parameter is the heat change upon interaction and this parameter is not necessarily linked to a particular species “M”, “A” or “B”.

An illustrative example is the evaluation of a monomer-dimer self-association process using NMR or ITC. In NMR, the standard experimental setup would consist in the incremental dilution of the compound sample at high concentration in the NMR tube, to monitor dimer dissociation (Figure 2a). In ITC the standard experimental setup would consist in a titration of the compound sample at high concentration in the syringe (species “A” according to the AFFINImeter code) into the calorimetric cell filled up with solvent (Figure 2b).


Fig.2 Representation of experimental setup for a) NMR dilution experiment and b) ITC dilution experiments. The corresponding schemes of AFFINImeter binding models for data analysis are shown.



Would you like to know more about AFFINImeter for Nuclear Magnetic Resonance? Press the button below:



The course of Isothermal Titration Calorimetry data analysis: second part

In the second part of this course, we are going to show you how to perform the analysis of binding isotherms considering an independent sites approach.
This approach uses a reaction scheme based on the binding of the ligand to individual sites present in the receptor and considering that all the sites are independent; thus, it supplies site binding constants.
This approach offers a sole reaction scheme where a receptor with a certain number of sites “n” binds to the ligand.
The sites are grouped into sets to discern between sites that are non-equivalent.

If you want to know more about how to get the stoichiometry (number of sites) and site binding constants with the independent sites approach you can click here:

Stoichiometric and site constants – two approaches to analyze data with AFFINImeter

The first video tutorial presented is about how to use an independent sites approach to perform fittings:


Into another subject, to introduce the second fitting approach that can be performed with AFFINImeter (Stoichiometric equilibria), we will describe how to use the model builder.
This original tool allows to design and apply your own binding model in an easy way. Check the following video to know the versatility of the model builder:

Finally, if you want to try the Model Builder click here:

Model Builder


Expanding the range of applications of ITC in the Pharmaceutical Industry with AFFINImeter: A practical Case.

Many Drug–receptor interactions are characterized by complex binding modes that are far away from the behavior of a standard 1:1 model. This is the case of Heparin (Hp), one of the most commonly prescribed anticoagulant drugs, which exerts its effect through its interaction with the serine protease Antithrombin (AT-III). Hp is a linear heterogeneous polysaccharide containing a specific pentasaccharide sequence that binds AT-III with high nanomolar affinity (responsible for the anticoagulant activity); but AT-III also binds other Hp sequences with lower affinity. Determining the content of AT-III binding pentasacchride in Low Molecular Weight (LMW) Heparins is a requirement for Pharmaceutical companies that manufacture this type of anticoagulants; due to the intrinsic heterogeneity of Hp, obtaining this information it is not straightforward (1).

We have developed a new protocol based on ITC and AFFINImeter to determine the content of AT-III binding pentasaccharide in Heparins, which is summarized in the following scheme:

New method based on AFFINImeter to determine the content of AT-III binding pentasacchride in LMW Hp: 1) use of a tailored binding model that describes the competitive binding between the pentasaccharide (A) and other low affinity sequences (B) with AT-III (M); 2) global fitting of several isotherms registered under different Hp and or AT-III concentrations where the parameters rA and rB (that account for the fraction of A and B in the Hp sample) are fitting parameters and common among the different isotherms.
New method based on AFFINImeter to determine the content of AT-III binding pentasacchride in LMW Hp: 1) use of a tailored binding model that describes the competitive binding between the pentasaccharide (A) and other low affinity sequences (B) with AT-III (M); 2) global fitting of several isotherms registered under different Hp and or AT-III concentrations where the parameters rA and rB (that account for the fraction of A and B in the Hp sample) are fitting parameters and common among the different isotherms.


This method illustrates the great potential of the model builder and global fitting AFFINImeter tools to develop protocols of practical utility in the Pharmaceutical industry (2). We have successfully validated the protocol in the analysis of unfractionated Hp and a series LMW Hp in collaboration with the Pharmaceutical company Laboratorios Rovi (


  1. Nandurkar H., Chong B, Salem H, Gallus A, Ferro V, McKinnon R. Low-molecular-weight heparin biosimilars: potential implications for clinical practice. Internal Medicine Journal, 2012, 44(5), pp 497–500.

  2. For a detailed description of the protocol contact us at

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.

The Model Builder is a versatile tool to translate binding interactions into mathematical models

The Model Builder is one of the novel features of AFFINImeter.  Through the model builder AFFINImeter offers an unlimited amount of thermodynamic models for ITC data analysis. The overall binding equilibria within the species involved in the experiments is easily drawn by the user directly in chemical language. Then AFFINImeter translates the resulting reaction scheme into robust  binding  models to be used to isotherm ITC simulation or to perform Isothermal Titration Calorimetry curve fitting.

The model builder is a versatile tool, it allows to design models involving up to three different species (i.e. the case of two ligands that compete with each other to bind a macromolecule) and has the advantage to selectively place them in the  syringe cell and/or in the calorimetric cell. 

Binding Reaction Scheme of a competitive interaction
Reaction Scheme of a Competitive Binding Interaction

It also allows the design of models for dissociation, ranging from simple homodimers to higher-order oligomers. During the model construction no mathematical equations are required, once the whole set of binding interactions is defined by the user in the reaction builder, AFFINImeter internally generates the system of equations that define the reaction scheme proposed. The new model (reaction scheme and equations) is saved  internally by AFFINImeter and listed in the user’s database so that can be utilized anytime so simulate or fit data.

AFFINImeter Reaction Builder
Competitive binding model builded with the AFFINImeter tool

AFFINImeter-ITC offers an exclusive unlimited amount of personalized model families including

  • Unrestricted Competitive Sequential Binding with no limitation in the stoichiometry of the binding model.
  • Competitive Multiple and Independent Sets of Identical and Independent Sites.
  • Dissociation of any chemical species including homogeneous n-mers, heterogeneous complexes and even micelles.

With this extensive offer of model families the user will be able to perform the thermodynamic characterization of a vast variety of biological and physicochemical processes from ITC measurements. A few examples of classical and new applications of ITC experiments that you can analyze with AFFINImeter are:

  • Protein-ligand or host-guest complex formation with unlimited stoichiometries
  • Competition of different molecules to occupy a given binding site even for high order complexes
  • Binding between ligands and polymers or large macromolecules with any number of (independent) sets and/or sites
  • Dissociation/aggregation of supramolecular heterogeneous species including protein oligomers
  • Structural and thermodynamic information of large aggregates, including micelles: aggregation number, enthalpy of formation, Gibbs energy and dilution heat of monomers and aggregates