5 Tips to optimize your ITC experiments for kinetic analysis.

Since the method KinITC was implemented in AFFINImeter many researchers have been using it to obtain kinetic information of binding interactions from ITC data; the good news is that no special experimental setup different from the standard ITC experiment is required to register data for kinetic analysis! The information is derived from analysis of the thermogram of regular ITC titrations and therefore one can obtain kinetic information from old ITC data right away.

There are few recommendations though if you are planning to perform new ITC experiments, focused on getting high-quality data for kinetic analysis:

1) Set the time between successive power measurements to 1s or 2s. This will give a better definition of the thermogram peaks and therefore a more precise calculation of the equilibration times.

2) Set the time recording the baseline before the first injection to 1 or 2 minutes. In order to have a good reference when determining the signal baseline.

3) Leave enough time between injections so that a full equilibration for the overall set of injections is registered.

4) Clean thoroughly the instrument before the experiment. This is fundamental to optimize the response time of the instrument, which strongly determines the sensitivity of the kinetic analysis.

5) A high gain feedback mode is recommended in order provide the fastest response time (but, be careful because a high feedback mode can also generate signal overshooting after injection, which greatly difficulties the kinetic analysis! If overshooting happens, don´t use high gain model).

Need more information about this subject? Contact the Scientific team of AFFINImeter at info@affinimeter.com.

Follow these simple tips to increase the quality of your ITC data for kinetic analysis

Figure Junio2016

Variable Temperature ITC analysis with AFFINImeter

Analysis of Variable Temperature Isothermal Titration Calorimetry experiments with AFFINImeter

Monitoring a binding event with Isothermal Titration Calorimetry at different temperatures provides a powerful framework for elucidating interesting characteristics of the interaction. Analysis of the isotherms obtained determines the dependence of the association constant (KA) and binding enthalpy (ΔH) with temperature, information that can reveal mechanistic aspects of the interactions, i.e. the existence of allosteric effects and conformational changes (1).

Moreover, kinetic characterization of the interaction at various temperatures gives information about transition state thermodynamics, by means of the dependence of the association and dissociation rate constants (kon and koff) with the temperature. This way, activation free energies of association and dissociation are resolved into its enthalpic and entropic components (2).

Obtaining the full thermodynamic and kinetic profile of 1:1 interactions in a single ITC experiment is now possible with AFFINImeter and KinITC; in order to further exploit the potential of our analytical tools we have recently incorporated a new functionality in AFFINImeter that automatically analyzes variable temperature isothermal Titration Calorimetry assays through Van´t Hoff Plot (Ln(KA) vs 1/T), temperature dependence of ΔH (that determines changes in heat capacity, ΔCp) and Eyring plots (Ln(kon) vs 1/T and Ln(koff) vs 1/T) (3).

AFFINImeter is the only software that provides thermodynamic and kinetic information from a single ITC titration; now incorporates the automatic analysis of variable-temperature experiments.  

You can use this feature for free during one month, go to AFFINImeter webpage.

References

  • Freiburger L, Auclair K, Mittermaier A. Global ITC fitting methods in studies of protein allostery. Methods 2015, 76, pp 149-161.
  • GE Healthcare application note 80. Transition state thermodynamics using Biacore T100, (2007).
  • Ladbury, J. and Doyle, M. (2004). Biocalorimetry 2. Chichester: Wiley.

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 (http://www.rovi.es/).

References

  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 support@affinimeter.com

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:

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.

affinimeter-approaches-small

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

 

DOWNLOAD PDF FILES HERE:

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

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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.

References:
(1) Methods 2007, 42, 162–172.

How to design an Isothermal Titration Calorimetry experiment?

Isothermal Titration Calorimetry Experiment Simulation

 

 theoretical-isotherm-Microcal-VP-ITCspecies-concentration-isothermal-titration-calorimetry-experiment

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.