Tag: Kinetic

About the disuses of  Isothermal Titration Calorimetry in drug discovery research

Juan Sabín

Isothermal Titration Calorimetry (ITC) is the gold standard for the calculation of affinity in molecular interactions. Many times, researchers claim that the high consumption of sample does not offset the use of ITC for Kd calculation.
Conversely, ITC hides many surprises in the acquisition data that can provide more information in a single experiment that other techniques that are more expensive and more complicated to use.

Download the PDF file of Implementation of kinITC into AFFINImeter

 

1. ITC collects data from the interaction as a function of time that can be analyzed to obtain kinetic information (kon and koff values). It can cover a very similar range as Surface Plasmon Resonance in a “label-free” and “in-solution” manner (Fig 1).

2. ITC can also provide valuable information about the mechanism of interaction. The high sensitivity of the ITC sensor makes it sensitive to more intriguing interactions as conformational changes, cooperativity…

Using a global fitting approach for the analysis of the isotherms and a model builder to create tailored binding models, the different mechanisms of interaction can be confirmed and characterized.

Find attached a couple of publications describing the application of this new method for ITC data analysis:

Download the PDF file of Implementation of kinITC into AFFINImeter

 

How to get the most out of biophysical techniques to address binding interactions?

Juan Sabín

The analysis of isotherms is the more direct way to calculate binding constants for molecular interactions. Isotherms can be obtained using different techniques (ITC, SPR, NMR, Uv-vis, IR, Fluorescence, Circular Dichroism…) and at different experimental conditions.

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1. The global fitting approach allows to simultaneously analyze several isotherms obtained by different biophysical techniques and/or at different experimental conditions in a very accurate manner.

 

 

2. Many interacting systems do not bind with a simple 1:1 model, more complex binding model can be designed to address complex interaction

3. Using tools to globally analyze isotherms obtained by different biophysical techniques is the most reliable method to characterize binding interactions by the orthogonal approach.

Click here to star using AFFINImeter and to start to create your own binding model:

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5 Tips to optimize your ITC experiments for kinetic analysis.

Eva Muñoz

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

Extending ITC to Kinetics with kinITC

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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.

Abstract:

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 kinITC–ETC 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

 

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

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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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Titration of a

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.

 

 

 

kinetics-analysis-in-affinimeter-2
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!

 

References:

(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.

 

 

Thermodynamic and kinetic aspects of Molecular Recognition Processes

Eva Muñoz

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).

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