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Thermodynamic and Kinetic Analysis of Isothermal Titration Calorimetry Experiments by Using KinITC in AFFINImeter

  • Eva Muñoz
  • Juan Sabín
  • Javier Rial
  • Daniel Pérez
  • Eric Ennifar
  • Philippe DumasEmail author
  • Ángel PiñeiroEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1964)

Abstract

Standard molecular binding isothermal titration calorimetric (ITC) experiments are designed to get thermodynamic information: changes in Gibbs energy, enthalpy, and entropy associated to the studied process. Traditionally, the kinetic information contained in the ITC raw signal has been ignored. For a usual one-step process, this corresponds to the rate constants for the association and the dissociation of the complex (kon and koff). The availability of highly sensitive ITC instruments with low response time, together with the development of theoretical methods and of public software for the proper analysis of the signal, cancels any reason for not retrieving this kinetic information. Here we describe how to further exploit ITC experiments of simple one-step interactions by using the software AFFINImeter.

The method is exemplified using a standard reference system for thermodynamic and kinetic molecular binding analysis: the interaction of carbonic anhydrase (CA) with its inhibitor 4-carboxybenzenesulfonamide (4-CBS) at several temperatures. It is to be emphasized that old experiments initially designed and executed just for thermodynamic analysis can be readily recycled by using AFFINImeter to retrieve the previously ignored kinetic information.

Key words

ITC AFFINImeter KinITC Carbonic anhydrase Data analysis Kinetics Thermodynamics Molecular binding 

Notes

Acknowledgment

Á. P. thanks the financial support of the Spanish Ministry of Economy and Competitiveness (Project MAT2015-71826-P) and Xunta de Galicia (AGRUP2015/11).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Eva Muñoz
    • 1
  • Juan Sabín
    • 1
  • Javier Rial
    • 1
  • Daniel Pérez
    • 1
  • Eric Ennifar
    • 2
  • Philippe Dumas
    • 2
    Email author
  • Ángel Piñeiro
    • 1
    • 3
    Email author
  1. 1.AFFINImeter Scientific & Development Team, Software 4 Science DevelopmentsS. L. Ed. Emprendia, Campus VidaSantiago de CompostelaSpain
  2. 2.Institut de Biologie Moléculaire et CellulaireUniversité de Strasbourg, CNRSStrasbourgFrance
  3. 3.Soft Matter & Molecular Biophysics Group, Departamento de Física Aplicada, Facultad de FísicaUniversidade de Santiago de Compostela, Campus Vida s/nSantiago de CompostelaSpain

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