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Characterization of Enzymatic Reactions Using ITC

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Microcalorimetry of Biological Molecules

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1964))

Abstract

Life is governed by a complex and tightly regulated sequence of biochemical reactions, catalyzed by enzymes. Characterizing enzyme activity is extremely important both to understand biological processes and to develop new industrial applications. Calorimetry represents an ideal system to measure kinetics of biochemical transformations, because it uses heat, always produced or absorbed during chemical reactions, as a probe.

The following protocol describes the details of experimental setup and data analysis of isothermal titration calorimetry (ITC) experiments aimed to quantify the thermodynamic (ΔH) and kinetic (KM and kcat) parameters of enzyme catalysis. A general guideline to choose the right procedure according to the system under analysis is given, together with some instructions on how to adjust the experimental conditions for obtaining reliable data. The method to analyze the obtained raw ITC curves and to derive the kinetic parameters is described.

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Authors and Affiliations

  1. Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy

    Barbara Zambelli

Authors
  1. Barbara Zambelli
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Corresponding author

Correspondence to Barbara Zambelli .

Editor information

Editors and Affiliations

  1. Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, CNRS, Strasbourg, France

    Eric Ennifar

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Zambelli, B. (2019). Characterization of Enzymatic Reactions Using ITC. In: Ennifar, E. (eds) Microcalorimetry of Biological Molecules. Methods in Molecular Biology, vol 1964. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9179-2_18

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  • DOI: https://doi.org/10.1007/978-1-4939-9179-2_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9178-5

  • Online ISBN: 978-1-4939-9179-2

  • eBook Packages: Springer Protocols

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