Advertisement

Enzyme Kinetics Determined by Single-Injection Isothermal Titration Calorimetry

  • Colette F. QuinnEmail author
  • Lee D. Hansen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1964)

Abstract

This chapter describes how to collect Michaelis-Menten kinetic data on an enzyme-catalyzed reaction with the isothermal titration calorimetry (ITC) and the single-injection method. ITC measures the heat rate which is directly proportional to the reaction rate. The enthalpy change (ΔrH), Km, kcat, and vmax are determined in a single assay that does not require labeling or immobilization.

Key words

Kinetics ITC Michaelis-Menten Enzyme Single-injection method Calorimetry Enthalpy 

References

  1. 1.
    Todd M, Gomez J (2001) Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Anal Biochem 296:179–187CrossRefGoogle Scholar
  2. 2.
    Morin PE, Friere E (1991) Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase. Biochemist 30:8494–8500CrossRefGoogle Scholar
  3. 3.
    Olsen S (2006) Applications of isothermal titration calorimetry to measure enzyme kinetics and activity in complex solutions. Thermochim Acta 448:12–18CrossRefGoogle Scholar
  4. 4.
    Choma CT (2006). Characterizing enzyme kinetics by ITC. Applications Note M125, TA Instruments. http://www.tainstruments.com. Accessed 06 Sept 2012
  5. 5.
    Bianconi ML (2003) Calorimetric determination of thermodynamic parameters of reaction reveals different enthalpic compensations of the yeast hexokinase isozymes. J Biol Chem 278(21):18709–18713CrossRefGoogle Scholar
  6. 6.
    Transtrum M, Hansen L, Quinn C (2015) Enzyme kinetics determined by single-injection isothermal titration calorimetry. Methods 76:194–200CrossRefGoogle Scholar
  7. 7.
    Transtrum M, Hansen L, Quinn C, Demarse N (2016) Enzyme-catalyzed and binding reaction kinetics determined by titration calorimetry. Biochim Biophys Acta 1860:957–966CrossRefGoogle Scholar
  8. 8.
    Hansen CW, Hansen LD, Nicholson AD et al (2011) Correction for instrument time constant and baseline in determination of chemical kinetics. International J Chem Kinetics 43(2):53–61CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Applications LabTA InstrumentsLindonUSA
  2. 2.Department of Chemistry and BiochemistryBrigham Young UniversityProvoUSA

Personalised recommendations