European Biophysics Journal

, Volume 48, Issue 2, pp 139–152 | Cite as

Repeatability, precision, and accuracy of the enthalpies and Gibbs energies of a protein–ligand binding reaction measured by isothermal titration calorimetry

  • Vaida Paketurytė
  • Vaida Linkuvienė
  • Georg Krainer
  • Wen-Yih Chen
  • Daumantas MatulisEmail author
Original Article


In rational drug design, it is important to determine accurately and with high precision the binding constant (the affinity or the change in Gibbs energy, ∆G), the change in enthalpy (ΔH), and the entropy change upon small molecule drug binding to a disease-related target protein. These thermodynamic parameters of the protein–ligand association reaction are usually determined by isothermal titration calorimetry (ITC). Here, the repeatability, precision, and accuracy of the measurement of the affinity and the change in enthalpy upon acetazolamide (AZM) interaction with human carbonic anhydrase II (CA II) are discussed based on the measurements using several ITC instruments. The AZM–CA II reaction was performed at decreasing protein–ligand concentrations until the determination of ∆G and ΔH was not possible, indicating a lower limit for accuracy. To obtain the confidence intervals (CI) of the ∆G and ΔH of AZM binding to CA II, the binding reaction was repeated numerous times at the optimal concentration of 10 µM and 25 °C temperature. The CI (at a confidence level α = 0.95) for ΔH = − 51.2 ± 1.0 kJ/mol and ∆G = − 45.4 ± 0.5 kJ/mol was determined by averaging the results of multiple repeats.


Isothermal titration calorimetry Repeatability Precision Accuracy NITPIC software SEDPHAT software 



This research was funded by Grant no. TAP LLT-1/2016 from the Research Council of Lithuania. The authors acknowledge the COST projects CM1406, CM1407, CA15126, and CA15135.

Supplementary material

249_2018_1341_MOESM1_ESM.docx (543 kb)
Supplementary material 1 (DOCX 542 kb)


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Copyright information

© European Biophysical Societies' Association 2018

Authors and Affiliations

  1. 1.Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences CenterVilnius UniversityVilniusLithuania
  2. 2.Molecular BiophysicsTechnische Universität KaiserslauternKaiserslauternGermany
  3. 3.B CUBE – Center for Molecular BioengineeringTechnische Universität DresdenDresdenGermany
  4. 4.Department of Chemical and Materials EngineeringNational Central UniversityTaoyuan CityTaiwan

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