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European Biophysics Journal

, Volume 48, Issue 8, pp 825–835 | Cite as

Obtaining precise and accurate results by ITC

  • Lee D. HansenEmail author
  • Colette Quinn
Methods Paper
First Online:
  • 131 Downloads

Abstract

Acquisition of precise and accurate results by isothermal titration calorimetry (ITC) can be achieved through thoughtful experimental design and modeling and careful experimental operations. Large reported errors in ITC results in determinations of stoichiometries, equilibrium constants and enthalpy changes for ligand binding to proteins are the consequence of poor experiment design, failure to properly calibrate and test instruments and protocols, lack of controls, errors in solution preparation, and incorrect data analyses. Analysis of a recent report that claimed to have determined the “repeatability, precision, and accuracy of the enthalpies and Gibbs energies of a protein–ligand binding reaction” by ITC is used to illustrate how to improve ITC operations and results. The analysis shows that the reported results are misleading because calorimeters were not calibrated, operating parameters were not optimized, errors were made in solution preparations, and data analysis was not optimized. As a consequence, the results do not provide a valid comparison of the capabilities of the calorimeters included in the study. A proposal that reaction of acetazolamide with carbonic anhydrase II be used as a comparison standard for testing ITCs and procedures is problematic because the binding constant is too large and for several other reasons discussed in the paper. Requirements for obtaining precise and accurate results by ITC are discussed and experimental results are presented to illustrate the precision and accuracy attainable with low volume ITCs. The problem of the blank correction is identified as the limiting factor in obtaining accurate results by ITC.

Keywords

ITC Standards Calibration Accuracy Precision Data analysis 
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Notes

References

  1. Baranauskienė L, Petrikaitė V, Matulienė J, Matulis D (2009) Titration calorimetry standards and the precision of isothermal titration calorimetry data. Int J Mol Sci 10:2752–2762CrossRefGoogle Scholar
  2. Berg RL, Vanderzee CE (1978) Enthalpies of dilution of sodium carbonate and sodium hydrogen carbonate solutions, and standard enthalpies of ionization of aqueous carbonic acid at 298.15 K. J Chem Thermodyn 10:1049–1075CrossRefGoogle Scholar
  3. Box GEP, Hunter WG, Hunter JS (1978) Statistics for Experimenters: An Introduction to Design, Data Analysis, and Model Building. John Wiley & Sons, NYGoogle Scholar
  4. Broecker J, Vargas C, Keller S (2011) Revisiting the optimal c value for isothermal titration calorimetry. Analyt Biochem 418:307–308CrossRefGoogle Scholar
  5. Castellano BM, Eggers DK (2013) Experimental support for a desolvation energy term in governing equations for binding equilibria. J Phys Chem B 117:8180–8188CrossRefGoogle Scholar
  6. Christensen T, Gooden DM, Kung JE, Toone EJ (2003) Additivity and the physical basis of multivalency effects: A thermodynamic investigation of the calcium EDTA interaction. J Am Chem Soc 125:7357–7366CrossRefGoogle Scholar
  7. Demarse NA, Quinn CF, Eggett DL, Russell DJ, Hansen LD (2011) Calibration of nanowatt isothermal titration calorimeters with overflow reaction vessels. Analyt Biochem 417:247–255CrossRefGoogle Scholar
  8. Goldberg RN, Kishore N, Lennen RM (2002) Thermodynamic quantities for the ionization reactions of buffers. J Phys Chem Ref Data 31:231–370CrossRefGoogle Scholar
  9. Griko YV (1999) Energetics of Ca2+-EDTA interactions: calorimetric study. Biophys Chem 79:117–127CrossRefGoogle Scholar
  10. Hansen LD, Transtrum MK, Quinn CF, Demarse NA (2016) Enzyme-catalyzed and binding reaction kinetics determined by titration calorimetry. Biochim Biophys Acta 1860(5):957–966CrossRefGoogle Scholar
  11. Hansen LD, Christensen JJ, Izatt RM (1965) Entropy Titration A Calorimetric Method for the Determination of ∆G° (K), ∆H° and ∆S°. J Chem Soc Chem Commun 3:36–37Google Scholar
  12. Hansen LD, Fellingham GW, Russell DJ (2011) Simultaneous determination of equilibrium constants and enthalpy changes by titration calorimetry: Methods, instruments, and uncertainties. Analyt Biochem 409:220–229CrossRefGoogle Scholar
  13. Hansen LD, Transtrum MK, Quinn CF (2018) Titration calorimetry from concept to application. Springer Briefs in Molecular Science, Springer International PublishingGoogle Scholar
  14. Kantonen SA, Henriksen NM, Gilson MK (2017) Evaluation and minimization of uncertainty in ITC binding measurements: Heat error, concentration error, saturation, and stoichiometry. Biochim Biophys Acta 1861:485–498CrossRefGoogle Scholar
  15. Marini MA, Evans WJ, Berger RL (1985) Use of the twin-cell differential titration calorimeter for binding studies I EDTA and its calcium complex. J Biochem Biophys Methods 10:273–285CrossRefGoogle Scholar
  16. Mizoue LS, Tellinghuisen J (2004) Calorimetric vs. van’t Hoff binding enthalpies from isothermal titration calorimetry: Ba2+-crown ether complexation. Biophys Chem 110:15–24CrossRefGoogle Scholar
  17. Myszka DG, Abdiche YN, Arisaka F, Byron O, Eisenstein E, Hensley P, Thomson JA, Lombardo CR, Schwarz F, Stafford W, Doyle ML (2003) The ABRF-MIRG’02 Study: Assembly State, Thermodynamic, and Kinetic Analysis of an Enzyme/Inhibitor Interaction. J Biomolecular Techniques 14:247–269Google Scholar
  18. O’Brien L, Root H, Wei C, Eder D (2015) M2+-EDTA binding affinities: A modern experiment in thermodynamics for the physical chemistry laboratory. J Chem Ed 92(9):1547–1551CrossRefGoogle Scholar
  19. Paketurytė V, Linkuvienė V, Krainer G, Chen W-Y, Matulis D (2019) Repeatability, precision, and accuracy of the enthalpies and Gibbs energies of a protein–ligand binding reaction measured by isothermal titration calorimetry. European Biophys J Biophys Letters 48(2):139–152CrossRefGoogle Scholar
  20. Rafols C, Bosch E, Barbas E, Prohens R (2016) The Ca2+-EDTA chelation as standard reaction to validate isothermal titration calorimeter measurements (ITC). Talanta 154:3554–3559CrossRefGoogle Scholar
  21. Tellinghuisen J (2003) A study of statistical error in isothermal titration calorimetry. Analyt Biochem 321:79–88CrossRefGoogle Scholar
  22. Tellinghuisen J (2004) Volume errors in isothermal titration calorimetry. Analyt Biochem 333:405–406CrossRefGoogle Scholar
  23. Tellinghuisen J (2007) Calibration in isothermal titration calorimetry: Heat and cell volume from heat of dilution of NaCl(aq). Analyt Biochem 360:47–55CrossRefGoogle Scholar
  24. Tellinghuisen J (2007) Optimizing experimental parameters in isothermal titration calorimetry: Variable volume procedures. J Phys Chem B 111:11531–11537CrossRefGoogle Scholar
  25. Tellinghuisen J (2008) Isothermal titration calorimetry at very low c. Analyt Biochem 373:395–397CrossRefGoogle Scholar
  26. Tellinghuisen J (2012) Designing isothermal titration calorimetry experiments for the study of 1:1 binding: Problems with the “standard protocol. Analyt Biochem 424:211–220CrossRefGoogle Scholar
  27. Tellinghuisen J (2016) Optimizing isothermal titration calorimetry protocols for the study of 1:1 binding: Keeping it simple. Biochim Biophys Acta 1860:861–867CrossRefGoogle Scholar
  28. Tellinghuisen J (2018) Critique of methods for estimating heats in isothermal titration calorimetry. Analyt Biochem 563:79–86CrossRefGoogle Scholar
  29. Tellinghuisen J, Chodera JD (2011) Systematic errors in isothermal titration calorimetry: Concentrations and baselines. Analyt Biochem 414:297–299CrossRefGoogle Scholar
  30. Turnbull WB, Daranas AH (2003) On the value of c: Can low affinity systems be studied by isothermal titration calorimetry? J Am Chem Soc 125:14859–14866CrossRefGoogle Scholar
  31. Wadso I, Goldberg RN (2001) Standards in isothermal microcalorimetry (IUPAC technical report). Pure Applied Chemistry 73(10):1625–1639CrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2019

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryBrigham Young UniversityProvoUSA
  2. 2.TA InstrumentsLindonUSA

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