JBIC Journal of Biological Inorganic Chemistry

, Volume 15, Issue 8, pp 1183–1191 | Cite as

Application of isothermal titration calorimetry in bioinorganic chemistry

  • Nicholas E. Grossoehme
  • Anne M. Spuches
  • Dean E. WilcoxEmail author


The thermodynamics of metals ions binding to proteins and other biological molecules can be measured with isothermal titration calorimetry (ITC), which quantifies the binding enthalpy (ΔH°) and generates a binding isotherm. A fit of the isotherm provides the binding constant (K), thereby allowing the free energy (ΔG°) and ultimately the entropy (ΔS°) of binding to be determined. The temperature dependence of ΔH° can then provide the change in heat capacity (ΔC p°) upon binding. However, ITC measurements of metal binding can be compromised by undesired reactions (e.g., precipitation, hydrolysis, and redox), and generally involve competing equilibria with the buffer and protons, which contribute to the experimental values (K ITC, ΔH ITC). Guidelines and factors that need to be considered for ITC measurements involving metal ions are outlined. A general analysis of the experimental ITC values that accounts for the contributions of metal–buffer speciation and proton competition and provides condition-independent thermodynamic values (K, ΔH°) for metal binding is developed and validated.


Calorimetry Enthalpy Thermodynamics Heat capacity Binding affinity 



Isothermal titration calorimetry







We thank Robert Cantor for assistance in developing the coupled-equilibria fitting model, and are grateful to Ann Valentine and Tim Elgren for valuable feedback on the manuscript and to former members of the Wilcox group for helpful discussions. We also thank a reviewer for pointing out the utility of HypDH. Research that led to the development of these guidelines has been supported by NIH (P42 ES07373) and is currently supported by NSF (CHE 0910746).

Supplementary material

775_2010_693_MOESM1_ESM.pdf (91 kb)
Supplementary material 1 (PDF 90 kb)


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Copyright information

© SBIC 2010

Authors and Affiliations

  • Nicholas E. Grossoehme
    • 1
    • 2
  • Anne M. Spuches
    • 1
    • 3
  • Dean E. Wilcox
    • 1
    Email author
  1. 1.Department of Chemistry, 6128 Burke LaboratoryDartmouth CollegeHanoverUSA
  2. 2.Department of ChemistryWinthrop UniversityRock HillUSA
  3. 3.Department of ChemistryEast Carolina UniversityGreenvilleUSA

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