Isothermal titration calorimetry of protein-protein interactions

Michael M. Pierce, C. S. Raman, Barry T. Nall

Research output: Contribution to journalArticlepeer-review

392 Scopus citations


The interaction of biological macromolecules, whether protein-DNA, antibody-antigen, hormone-receptor, etc., illustrates the complexity and diversity of molecular recognition. The importance of such interactions in the immune response, signal transduction cascades, and gene expression cannot be overstated. It is of great interest to determine the nature of the forces that stabilize the interaction. The thermodynamics of association are characterized by the stoichiometry of the interaction (n), the association constant (K(a)), the free energy (ΔG(b)), enthalpy (ΔH(b)), entropy (ΔS(b)), and heat capacity of binding (ΔC(p)). In combination with structural information, the energetics of binding can provide a complete dissection of the interaction and aid in identifying the most important regions of the interface and the energetic contributions. Various indirect methods (ELISA, RIA, surface plasmon resonance, etc.) are routinely used to characterize biologically important interactions. Here we describe the use of isothermal titration calorimetry (ITC) in the study of protein-protein interactions. ITC is the most quantitative means available for measuring the thermodynamic properties of a protein-protein interaction. ITC measures the binding equilibrium directly by determining the heat evolved on association of a ligand with its binding partner. In a single experiment, the values of the binding constant (K(a)), the stoichiometry (n), and the enthalpy of binding (ΔH(b)) are determined. The free energy and entropy of binding are determined from the association constant. The temperature dependence of the ΔH(b) parameter, measured by performing the titration at varying temperatures, describes the ΔC(p) term. As a practical application of the method, we describe the use of ITC to study the interaction between cytochrome c and two monoclonal antibodies.

Original languageEnglish (US)
Pages (from-to)213-221
Number of pages9
JournalMethods: A Companion to Methods in Enzymology
Issue number2
StatePublished - Oct 1999

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)


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