Isothermal titration calorimetry to determine association constants for high-affinity ligands

Adrian Velazquez-Campoy, Ernesto Freire

Research output: Contribution to journalArticlepeer-review

192 Scopus citations


An important goal in drug development is to engineer inhibitors and ligands that have high binding affinities for their target molecules. In optimizing these interactions, the precise determination of the binding affinity becomes progressively difficult once it approaches and surpasses the nanomolar level. Isothermal titration calorimetry (ITC) can be used to determine the complete binding thermodynamics of a ligand down to the picomolar range by using an experimental mode called displacement titration. In a displacement titration, the association constant of a high-affinity ligand that cannot be measured directly is artificially lowered to a measurable level by premixing the protein with a weaker competitive ligand. To perform this protocol, two titrations must be carried out: a direct titration of the weak ligand to the target macromolecule and a displacement titration of the high-affinity ligand to the weak ligand - target macromolecule complex. This protocol takes approximately 5 h.

Original languageEnglish (US)
Pages (from-to)186-191
Number of pages6
JournalNature protocols
Issue number1
StatePublished - Jun 2006

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)


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