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Measurement of Tight Binding Affinities with ITC

The range of binding constants which can be directly measured with Isothermal Titration Calorimetry (ITC) range from millimolar to nanomolar.  With tight binding (K_B >10⁹ M^-1 or K_d tighter than nanomolar), ITC titrations lose their curvature and affinity cannot be accurately calculated (Figure1, panel A - Wiseman et al, 1989). A method to measure K_B greater than 10⁹ M^-1 has been developed (Sigurskjold, 2000, Velazquez-Campoy and Freire, 2006). This method requires two ligands: a tight binding ligand (A) and a second ligand (B) which has a weaker binding affinity and binds competitively to the same site as ligand A. In the first ITC experiment (Figure 1, panel B), ligand B is titrated into the protein solution, and K_B and ΔH are determined.  In the second ITC experiment (Figure 1, panel C), ligand A is titrated into the protein-ligand B complex and ligand A displaces ligand B.  Using appropriate curve-fitting models (Sigurskjold, 2000) the binding constant of the tight-binding ligand can be calculated.  Velazquez-Campoy et al (2001) have used this method to study several inhibitors of HIV-1 protease.

Panel A:  Macromolecule in ITC cell, tight binding ligand A in syringe.  Titration is not sigmoidal curve, so unable to determine K_B for ligand A.

Panel B:  Macromolecule in ITC cell, weak binding ligand B in syringe

Panel C: Macromolecule plus ligand B in ITC cell, ligand A in syringe. During titration, ligand A binds to macromolecule, displacing ligand B. Using model of Sigurskjold, K_B for ligand A is 1.2 x 10⁹ M^-1