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

Abstract

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.

Thermodynamic Analysis of the Binding of 2F5 (Fab and Immunoglobulin G Forms) to Its gp41 Epitope Reveals a Strong Influence of the Immunoglobulin Fc Region on Affinity

“…To overcome such a drawback, ITC displacement experiments can extend the useful range for the association constant determination (29,30).…”

Enhanced Mechanical Properties in Cellulose Nanocrystal–Poly(oligoethylene glycol methacrylate) Injectable Nanocomposite Hydrogels through Control of Physical and Chemical Cross-Linking

“…Data from the first injection was disregarded, omitting errors originating from the diffusion of titrant into the calorimetric cell. 49 The heat of dilution of adding each POEGMA precursor polymer solution (prepared in PBS) into water not containing CNCs was used as a blank, with those measured heats of dilution subtracted from the enthalpies measured for each run. The molar heat of injection (ΔH, kJ mol -1 ) was determined by integrating each individual injection peak, as demonstrated previously for other polymer systems adsorbing onto the surface of CNCs.…”

Thermodynamics of Natural and Synthetic Inhibitor Binding to Human Hsp90

“…ITC was useful to provide the enthalpy of binding. However, as seen in radicicol binding ITC curves in Figure 5, the binding is too tight and would require the displacement assay as described previously [Velazquez-Campoy and Freire, 2006]. Our experience shows that the TSA is much easier and yields more precise results than the displacement ITC assay .…”