The direct determination of very weak association constants can require such high concentrations of reactants that the experiment may be impractical or encounter solubility problems. Measuring very tight association constants, on the other hand, may require such low concentrations of reactants that the heat signal from the binding event cannot be measured accurately.
Very weak or very tight association constants can be conveniently determined by ITC using competitive binding. In a competition experiment, two ligands with different affinities compete for the same binding site on a macromolecule (e.g., a characterized drug and a new drug candidate competing for the same protein target binding site). Since the binding constant of the characterized drug is known, the change in its binding constant due to the presence of the new drug candidate allows the binding constant of the uncharacterized ligand to be calculated, even if its binding constant falls outside the range normally accessible by ITC.
In a typical experiment to measure the Ka of a weakly binding ligand, for example, sufficient weak ligand is added to the target macromolecule to occupy approximately 50% of the binding sites. Using ITC, the binding sites are then incrementally filled by the characterized, stronger-binding ligand, displacing the weaker ligand until the protein is saturated. The extent to which the second ligand displaces the first will be dependant on the relative affinities and concentrations of the competing compounds.
A similar approach can be used to measure the Ka of very strongly-binding ligands. An application of the competitive binding approach for determining the Ka of a weakly binding inhibitor is presented in Figure 3. RNase A binds both 2´-CMP and 5´-CMP in the same binding pocket, but shifting the hydroxyl group from the 2´ to the 5´ position significantly decreases the binding affinity of the ligand. The single binding site of RNase A is so weakly inhibited by 5´-CMP that there is little curvature in the titration plot, so the affinity of 5´-CMP can only be roughly estimated from a direct titration experiment without resorting to high reactant concentrations (red dataset, Figure 3).
However, if sufficient 5´-CMP is added to RNase A to occupy about half the binding sites (in this example, 1.0 mL of 70-µM RNase A prebound with 0.32-µM 5´-CMP), and then 2´-CMP is titrated in (100 µL total of 1.3-mM 2´-CMP, titrated in 5-µL increments), significant heats of binding are obtained (blue dataset, top panel). This provides a graph of integrated heats (bottom panel) that can be accurately fit to provide the stoichiometry (n = 1), binding constant (3.1 x 103), and enthalpy (-47 kJ/mol) of the 5´-CMP binding reaction.
ITC is rapidly becoming the method of choice for characterizing binding reactions. The approach is completely general: small molecules binding to proteins, DNA, RNA, and polysaccharides can all be studied in an analogous manner, as can the binding of one macromolecule to another (by placing the second macromolecule rather than a small molecule ligand in the syringe).
The increasing prominence of ITC as a fundamental tool in biomedical research is due to the accurate assessment of binding interactions that can be obtained in several 1–2 hour automated experiments using just nanomoles of material and without the need to develop new assay protocols for each biomolecule or ligand. In addition, ITC’s direct nature and high precision allows users to validate the results of less rigorous, high-throughput assay protocols.