The use of D2O as an exchange reagent produces the least perturbation of any chemical method. No additional reagents are needed. No physical separations of the free ligand or protein from the protein-ligand binding system are required as in affinity chromatography, size exclusion chromatography, and ultra-filtration. Certain methods that track stability of protein-ligand interactions (e.g. circular dichroism and other spectroscopy methods [98-100] as well as SUPREX [20, 101]) require denaturants, and they may perturb the original binding equilibrium. ESI- or MALDI-based methods that attempt to measure directly the solution concentrations may also perturb the equilibrium during the ionization process, causing inaccuracies in the determination [76, 87, 88].
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