Abstract
NMR diffusion measurements are a useful tool for probing molecular interactions, especially ligand–protein binding, because the diffusion coefficient measured for the ligand can reflect the binding affinity. This technique is useful for determining relative affinities of multiple ligands and quantitating binding constants to a specific biomolecular target, such as an enzyme. In this work, binding of two TFA‐derivitized inhibitors to the enzyme elastase was measured by ^19^F pulsed field gradient NMR spectroscopy. The differing thermodynamic (K~d~) and kinetic (chemical exchange) properties of the elastase inhibitors introduced unique challenges to the NMR diffusion analysis. Line broadening due to fast T~2~ relaxation, differential T~1~ relaxation on the diffusion time‐scale and chemical exchange severely compromised the spectral signal‐to‐noise ratio and forced the use of extensive signal averaging. The resultant experimental time caused inhibitor hydrolysis to occur on the time‐scale of the measurement. Modulation of resonance intensity in LED spectra measured as a function of the diffusion delay time was explored as a function of the population ratio of the free and bound ligands. The intensity modulation introduced by chemical exchange during the diffusion delay of the LED experiment can be removed by choosing a pulse sequence such as BPPLED that incorporates bipolar gradient pulse pairs separated by 180° pulses that refocus chemical shift evolution. Copyright © 2002 John Wiley & Sons, Ltd.