ANALYSIS OF DRUG-PROTEIN BINDING USING NUCLEAR-MAGNETIC-RESONANCE BASED MOLECULAR-DIFFUSION MEASUREMENTS

H-1 and F-19 NMR spectroscopic approaches are presented for investigat ion of protein-ligand binding using the measurement of molecular diffu sion coefficients, This has been exemplified by investigation of the b inding of 4-trifluoromethylbenzoic acid (TFBA), and the non-steroidal anti-inflammatory drugs ibuprofen and flurbiprofen to human serum albu min (HSA). To avoid the need to define the number of independent bindi ng sites and their individual dissociation constants, the relative bin ding of the ligands to HSA was quantified using a binding capacity, de fined as log (ratio of ligand to HSA) required to give 50% of the liga nd bound, The diffusion-based approach to the study of protein-ligand binding offers a number of advantages over previous NMR methods, which rely on the error-prone determination of bound-ligand chemical shifts , relaxation times or linewidths. The comparative simplicity of the di ffusion method may allow its widespread use in the investigation of dr ug-macromolecule interactions, particularly for the weak but extensive binding of drugs to HSA in blood, which can affect biological activit y in vivo.