Determining molecular binding sites on human serum albumin by displacement of oleic acid

An NMR method was developed for determining binding sites of small molecules on human serum albumin (HSA) by competitive displacement of ¹³C-labeled oleic acid. This method is based on the observation that in the crystal structure of HSA complexed with oleic acid, two principal drug-binding sites, Sudlow's sites I (warfarin) and II (ibuprofen), are also occupied by fatty acids. In two-dimensional [¹H,¹³C] heteronuclear single quantum coherence NMR spectra, seven distinct resonances were observed for the ¹³C-methyl-labeled oleic acid as a result of its binding to HSA. Resonances corresponding to the major drug-binding sites were identified through competitive displacement of molecules that bind specifically to each site. Thus, binding of molecules to these sites can be followed by their displacement of oleic acids. Furthermore, the amount of bound ligand at each site can be determined from changes in resonance intensities. For molecules containing fluorine, binding results were further validated by direct observations of the bound ligands using ¹⁹F NMR. Identifying the binding sites for drug molecules on HSA can aid in determining the structure-activity relationship of albumin binding and assist in the design of molecules with altered albumin binding.