Correspondence of Fatty Acid and Drug Binding Sites on Human Serum Albumin: A Two-Dimensional Nuclear Magnetic Resonance Study

The ability of human serum albumin (HSA) to bind fatty acids (FA) in multiple sites has been revealed by many studies. Here we detect and characterize nine individual binding sites by two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy of 18-[13C]-oleic acid (OA) complexed with HSA. We characterize site-specific FA binding by addition of (i) different FA molar ratios (from 1:1 to 4:1 OA:HSA) to observe the order of filling and occupancy of binding sites; (ii) methyl-β-cyclodextrin, as a FA acceptor, to observe the dissociation of FA; and (iii) drugs (with known binding sites in the crystal structure) to reveal the correspondence of three NMR peaks with sites in the crystal structure. At 1:1 and 2:1 OA:HSA ratios, three sites were shown to fill sequentially. These high-affinity sites were well resolved from additional sites (one medium-affinity and five low-affinity) observed at 3:1 and 4:1 OA:HSA ratios. Methyl-β-cyclodextrin extracted OA from individual sites in the reverse order of fillin...