3 research outputs found
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-[<sup>13</sup>C]-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 filling.
FA bound in three low-affinity sites were displaced by drugs shown
to bind in crystalline HSA to FA sites 7 and 3 (Sudlow’s drug
sites I and II, respectively) and FA site 6. With this strategy, 2D
NMR spectral analysis permits site-specific characterization of the
binding of drugs and FA and provides a sensitive probe of the mutual
effects of FA and ligand binding