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Correspondence of fatty acid and drug binding sites on HSA: A 2D NMR Study.


Biochemistry. 2013 Jan 29;


Authors: Krenzel ES, Chen Z, Hamilton JA


Abstract

The ability of human serum albumin (HSA) to bind fatty acids (FA) in multiple sites has been revealed by many studies Here we characterize 9 individual binding sites and quantify their relative affinities by 2D NMR spectroscopy of 18-13C-oleic acid (OA) complexed with HSA. We probed site-specific FA binding by addition of: (i) different FA molar ratios to observe the order of filling and occupancy of binding sites; (ii) cyclodextrin as FA acceptor to observe the dissociation of FA; and (iii) drugs that are known to bind to low affinity FA sites. At 1:1 and 2:1 OA:HSA, 3 sites filled sequentially. These high affinity sites were well resolved from 1 medium and 5 lower affinity sites observed at 3:1- 4:1 OA:HSA. At the 4:1 ratio, the highest affinity-binding sites had similar equilibrium constants, with the others having constants ~5 times lower. Methyl-β-cyclodextrin extracted OA from individual sites, in the reverse order of filling. Three primary drug sites, Sudlow's Drug Sites 1 and 2, and FA Site 6, were identified by drug competition as lower affinity FA sites. Our strategies permit site-specific characterization of drug binding, and provide a sensitive probe of the mutual effects of FA and ligand binding.

PMID: 23360066 [PubMed - as supplied by publisher]