Relative Affinities of Fatty Acid Binding Sites on Human Serum Albumin Probed by 2D-NMR

In circulation HSA is the principal carrier for endogenous lipophilic compounds, primarily non-esterified long chain fatty acids (FA). Since FA bind to multiple binding sites with varying affinities, it would be useful to probe the relative affinities for these FA binding sites with a method that distinguishes individual binding sites. As visualized in crystal structures FA binding distributes asymmetrically throughout the protein. In the physiologically relevant solution state, these sites were partially distinguished by 1D-13C-NMR spectroscopy and subsequently correlated with crystal structure sites. Here we show nine, well-resolved peaks in 1H-13C-NMR spectra of 18-13C-oleic acid (OA) / HSA complexes. Different NMR signals represent FA bound at different sites throughout the protein, with the varying intensities corresponding to the different affinities HSA has for OA. This investigation probes the relative affinities of FA with three approaches: (i) addition of different FA molar ratios to HSA, to observe the order of filling of binding sites; (ii) addition of FA acceptors to observe the dissociation of FA from HSA; and (iii) addition of drugs that are known to bind to low affinity FA sites. From the order of filling, the three highest affinity-binding sites are clearly differentiated from the six lower/medium affinity-binding sites at the physiologically relevant FA:HSA molar ratio- 4:1. Methyl-β-cyclodextrin (MβCD) extracted FA from individual sites, in a concentration dependent manner, with the highest concentrations removing FA from the highest affinity sites. Relative affinities determined as above were consistent with the binding of drugs to Sudlow's drug binding sites, which displaced bound FA from specific lower affinity sites. 2D-NMR spectroscopy is a powerful approach for studying interactions of FA with HSA and FA-competitive drugs in a site-specific manner, providing a unique view of HSA-ligand binding.