13C NMR studies of the binding of medium-chain fatty acids to human serum albumin

Abstract

Binding of the medium-chain fatty acids (MCFA), octanoic (OCT) and decanoic (DEC) acid, to human serum albumin (HSA) has been studied by 13C NMR spectroscopy. NMR spectra at 35 degrees C showed an apparently homogeneous binding environment (a single, narrow resonance for the 13C-enriched carboxyl carbon) at different mole ratios and pH values. Changes in the chemical shift of this peak with mole ratio and protein concentration demonstrated rapid equilibration (< or = msec) of bound and unbound MCFA and permitted a direct quantitation of bound/unbound MCFA. Spectra of OCT/HSA mixtures at 6 degrees C revealed at least three distinct binding sites that fill sequentially. The observed heterogeneity of binding at low temperature, compared to 35 degrees C, is attributed to a slower exchange rate of OCT between binding sites. The highest affinity sites for both OCT and DEC have properties similar to those of binding sites for longer-chain fatty acids, such as the close proximity of the fatty acid carboxylate to basic amino acid residue(s). Interestingly, chemical shift data showed that the first mole of OCT and DEC either bind differently to the same site or bind to different sites on HSA. The rapid desorption of MCFA from HSA binding sites has implications for dietary regimens with medium chain triglycerols.