1H-NMR study on the interactions of human serum albumin with free fatty acid.

Binding of free fatty acid (FFA) to human serum albumin (HSA) was studied by 1H-NMR spectroscopy. Addition of FFA to defatted HSA at a mole ratio (FFA/HSA) up to 4 caused a small change in the NMR spectrum of HSA. The integrated intensity of sharp signals of the histidine C2 proton region of HSA decreased as the mole ratio was increased from 0 to 4 for both medium chain (lauric acid) and long chain (palmitic acid, stearic acid, and oleic acid) FFA's. By contrast, when the mole ratio was increased above 4, several histidine C2 proton signals coalesced and sharpened. Therefore, the HSA molecule appears to have a different conformation on binding with more than 4 FFA molecules, which allows increased local motions of HSA. By analyzing the NMR difference spectra of HSA with various amounts of FFA, the conformational change of HSA was investigated in more detail. The difference spectrum between [HSA + 2FFA] and [HSA + FFA] was almost the same as the difference spectrum between [HSA + FFA] and [HSA], which suggests that one primary site binds a pair of FFA molecules. These results are consistent with those of a spectroscopic study with polyene fatty acids (Berde, C.B., et al. (1979) J. Biol. Chem. 254, 391-400). The existence of a bimolecular complex of FFA molecules in aqueous solution may facilitate this type of binding. Similarly, it was found that the third and fourth FFA molecules were bound to a secondary site on HSA, because the difference spectrum between [HSA + 4FFA] and [HSA + 3FFA] was nearly equal to the difference spectrum between [HSA + 3FFA] and [HSA + 2FFA]. Further addition of FFA resulted in a drastic spectral change of HSA. The NMR difference spectrum between HSA solutions with perdeuterated FFA and those with undeuterated FFA gave the 1H-NMR spectra of FFA molecules bound to HSA. Titration of FFA revealed that, in the binding to the primary site of HSA, the carboxyl group of FFA is tightly bound to the protein, whereas the methyl group is not so firmly bound. In contrast, in the binding to low affinity sites, the methyl group is bound to HSA as tightly as other portions of the molecule.