Abstract
The interactions of the carboxyl group of oleic acid with bovine serum albumin (BSA) were studied by 13C NMR spectroscopy at 50.3 MHz using 90% isotopically substituted [1-13C]oleic acid. 13C NMR spectra were obtained as a function of the mole ratio of oleic acid to BSA (from 0.5-10.0) and, for selected mole ratios, as a function of pH (between pH 3.0 and 10.6) and temperature (between 15 and 55 degrees C and thermally denatured at 95 degrees C). Except for spectra of highly acidic (pH less than or equal to 3.9) and denatured samples, spectra of oleic acid/BSA complexes showed multiple narrow resonances from the oleic acid carboxyl carbon in a region (179-184 ppm) downfield from protein carbonyl and carboxyl carbon resonances. At low oleic acid/BSA ratios (0.5 and 1.0), at least two oleic acid carboxyl carbon peaks were observed; at high ratios (greater than or equal to 3.0), at least four peaks were present. The intensities of individual peaks, but not their chemical shifts, varied with the oleic acid/BSA ratio. The chemical shift of individual oleic acid peaks was invariant between pH 6.0 and 10.6; below pH 6.0, one of the oleic acid resonances exhibited an NMR titration curve with an apparent pKa of approximately 4. Thus, BSA binding sites for oleic acid are heterogeneous as monitored by the magnetic microenvironment of the oleic acid carboxyl carbon. The number of different oleic acid environments and the relative population of oleic acid molecules in these environments is dependent on the mole ratio of oleic acid/BSA. Our results suggested that the anionic form of oleic acid is bound to BSA at physiological pH and that the multiplicity of NMR peaks for [1-13C]oleic acid resulted from, at least in part, different electrostatic and hydrogen bonding interactions between the oleic acid carboxyl group and specific amino acid residues of BSA.
Highlights
NMR spectroscopyat 50.3 MHz using 90%isotopically In spite of the obvious importance of fatty acid/albumin substituted [l-’3C]oleic acid
Our results suggested that the anionic form of oleic acid is bound to bovine serum albumin (BSA) at physiological pH and that the multip~~citoyf NMR peaks for [f-‘3C]oleic acid resulted from, at least in part, different electrostatic and hydrogen bonding interactions between the oleic acid carboxyl group and specific amino acid residues of that thefirst two fatty acids bind to a single specific domain[11]and thateach of the remaining two domains can bind two fatty acid molecules [11, 18]
This study demonstrates the feasibility of usingNMR spectroscopy to probe interactions between albumin and a biological fatty acid, oleic acid
Summary
BSA (420 mg in 3.5 ml of HzO) to 8.8 mg of potassium oleate. The gram. On selected spectra with good signal-to-noise ratios, broad pH was adjusted to 7.0 and a 2-ml aliquot (240 mg of BSA; 5 mg of spectral components wereremovedby the convolution difference fatty acid) was applied to a Sephadex G-150 column (90 x 1.5 cm). 1“C) with the Bruker B-VT-1000 variable temperatureunit and Interactions of Oleic Acid withAlbumin. Spectra shown in the middle row are from samples with mole ratios indicated in the ~~~0~ line of the figure. The spectra (middle row) for 0.5, 2, and 3 mol of oleic acid/mol of BSA were obtained following 20,000, 24,000, and 31,327 accumulations, respectively, and are printed with higher vertical gain. The spectrum (middle row) for 1mol of oleic acidlmol of BSA is a 2-fold vertical expansion of the bottom spectrum (4,000 accumulations). Major oleic acid carboxyl peaks are labeled as a-d. The broad signal is primarily from protein backbone carbonyl groups
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