Carbon 13 NMR studies of saturated fatty acids bound to bovine serum albumin. I. The filling of individual fatty acid binding sites

Abstract

13C NMR chemical shift and intensity results for a series of carboxyl 13C-enriched saturated fatty acids (8-18 carbons) bound to bovine serum albumin (BSA) are presented as a function of increasing fatty acid (FA)/BSA mole ratio. Spectra for long-chain (greater than or equal to 12 carbons) FA X BSA complexes exhibited up to five FA carboxyl resonances, designated a, b, b', c, and d. Only three resonances (peaks b, b', and d) were observed below 3:1 FA X BSA mole ratio, and at greater than or equal to 3:1 mole ratio, two additional resonances were observed (peaks c and a). In a spectrum of 5:1 stearic acid X BSA complexes, peaks b, b', and d each represented approximately one-fifth, and peak c approximately two-fifths, of the total FA carboxyl intensity. Plots of total carboxyl/carbonyl intensity ratio as a function of FA X BSA mole ratio were linear up to 7-9 mole ratio. Deviation from linearity at mole ratios greater than or equal to 7 was accompanied by the detection of crystalline unbound FA (as 1:1 acid/soap) by X-ray diffraction. In contrast to long-chain FA X BSA complexes, 13C NMR spectra of octanoic acid X BSA complexes yielded only one FA carboxyl resonance (peak c) at FA X BSA mole ratios between 1 and 20. We conclude: peaks b, b', and d represent FA bound to three individual high affinity (primary) long-chain FA binding sites on BSA; peak c represents FA bound to several secondary long-chain (or primary short-chain) FA binding sites on BSA; peak a represents long-chain FA bound to an additional lower affinity binding site. We present a model that correlates the observed 13C NMR resonances with individual binding site locations predicted by a recent three-dimensional model of BSA.