Equilibrium binding of spin-labeled fatty acids to bovine serum albumin: suitability as surrogate ligands for natural fatty acids

Abstract

Electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopies were used to characterize the binding of spin-labeled fatty acid (SLFA) to bovine serum albumin (BSA). Association constants of three stearic acid derivatives labeled with a nitroxyl radical at C-5, C-12, or C-16 were estimated by EPR spectroscopy as the ratio of SLFA to BSA was increased from about 0 to 9. The values were compared to those for unmodified stearate. With all three SLFA, it was apparent that the nitroxyl residue modified the binding pattern. For SLFA:BSA ratios up to 1, which probably involves the site(s) on BSA most specific for long-chain FA, the C-16 derivative bound with an affinity similar to that of the natural FA. At higher ratios, the association constants for this SLFA were lower than those for stearate. The C-12 and C-5 derivatives showed only low-affinity binding relative to stearate. The spectral parameter, W, was constant for SLFA:BSA ratios between 0 and 1 in the case of C-16 compound, indicating physical homogeneity of the high-affinity binding site. At higher ratios, the spectra changed progressively, indicating inhomogeneity of the lower affinity binding sites although parallel changes in association constants were not observed. Changes in W due to Heisenberg spin exchange were ruled out. By examining the mobility profile of the bound SLFA by both EPR and ST-EPR techniques, it was shown that the nitroxyl group was maximally immobilized when attached near the center of the carbon chain of the bound SLFA.

ATP binding to bovine serum albumin

“…Four mol of fatty acid are bound per tool of BSA [23]. These compounds are amphiphilic and hence° resemble nucleotides in this respect.…”

Interaction of albumin with the endothelial cell surface

“…The nitroxyl moities of 5-and 12-DOXYL-stearic acid are differently attached to the protein resulting in different rotational correlation times (24,26) over a large temperature range (Fig. 4).…”

“…Well-established molecular probes for studies on biomembranes (5,13,23), on model membranes (1,7), and in lipoprotein research (9) are fatty acids with a nitroxide substitution at various C-positions (17,35). This class of compounds binds avidly to albumin (3,44) where the spin label shows restricted mobility depending on its position along the backbone of the fatty acid (26).…”

Interactions of myristic acid with bovine serum albumin: a 13C NMR study.

“…[24][25][26][27]. In contrast to the 13C results, stearic acid molecules containing nitroxide probes near the carboxyl group often appear to be immobilized (25,27,28). However, nitroxide probes may significantly alter both the FA binding environment and FA molecular motions whereas the 13C probes are nonperturbing.…”