Equilibrium Binding of Spin-Labeled Fatty Acids to Bovine Serum Albumin: Suitability as Surrogate Ligands for Natural Fatty Acids

Ray C. Perkins, Nada Abumrad, K. Balasubramanian, L. R. Dalton, Albert H. Beth, Jane H. Park, Charles R. Park

Research output: Contribution to journalArticle

27 Scopus citations

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 the 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 Wdue 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.

Original languageEnglish
Pages (from-to)4059-4064
Number of pages6
JournalBiochemistry
Volume21
Issue number17
DOIs
StatePublished - Aug 1982
Externally publishedYes

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