ESR studies of stearic acid binding to bovine serum albumin.
The ESR spectra of a series of chain-labelled doxyl stearic acids (5-, 7-, 12- and 16-DSA) and doxyl methyl stearates (5-, 7-, 12- and 16-DMS) bound to the high-affinity binding sites of bovine serum albumin (BSA) have been analyzed using nonlinear least-squares fitting of slow-motional ESR stimulation. The motional analysis reveals that the rotational diffusion of these stearates around the axis perpendicular to the long hydrocarbon chain is greatly hindered, suggesting that they are held tightly in a channel of the protein. Comparison of the isotropic hyperfine splitting, A0, among each series shows that 5- and 16-DSA and 16-DMS have larger A0 values than the other spin labels. In addition, labels at the 16-C position of both DSA and DMS exhibit significantly increased motion relative to the other positions. These observations suggest that the channel starts at 5-C of the chain and ends somewhere between 13-C and 15-C, leading to an estimate of 11 +/- 1 A for the length of the channel. The methyl stearate labels exhibit significantly faster rotation around the chain axis than the analogous stearic acid labels, suggesting a double hydrogen-bonding mechanism for fatty acid binding to BSA. The ability of the acid to form two hydrogen bonds apparently fixes it more rigidly in the protein, preventing rotation about either single hydrogen bond. A double-hydrogen bonding mechanism is most consistent with the formation of a salt bridge between the negatively charged carboxylate of the acid and either a positively charged guanidino group of arginine, or the positively charged omega-amino groups of two lysine residues. An ESR study of the pH dependence of DSA binding indicates that salt bridge formation with lysine is responsible for at least some of the long chain fatty acid binding sites of BSA.
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Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, NY 14853.
Rananavare
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SB
Freed
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004058:Diffusion; D004578:Electron Spin Resonance Spectroscopy; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009589:Nitrogen Oxides / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D013229:Stearic Acids / Q000378:metabolism*
D009589:Nitrogen Oxides; D013229:Stearic Acids; D012710:Serum Albumin, Bovine; C039900:nitroxyl