Location of long chain fatty acid-binding sites of bovine serum albumin by affinity labeling.
Affinity labeling with palmitic acid was used to identify long chain fatty acid-binding sites of bovine serum albumin. [1-14C]Palmitic acid was activated by esterification with N-ethyl-5-phenyl-isoxazolium-3'-sulfonate (Woodward's Reagent K). The product was purified by chromatography and shown to compete with unesterified fatty acids for binding sites on bovine serum albumin. Activated [14C]palmitic acid coupled covalently to albumin producing [14C]palmitoyl-albumins containing from 0.12 to a maximum of 6.9 mol of attached label per mol of albumin. The presence of the covalently attached affinity label depressed binding of other long chain fatty acids to albumin. Albumin carrying 1 eq. of [14C]palmitate was cleaved using cyanogen bromide, pepsin, and trypsin. Radioactive peptides were isolated by high pressure liquid chromatography. Three peptides accounted for greater than 90% of the label. Residues labeled with [14C]palmitate were identified as Lys-116, Lys-349 and Lys-473, and the relative distribution of label was 10, 45, and 45% respectively, consistent with the presence of two strong binding sites in the COOH-terminal half of albumin and a somewhat weaker site in the NH2-terminal half.
D000345:Affinity Labels / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D003488:Cyanogen Bromide; D004951:Esterification; D005227:Fatty Acids / Q000378:metabolism*; D007555:Isoxazoles; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D010434:Pepsin A; D010446:Peptide Fragments / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D014357:Trypsin
D000345:Affinity Labels; D005227:Fatty Acids; D007555:Isoxazoles; D010169:Palmitic Acids; D010446:Peptide Fragments; D012710:Serum Albumin, Bovine; D019308:Palmitic Acid; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D014357:Trypsin; D010434:Pepsin A; D003488:Cyanogen Bromide