Fragments of bovine serum albumin produced by limited proteolysis. Conformation and ligand binding.
Twelve fragments of bovine serum albumin, isolated following limited tryptic or peptic hydrolysis, have been studied to define secondary structure and locate ligand-binding sites. Based on circular dichroism, the conformational pattern of albumin (68% alpha helix and 18% beta structure) is substantially retained by individual fragments, indicating that secondary configuration is locally determined and is not destroyed during the cleavage process nor during fragment purification. The strong bilirubin-binding site of bovine serum albumin is present in 3 of the 12 fragments. Residues 186-238 are common to the three fragments and absent from those fragments which do not bind bilirubin; consequently the strong bilirubin-binding site is suggested to involve this region. By similar reasoning, the presence of palmitate-binding sites in some fragments and not in others indicates that the three strongest sites for the binding of palmitate are located in the carboxyl-terminal two-thirds of the molecule. The first site (KA approximately 2 X 10(7) M-1) is suggested as residues 377-503; the second site (KA approximately 8 X 10(6) M-1), residues 239-306; the third site (KA approximately 2 X 10(6) M-1), residues 307-377. Bromocresol Green, a reagent used in the assay of ablumin, was bound by fragments rougly in proportion to their size but showed particular affinity for the region of the strong bilirubin-binding site. The fluorescent probe, 8-anilino-1-naphthalensulfonate, was in general bound by large fragments, supporting the concept that this ligand is held principally in clefts between domains of the macromolecule.
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001663:Bilirubin / Q000378:metabolism; D001665:Binding Sites; D001961:Bromcresol Green / Q000378:metabolism; D002417:Cattle; D008024:Ligands; D010168:Palmitates / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013329:Structure-Activity Relationship
D000817:Anilino Naphthalenesulfonates; D008024:Ligands; D010168:Palmitates; D010446:Peptide Fragments; D012710:Serum Albumin, Bovine; D001961:Bromcresol Green; D001663:Bilirubin