RT Journal Article SR Electronic T1 Binding of Anionic Ligands to Peptic Fragments of Bovine Serum Albumin JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 224 OP 229 VO 18 IS 2 A1 HERMAN MEISNER YR 1980 UL http://molpharm.aspetjournals.org/content/18/2/224.abstract AB The binding of anionic ligands was determined in fragments of bovine serum albumin prepared by pepsin digestion. The two fragments were the —NH2 terminal (amino acids 1-306) and the —COOH terminal half (amino acids 307-582), originally described by T. King [Arch. Biochem. Biophys. 156: 509-520 (1973)]. Ligand binding and competitive interactions were described by a Scatchard model having multiple independent sites. Equilibrium partitioning at 37°C showed that 14C-palmitate was bound to the —COOH terminal fragment (P-A) at one high-affinity site, k1 = 5.2 x 106 M-1, while the —NH2 terminal fragment (P-B) bound up to 4 mol with a lower affinity. The weaker binding ligands octanoate and chlorophenoxyisobutyrate (CPIB) displaced 14CC-palmitate competitively from fragments P-A and P-B, respectively, with apparent competition constants (ki') 10-1 less than the respective association constants (ki). Fragment P-A contained 0.4-0.8 high-affinity sites for 14C-CPIB and 3H-ANS, with a k1 of 105 M-1, and fragment P-B bound these ligands at several low-affinity sites (ki ≅ 103 M-1). The binding curve of 3H-ANS to reconstituted albumin, prepared by mixing equimolar amounts of P-A and P-B, was greater than to either peptic fragment. The profile equaled that obtained by summing the affinity constants of P-A and P-B. It is concluded that P-A is the fragment with a strong binding site for anionic ligands, but few if any weak sites, while P-B contains multiple weak sites and no strong sites. These data were explained in terms of a model with initially independent sites of unequal affinity for the ligand. ACKNOWLEDGMENTS I want to thank Dr. Kenneth Neet for many stimulating discussions and Ms. Susan Ruzich for technical assistance.