TY - JOUR T1 - Binding of Anionic Ligands to Peptic Fragments of Bovine Serum Albumin JF - Molecular Pharmacology JO - Mol Pharmacol SP - 224 LP - 229 VL - 18 IS - 2 AU - HERMAN MEISNER Y1 - 1980/09/01 UR - http://molpharm.aspetjournals.org/content/18/2/224.abstract N2 - 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. ER -