Molecular Pharmacology, Vol 18, 224-229, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

Binding of Anionic Ligands to Peptic Fragments of Bovine Serum Albumin

¹ Department of Pharmacology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106

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 (k_i') 10^-1 less than the respective association constants (k_i). 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 (k_i 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.

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ACKNOWLEDGMENTS I want to thank Dr. Kenneth Neet for many stimulating discussions and Ms. Susan Ruzich for technical assistance.