The Non‐Covalent Binding of Small Molecules by Ligandin

Authors:
Publisher website
Google Scholar
Abstract
Equilibrium dialysis studies have been made of the binding of a number of small molecules by rat ligandin. Direct measurements of binding together with competition experiments indicated that bromosulphophthalein, oestrone sulphate and dehydroepiandrosterone sulphate each bind at the same single primary binding site with association constants of 1.1 × 107. 6.6 × l05 and 2.6 × 105 l/mol respectively at pH 7.0, 10.16 M, 4 oC. As well as bromosulphophthalein and dehydroepiandrosterone sulphate, a number of strucurally similar organic anions including 2‐hydroxyoestradiol‐glutathione. oestrone glucuronide, N‐methyl‐4‐aininoazo benzene‐glutathione and several bile acids, were able to displace oestrone sulphate from ligandin in a manner consistent with competition at a single binding site. From these experiments association constants for the competing ligands were derived; these were in the range 1 × 104‐1 × 106 l/mol. Ligandin was found to bind a number of compounds for which, because of their low aqueous solubilities relative to their binding affinities, complete binding isotherms could not be obtained. These included several steroids (but not cortisol), 20‐methylcholanthrene, diethylstilboestrol, oleate and palmitate. Oestrone sulphate was able to compete with these ligands for binding and the results of the competition experiments were interpretable in terms of 1 :1 competition at a single binding site. In general the conjugation of non‐polar ligands with sulphate or glutathione resulted in increased affinities, but such increases were relatively small (∼ 15% in terms of free energy) implying that the main driving force for the binding of both the conjugated and unconjugated species was the hydrophobic effect. This conclusion is borne out by the oobservations that both oestrone and its sulphate showed slight increases in affinity with increase in ionic strength, as would be expected for hydrophobic interactions. As well as non‐polar compounds and organic anions, ligandin was also found to bind sulphate and glucuronate to a measurable degree, and to interact quite strongly with glutathione. For the latter compound a single binding site was found with an associat.ion.constant of 1 × 105 l/mol. Glutathione was able to cause the dissociation of the ligandin‐oestrone sulphate complex, but this effect was not explicable in terms of simple 1 : 1 competition. Both oestrone and oestrone sulphate were bound most strongly at pH 6‐ 7, the affinity of the protein for these ligands falling off quite sharply on either side of this maximum. The affinities of ligandin for bromosulphophthalein, steroids and their conjugates, diethylstilboestrol and N,N‐dimethyl‐4‐aminoazohenzenc are similar in magnitude to those of serum albumin and aminoazodye‐binding protein A (B. Ketterer, E. Tipping, J. F. Hackney and D. Beale, 1976).

This publication has 36 references indexed in Scilit: