The Non‐Covalent Binding of Small Molecules by Ligandin
- 1 August 1976
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 67 (2), 583-590
- https://doi.org/10.1111/j.1432-1033.1976.tb10724.x
Abstract
Equilibrium dialysis studies were made of the binding of a number of small molecules by rat ligandin. Direct measurements of binding together with competition experiments indicated that bromosulfophthalein, estrone sulfate and dehydroepiandrosterone sulfate each bind at the same single primary binding site with association constants of 1.1 .times. 107, 6.6 .times. 105 and 2.6 .times. 105 l/mol, respectively, at pH 7.0, I [ionic strength] 0.16 M, 4.degree. C. In addition to bromosulfophthalein and dehydroepiandrosterone sulfate, a number of structurally similar organic anions including 2-hydroxyestradiol-glutathione, estrone glucuronide, N-methyl-4-aminoazobenzene-glutathione and several bile acids, could displace estrone sulfate 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 .times. 104 - 1 .times. 106 l/mol. Ligandin bound 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, diethylstilbestrol, oleate and palmitate. Estrone sulfate competed 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 sulfate or glutathione increased affinities, but such increases were relatively small (.apprxeq. 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 observations that estrone and its sulfate showed slight increases in affinity with increase in ionic strength, as expected for hydrophobic interactions. In addition to non-polar compounds and organic anions, ligandin bound sulfate and glucuronate to a measurable degree and interacted strongly with glutathione. For the latter compound a single binding site was found with an association constant of 1 .times. 105 l/mol. Glutathione caused the dissociation of the ligandin-estrone sulfate complex, but this effect was not explicable in terms of simple 1:1 competition. Both estrone and estrone sulfate 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 bromosulfophthalein, steroids and their conjugates, diethylstilbestrol and N,N-dimethyl-4-aminoazobenzene 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:
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