Steroids as Immunochemical Probes; Thermodynamic and Kinetic Data with Special Regards to the 'Bridge Problem' in Estrogen Radioimmunoassay

摘要

The binding sites of antibodies raised against estrogen-6-one-oxime-0-carboxymethyl-derivatives attached to epsilon-amino groups of lysine residues in albumin were mapped thermodynamically and kinetically by reactions with labelled and unlabelled haptens. By equilibrium dialysis Gibbs energies were found to amount to about -14 kcal/mol, to be rather uniformly distributed, and to be predominantly enthalpy contributed. From unlabeled haptens their potencies to inhibit binding of labeled ligands were determined, which values are related to their binding energies. The structures of the series of systematically varied unlabelled haptens approached increasingly the structure of the immunodeterminant. Ranking the inhibition potencies of this series revealed that estrogen-6-one exhibited the highest potency, even higher than the inhibition potency of the estrogen-6-one-oxime-0-carboxymethyl-lysine derivative, which is obviously most closely related to the immunodeterminant group. To prove whether the inhibition potencies were actually restricted by the size of the antibody-binding site, both components of the equilibrium constants were determined, the association rate constants and the dissociation rate constants. Structure dependent variation of both indicated that explanation of the binding processes in terms of only the equilibrium constants was insufficient. Association reaction rates decreased with increasing length of the hapten molecules. Inversely dissociation rates decreased, but the slowest dissociation rate was observed on complexes of antibodies with estrogen-6-one-oxime-0-methylether. Further lengthening of the hapten had no effect on the dissociation rate. It was concluded that this ligand reflects the size of antibody binding site. (Atomindex citation 09:382805)