Steroids as immunochemical probes. Thermodynamic and kinetic data with special regard to the ''bridge problem'' in estrogen radioimmunoassay

The binding sites of antibodies raised against estrogen-6-one-oxime-O-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 with labelled estrogens, Gibbs energy changes were found to be about - 14kcal/mol, to be rather uniformly distributed, to be predominantly enthalpy contributed and to depend in some instances on the binding-site concentration. The potency of unlabelled haptens to inhibit binding of labelled ligands was determined, the values being related to their binding energies. The structures of the series of systematically varied unlabelled haptens increasingly approached the structure of the immunodeterminant. Ranking the inhibition potencies of this series revealed that estrogen-6-one exhibited the highest potency - a potency even higher than the inhibition potency of the estrogen-6-one-oxime-O-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 the binding processes could not be explained simply in terms of equilibrium constants. Association reaction rates decreased with increasing length of the hapten molecules. Conversely, dissociation rates decreased, but the slowest dissociation rate was observed with complexes of antibodies with estrogen-6-one-oxime-O-methylether. Further lengthening of the hapten had no effect on the dissociation rate. It was concluded that this ligand reflects the size of the antibody binding site. (author)