BINDING OF PHENYLPHOSPHOCHOLINE-CARRIER CONJUGATES TO THE COMBINING SITE OF ANTIBODIES MAINTAINS A CONFORMATION OF THE HAPTEN

The structural basis of the binding of phenylphosphocholine haptens to antibodies was studied, This was done by preparing antibodies and tes ting binding to conjugates of phenylphosphocholine. The choice of hapt ens was made in order to evaluate the contribution of the carrier to b inding, and its effect on hapten conformation in the active site. Thus , phosphocholine (PC) was diazophenyl-linked to tyrosine or histidine as single amino acid carriers and to tripeptides or octapeptides conta ining tyrosine or histidine as central amino acids to which PC was att ached. Relative affinity was assessed by inhibition enzyme-linked immu nosorbent assay (ELISA) and binding constants were determined by fluor escence quenching. Fluorinated haptens were used to determine the kine tics of binding using F-19 nuclear magnetic resonance. The transferred nuclear Overhauser effect was used to characterize conformation of th e bound hapten. We had previously shown that nitrophenylphosphocholine unlinked to carrier is bound in the active site as a bent structure [ Bruderer, U., Peyton, D. H., Barbar, E., Fellman, J. H., & Rittenberg, M. B. (1992) Biochemistry 31, 584-589]. We show here that this same b ent conformation is retained in the active site regardless of the neig hboring carrier or the conformation of the hapten in the unbound conju gate. The presence of the carrier residues in the bound state does, ho wever, influence affinity.