Verification of a model of a F(ab) complex with phenylarsonate by oligonucleotide-directed mutagenesis.

A model of the combining site of a mouse antibody specific for p-azophenylarsonate was tested by oligonucleotide-directed mutagenesis of the proposed hapten-contacting residues, Arg96 in the L chain, and Asn35, Trp47, Tyr50, Ser95, and Tyr100b in the H chain. The affinity and relative affinity for p-azophenylarsonate-N-acetyl-L-tyrosine of mutant antibodies expressed in transfectomas were determined by fluorescence quenching and by inhibition ELISA, respectively. The results show that alteration of the proposed contacting residues has drastic effects on hapten binding, and that the hydroxyl groups of Tyr50 and Tyr100b appear to orient the phenyl rings for optimal aromatic-aromatic interactions with the phenyl ring of the hapten. They further indicate a tight packing of the contacting residues around the hapten, which cannot accommodate changes in the positions of the functional groups of Asn35 and Ser95.