On the role of Tyr 34_L in the antibody combining site of the dinitrophenyl binding protein 315

The role of Tyr 34_L in the combining site of the dinitrophenyl binding mouse myeloma protein 315 has been studied by chemical modification. Tyr 34_L was specifically nitrated by separating the peptide chains of the Fv fragment intoV_L and V_H (Hochman et al., 1973), nitrating V_L with tetranitromethane and reassociating the modified V_L with unmodified V_H Comparison of the binding properties of NO₂ Fv and Fv, and of NO₂V_L and V_L was performed by equilibrium dialysis with ³H-Dnp-lysine, dinitrophenol, Tnpamino caproic acid and Tnp-amino ethylamine at pH 5.0 and pH 8.0. Since the pK_a of the NO₂ Tyr in the above fragments is 6.2-6.6. these binding studies were designed to investigate the possibility of a steric effect of introduction of the nitro group, and to demonstrate the presence or absence of a hydrogen bond between Tyr 34_L and the ligand suggested in a recent paper (Dower et al., 1977), by observing the effect of ionization of the phenol group. Both the affinities and numbers of sites of the modified and unmodified fragments were found to be very similar. Nuclear magnetic resonance studies showed that the hapten occupied the same position in the combining sites of the modified and unmodified Fv fragments. Hence it is concluded that Tyr 34_L does not form a hydrogen bond to nitrophenyl ligands.