Active site contributions to fluorescein binding determined by in vitro heavy and light chain reassociations

In addition to prior crystallographic studies that determined antigen contact residues for high affinity murine monoclonal anti-fluorescein (Fl) antibody 4-4-20 (Ka = 1.7 x 10(10) M-1), primary structure comparisons revealed idiotypically cross-reactive monoclonal anti-Fl antibody 9-40 (Ka = 5.7 x 10(7) M-1) possessed identical Fl contact residues with the exception of L34 His for Arg. Mab 9-40 L34 His was germ-line encoded and 4-4-20 L34 Arg correlated with increased 4-4-20 affinity and enhanced Fl quenching. To better define L34 Arg and L96 Trp contributions to antigen binding, in vitro H and L chain reassociation experiments were performed. Following reassociations, affinity purified and homologous chain reassociated proteins 4-4-20 (H4-4-20 L4-4-20) and 9-40 (H9-40 L9-40) yielded identical idiotypes (greater than 91% related), Qmax values (91% and 44.7%), affinity constants (approximately 2.0 x 10(10) M-1 and 5.5 x 10(7) M-1) and iodide quenching values (1.2% and 2.1%), respectively. Although heterologous reassociated proteins were idiotypically related to prototypic proteins (greater than or equal to 87.1%), differences in Fl-binding characteristics were observed. Recombinant H4-4-20 L9-40 expressed Ka and Qmax values similar to 9-40 and implicated L34 Arg in increased 4-4-20 Ka and Qmax. Residue L34 Arg replacement in the 9-40 active site (H9-40 L4-4-20). however, exhibited low Qmax (44.4%) and slightly increased affinity (3.7 x 10(8) M-1). In addition, L96 Leu substitution into 4-4-20 (H4-4-20 L-04-01) and 9-40 (H9-40 L04-01) resulted in lower Qmax values (15.1% and 20.5%, respectively) and significantly reduced Fl affinity (approximately 10(3)-fold). These results demonstrated: (1) L34 Arg was responsible for increased 4-4-20 affinity, (2) L96 Trp was critical for an intermediate Fl affinity (approximately 10(7) M-1) and (3) active site Fl contact residue orientations were potentially affected by differences in H chain complementarity-determining region 3 and CH 1 residues.