title: Active site contributions to fluorescein binding determined by in vitro heavy and light chain reassociations.
abstract: 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.
authors.affiliation: Department of Microbiology, University of Illinois, Urbana 61801.
authors.forename: W D
authors.initials: WD
authors.lastname: Bedzyk
authors.forename: E W
authors.initials: EW
authors.lastname: Voss
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D019793:Fluorescein; D005452:Fluoresceins; D007130:Immunoglobulin Idiotypes / Q000276:immunology; D066298:In Vitro Techniques; D051379:Mice; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
chemical_list: D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D007130:Immunoglobulin Idiotypes; D019793:Fluorescein