Allosteric binding properties of a monoclonal antibody and its Fab fragment.
Detailed equilibrium binding studies were conducted on a monoclonal antibody directed against Pb(II) complexed with a protein conjugate of diethylenetriaminepentaacetic acid (DTPA). Binding curves obtained with DTPA and a cyclohexyl derivative of DTPA in the presence and absence of metal ions were consistent with the anticipated one-site homogeneous binding model. Binding curves obtained with aminobenzyl-DTPA or its complexes with Ca(II), Sr(II), and Ba(II) were highly sigmoidal, characterized by Hill coefficients of 2.3-6.5. Binding curves obtained with the Pb(II) and In(III) complexes of aminobenzyl-DTPA were hyperbolic, but in each case the apparent affinity of the antibody for the chelator-metal complex was higher in the presence of excess chelator than it was in the presence of excess metal ion. In the presence of excess chelator, the equilibrium dissociation constant for the binding of aminobenzyl-DTPA-Pb(II) to the antibody was 9.5 x 10(-)(10) M. Binding curves obtained with the Hg(II) and Cd(II) complexes of aminobenzyl-DTPA were biphasic, indicative of negative cooperativity. Further binding studies demonstrated that aminobenzyl-DTPA-Hg(II) opposed the binding of additional chelator-metal complexes to the antibody more strongly than did aminobenzyl-DTPA-Cd(II). The Fab fragment differed from the intact antibody only in that the apparent affinity of the Fab was generally lower for a given chelator-metal complex. These data are interpreted in terms of a model in which (i) aminobenzyl-DTPA and its complexes bind both to the antigen binding site and to multiple charged sites on the surface of the compact immunoglobulin; and (ii) the bound, highly charged ligands interact in a complicated fashion through the apolar core of the folded antibody.
Blake
Robert C
RC
Department of Ophthalmology, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112, USA. blake@tulane.edu
Delehanty
James B
JB
Khosraviani
Mehraban
M
Yu
Haini
H
Jones
R Mark
RM
Blake
Diane A
DA
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002104:Cadmium / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006241:Haptens / Q000008:administration & dosage / Q000276:immunology; D006433:Hemocyanins / Q000008:administration & dosage / Q000276:immunology; D006825:Hybridomas; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000378:metabolism*; D007275:Injections, Intravenous; D007854:Lead / Q000008:administration & dosage / Q000276:immunology; D017669:Mercury Compounds / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D004369:Pentetic Acid / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000276:immunology
D000911:Antibodies, Monoclonal; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D017669:Mercury Compounds; C408383:cyclohexyldiethylenetriamine pentaacetic acid; D002104:Cadmium; C071640:1-(4-aminobenzyl)-DTPA; D007854:Lead; D004369:Pentetic Acid; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin