Complete disulfide bond assignment of a recombinant immunoglobulin G4 monoclonal antibody |
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Authors: | Zhang Wei Marzilli Lisa A Rouse Jason C Czupryn Marta J |
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Affiliation: | Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, 73104, USA. biji-kurien@omrf.ouhsc.edu |
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Abstract: | Surface plasmon resonance biosensor analysis was used to evaluate the thermodynamics and binding kinetics of naturally occurring and synthetic cobalamins interacting with vitamin B(12) binding proteins. Cyanocobalamin-b-(5-aminopentylamide) was immobilized on a biosensor chip surface to determine the affinity of different cobalamins for transcobalamin, intrinsic factor, and nonintrinsic factor. A solution competition binding assay, in which a surface immobilized cobalamin analog competes with analyte cobalamin for B(12) protein binding, shows that only recombinant human transcobalamin is sensitive to modification of the corrin ring b-propionamide of cyanocobalamin. A direct binding assay, where recombinant human transcobalamin is conjugated to a biosensor chip, allows kinetic analysis of cobalamin binding. Response data for cyanocobalamin binding to the transcobalamin protein surface were globally fitted to a bimolecular interaction model that includes a term for mass transport. This model yields association and dissociation rate constants of k(a) = 3 x 10(7) M(-1) s(-1) and k(d) = 6 x 10(-4) s(-1), respectively, with an overall dissociation constant of K(D) = 20 pM at 30 degrees C. Transcobalamin binds cyanocobalamin-b-(5-aminopentylamide) with association and dissociation rates that are twofold slower and threefold faster, respectively, than transcobalamin binding to cyanocobalamin. The affinities determined for protein-ligand interaction, using the solution competition and direct binding assays, are comparable, demonstrating that surface plasmon resonance provides a versatile way to study the molecular recognition properties of vitamin B(12) binding proteins. |
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Keywords: | Monoclonal antibody Disulfide bond Endoproteinase Lys-C peptide map Mass spectrometry Reversed-phase-HPLC N-terminal Edman sequencing |
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