title: Equilibrium and kinetic analyses of the interactions between vitamin B(12) binding proteins and cobalamins by surface plasmon resonance.
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.
authors.affiliation: Department of Chemistry, University of Utah, 315 S. 1400 E., Salt Lake City, UT 84112-0850, USA.
authors.forename: Michelle J
authors.initials: MJ
authors.lastname: Cannon
authors.forename: David G
authors.initials: DG
authors.lastname: Myszka
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Bagnato
authors.forename: David H
authors.initials: DH
authors.lastname: Alpers
authors.forename: Frederick G
authors.initials: FG
authors.lastname: West
authors.forename: Charles B
authors.initials: CB
authors.lastname: Grissom
mesh_terms: D001667:Binding, Competitive; D015374:Biosensing Techniques; D002138:Calibration; D018929:Cell Culture Techniques; D002851:Chromatography, High Pressure Liquid; D003911:Dextrans / Q000737:chemistry; D006801:Humans; D007437:Intrinsic Factor / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D020349:Surface Plasmon Resonance / Q000379:methods*; D014155:Transcobalamins / Q000737:chemistry / Q000378:metabolism*; D014805:Vitamin B 12 / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D003911:Dextrans; D011994:Recombinant Proteins; D014155:Transcobalamins; C061902:cyanocobalamin-b-monocarboxylic acid; D007437:Intrinsic Factor; C014392:carboxymethyl dextran; D014805:Vitamin B 12