Equilibrium and kinetic analyses of the interactions between vitamin B(12) binding proteins and cobalamins by surface plasmon resonance.
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.
Cannon
Michelle J
MJ
Department of Chemistry, University of Utah, 315 S. 1400 E., Salt Lake City, UT 84112-0850, USA.
Myszka
David G
DG
Bagnato
Joshua D
JD
Alpers
David H
DH
West
Frederick G
FG
Grissom
Charles B
CB
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*
D003911:Dextrans; D011994:Recombinant Proteins; D014155:Transcobalamins; C061902:cyanocobalamin-b-monocarboxylic acid; D007437:Intrinsic Factor; C014392:carboxymethyl dextran; D014805:Vitamin B 12