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 β«Ψβ¬ 3 Ψ 10 7 M Ψ1 s Ψ1 and k d β«Ψβ¬ 6 Ψ 10 Ψ4 s Ψ1 , respectively, with an overall dissociation constant of K D β«Ψβ¬ 20 pM at 30Β°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.