Binding of cobalamin and cobinamide to transcobalamin from bovine milk

Abstract

We have studied the interaction between transcobalamin (TC) and the ligands cobalamin (Cbl) and cobinamide (Cbi). Partially purified TC from bovine milk was depleted of endogenous Cbl by 8 M urea treatment. Unsaturated TC was adsorbed on CM-Sepharose in order to ensure fast separation of the matrix-bound protein from the reaction medium. The forward reaction TC+Cbl-->TC-Cbl (rate constant k+Cbl) and the backward reaction TC-Cbl-->TC+Cbl (k-Cbl) were followed in time. A single-step binding model (with no intermediate protein-ligand complex) was sufficient to fit the data. The calculated rate constants were k+Cbl = 0.6 nM-1 min-1 and k-Cbl = 1.3 x 10(-4) min-1, which corresponded to the TC-Cbl dissociation constant KDCbl = 0.2 pM. Reaction between TC and Cbl developed against electrostatic forces, and the effective charges of the interacting species were estimated as both +1 or both -1. The competition between Cbl and Cbi for TC was studied, which resulted in determination of the relevant rate constants for Cbi: k+Cbi = 0.03 nM-1 min-1, k-Cbi = 0.03 min-1, and KDCbi = 1 nM. Slow dissociation of TC-Cbl guarantees its stability in plasma for 5-10 h, while Cbi bound to TC would be transferred to haptocorrin in less than 1 h.

Equilibrium and Kinetic Analyses of the Interactions between Vitamin B12 Binding Proteins and Cobalamins by Surface Plasmon Resonance

“…In the present work, the dissociation rate constant for CNCbl bound to surface immobilized rhTCII is 6 ϫ 10 Ϫ4 s Ϫ1 , whereas the dissociation rate constant for this complex is estimated to be 2 ϫ 10 Ϫ6 s Ϫ1 using adsorption chromatography (32). The rates for surface immobilized and adsorbed rhTCII-CNCbl complexes lead to calculated K D values of 20 and 0.2 pM, respectively.…”

Comparative Analysis of Cobalamin Binding Kinetics and Ligand Protection for Intrinsic Factor, Transcobalamin, and Haptocorrin

“…6A (4,15). When the apo forms of recombinant IF and TC were saturated with Cbi and exposed to equal concentration of external Cbl⅐OH 2 , the complete substitution occurred in less than 1 min (Fig.…”

Crystallographic studies on B12 binding proteins in eukaryotes and prokaryotes

“…Cubilin is amulti-domain protein with 27 similar CUB domains [10,46–49]. The binding site for IF-B12 is located at CUB domains 5–8 of cubilin;finally, the released B12 binds to transcobalamin that mediates the final delivery of B12 into thecirculation and in turn into all other cells [11,50–52]. The phylogram tree shows that mammalian transport proteins are evolutionaryvery close to each other (Fig.…”