BINDING OF COBALAMIN AND COBINAMIDE TO TRANSCOBALAMIN FROM BOVINE-MILK

We have studied the interaction between transcobalamin (TC) and the li gands cobalamin (Cbl) and cobinamide (Cbi). Partially purified TC from bovine milk was depleted of endogenous Cbl by 8 M urea treatment. Uns aturated TC was adsorbed on CM-Sepharose in order to ensure fast separ ation of the matrix-bound protein from the reaction medium. The forwar d reaction TC + Cbl --> TC-Cbl (rate constant k(+)(Chl) and the backwa rd reaction and the backward reaction TC-Cbl-->TC+Cbl (k(-)(Cbl)) were followed in time. A single-step binding model (with no intermediate p rotein-ligand complex) was sufficient to fit the data. The calculated rate constant were k(+)(Cbl)=-0.6 nM(-1) min(-1) and k(-)(Cbl)=1.3 X 1 0(-4) min(-1), which corresponded to the TC-Cbl dissociation constant K-D(Cbl)=0.2 pM. Reaction between TC and Cbl developed against electro static forces, and the effective charges of the interacting species we re estimated as both +1 or both -1. The competition between Cbl and Cb i for TC was studied, which resulted in determination of the relevant rate constants for Cbi: k(+)(Cbi)=0.03 nM(-1), and K-D(Cbi)=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 tha n 1 h.