Transcobalamin from cow milk: isolation and physico-chemical properties

Abstract

The concentration of endogenous cobalamin (Cbl) in cow milk was 3.3 nM while the Cbl-binding capacity was 0.05 nM. Both endogenous and newly added Cbl showed similar quantitative distribution between a 280 kDa protein complex (45%) and a 43 kDa Cbl-binder (55%). Long time incubation, as well as urea treatment, was accompanied by a slow release of the 43 kDa Cbl-binder from the 280 kDa fraction. No other Cbl-binding proteins appeared after these procedures. The 43 kDa binder from cow milk, depleted of the ligand by urea treatment, ieacted with Cbl even in the presence of a B12-analogue cobinamide (Cbi) at the ratio Cbl:Cbi = 1:40. The Stokes radius of the binder changed from 2.7 nm for the Cbl-free protein to 2.5 nm for the Cbl-saturated form and the Cbl-saturated binder was able to displace human transcobalamin (TC) from the TC-receptor. The interaction between the protein and Cbl was significantly suppressed at pH 2.0. The N-terminal sequence of the purified 43 kDa Cbl-binder revealed homology with TC from human and rabbit plasma. In conclusion we have shown that TC is the main Cbl-binding protein in cow milk. This is surprising, since previous studies on human and rat milk have shown another Cbl-binder, apo-haptocorrin, to be the dominating Cbl-binding protein.

Fractionation of bovine whey proteins and characterisation by proteomic techniques

“…For example, transcobalamin-2, a vitamin B 12 binding protein, could only be tentatively assigned on the basis of a single peptide identification (15 amino acids, top 10 ions matched) in each of two spots (10a, 10c, 1.5 M basic fraction). However, it is known to occur in bovine whey (Fedosov, Petersen, & Nexo, 1996) and, given its high theoretical basicity and mass (pI 9.22, 46 kDa) combined with reported cation exchange behaviour, it is likely to occur in this fraction and to migrate on 2-DE in the region identified.…”

Sequence, S–S Bridges, and Spectra of Bovine Transcobalamin Expressed in Pichia pastoris

“…High amounts of apoHC (10 -50 nM) are present in many body fluids like saliva, tears, and milk (8,9). However, only holo-TC has been found in bovine milk (10).…”

“…Purification of TC and Sequence of Its Peptides-TC was isolated from bovine milk according to the method described earlier (10). 100 g of protein was treated with 2 g of trypsin in 100 l of 0.04 M NH 3 HCO 3 at 37°C for 4 h. The peptides in 200 l of 0.5% heptafluorobutyric acid were applied to an HPLC column and separated by elution with a linear gradient of 80% acetonitrile in 0.5% heptafluorobutyric acid.…”

“…Isolation of 1 mg of TC requires, for instance, 150 -300 liters of human plasma (17,18) or 100 liters of bovine milk (10). The available amount of pure TC is insufficient for investigations requiring high protein quantities.…”

Conformational Changes of Transcobalamin Induced by Aquocobalamin Binding

“…The characteristics of Cbl binding and receptor recognition observed for the recombinant TC were similar to those for the natural TCs (11,13,21) and for the recombinant bovine TC (15). Thus, the protein-ligand complex (TC⅐Cbl⅐OH 2 ) was very stable, and the procedure of apo-TC preparation required prolonged incubation in Gnd.…”