Characterization of the vitamin B12 - binding protein isolated from sow's milk and its affinity for cyanocobalamin and other corrinoids

ABSTRACT

1. Unsaturated vitamin B12-binding protein was isolated from sow's milk whey by affinity chromatography on a vitamin B12-Sepharose colmn with a yield of 54% (3920-fold purification). The purified binding protein was homogeneous was homogeneous by the criteria of polyacrylamide gel electrophoresis and high speed sedimentation equilibrium. 2. The isolated vitamin B12-biding protein was a glycoprotein was a glycoprotein with 24% carbohydrates (fucose, galactose, mannose, galactosamine, glucosamine and sialic acid_ and high levels of aspartic and glutamic acids. The protein which has a molecular weight of 61800 determined by ultracentrifugation, consisted of a single polypepide chain, bound vitamin B12 on an equimolar vasis and had a partial specific volume of 0.697 ml/g (mechanical oscillaltor technique). 3. Its dissociation constant, Kd'for cyanocoballamin was 8.8 x 10-10M. The binding protein showed similar affinities for hidroxo-, and adenosylcobalamin when compared to cyanocobalamin relative affinity ratios, but lower affinities (31-49%) for cobinamide, Co-alfa [adenyl] cobamide and Co-alfa-[2-methyl-adenyl]cobamide. Therefore, the substitution of axial ligand to the cobalt atom in the corin ring of vitamin B12 had no effect on affinity, whereas the differences in the nucleotide part of the molecule caused a small decrease in energy of the interation. 4. The present results indicate close similarity of the vitamin B12-binding protein from sow's milk to vitamin B12-biding proteins from other