Characterization of the vitamin B12-binding protein isolated from sow's milk and its affinity for cyanocobalamin and other corrinoids.
1. Unsaturated vitamin B12-binding protein was isolated from sow's milk whey by affinity chromatography on a vitamin B12-Sepharose column with a yield of 54% (3920-fold purification). The purified binding protein was homogeneous by the criteria of polyacrylamide gel electrophoresis and high speed sedimentation equilibrium. 2. The isolated vitamin B12-binding protein 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 61,800 determined by ultracentrifugation, consisted of a single polypeptide chain, bound vitamin B12 on an equimolar basis and had a partial specific volume of 0.697 ml/g (mechanical oscillator technique). 3. Its dissociation constant, Kd, for cyanocobalamin was 8.8 x 10(-10)M. The binding protein showed similar affinities for hydroxo-, methyl-, and adenosylcobalamin when compared to cyanocobalamin relative affinity ratios, but lower affinities (31-49%) for cobinamide, Co-alpha-[adenyl] cobamide and Co-alpha-[2-methyl-adenyl] cobamide. Therefore, the substitution of the axial ligand to the cobalt atom in the corrin 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 interaction. 4. The present results indicate close similarity of the vitamin B12-binding protein from sow's milk to vitamin B12-binding proteins from other sources, especially those from human milk and from porcine gastric mucosa (nonintrinsic factor). Whether this similarity extends to antigenic and physiological properties remains to be investigated.
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National Institute for Research in Dairying, Shinfield, Reading, United Kingdom.
D000818:Animals; D045728:Corrinoids; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008892:Milk / Q000032:analysis* / Q000378:metabolism; D008895:Milk, Human / Q000378:metabolism; D013552:Swine; D014155:Transcobalamins / Q000302:isolation & purification* / Q000378:metabolism; D014805:Vitamin B 12 / Q000378:metabolism
D045728:Corrinoids; D014155:Transcobalamins; D014805:Vitamin B 12