Characterization of Vitamin B12-Binding Proteins Isolated from Human Milk and Saliva by Affinity Chromatography

Human tissues and body fluids contain a number of immunologically related R type vitamin B12-binding proteins that can often be distinguished from one another by the use of ion exchange chromatography, electrophoresis, and gel filtration. The single major vitamin B12-binding proteins present in human milk and saliva are included in this group of proteins and have been isolated in homogeneous form with the use of affinity chromatography on vitamin B12-Sepharose. Both proteins bind approximately 20.5 µg of vitamin B12 per mg of protein and are indistinguishable in terms of absorption spectra, immunodiffusion with rabbit antihuman milk and anti-human saliva vitamin B12-binding protein sera, molecular weight (61,000 to 63,000) as determined by sedimentation equilibrium ultracentrifugation, total amino acid composition, migration on disc gel electrophoresis, and sequence of the first 13 NH2-terminal amino acids (Glu-Ile-Ser-Glu-Val-Ser-Glu-Glu-Asn-Tyr-Ile-Arg-Leu). Both proteins contain fucose, galactose, mannose, galactosamine, glucosamine, and sialic acid residues, although the saliva vitamin B12-binding protein contains more fucose and galactose and has a higher total carbohydrate content (39.9%) than the milk protein (34.5%). Elevated values for molecular weight were obtained for both proteins with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, although the respective values obtained for the saliva protein (95,000 and 148,000) were significantly greater than those obtained for the milk protein (90,000 and 136,000). These studies suggest that differences among the various human R type vitamin B12-binding proteins may be due to differences in carbohydrate content rather than to differences in amino acid composition or sequence.