Isolation of Gastric Vitamin B12-binding Proteins Using Affinity Chromatography

Two vitamin B12-binding proteins, designated hog intrinsic factor and hog nonintrinsic factor, have been isolated from hog gastric mucosa. Affinity chromatography on vitamin B12-Sepharose resulted in the removal of the bulk of protein present in a crude extract of hog gastric mucosa. The two vitamin B12-binding proteins were separated subsequently by a method of selective affinity chromatography with an affinity adsorbent containing covalently bound derivatives of vitamin B12 that lack the nucleotide portion of the native vitamin. Under appropriate conditions hog nonintrinsic factor was adsorbed to a column of this material, while hog intrinsic factor was not. After additional purification both proteins were isolated in homogeneous form based on polyacrylamide disc gel electrophoresis, sedimentation equilibrium ultracentrifugation, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Hog intrinsic factor (24 µg) corrected vitamin B12 malabsorption when given to a patient with pernicious anemia, while hog nonintrinsic factor (49 µg) had no effect. Hog intrinsic factor binds 30.3 µg of vitamin B12 per mg of protein. Molecular weight values of 52,300 to 58,600 were obtained by sedimentation equilibrium ultracentrifugation and amino acid and carbohydrate analysis. The protein contains 17.5% carbohydrate which accounts for the elevated molecular weight values (66,000 to 75,000) obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. In the presence of vitamin B12 hog intrinsic factor aggregates to form dimers and higher molecular weight oligomers. Hog nonintrinsic factor binds 25.1 µg of vitamin B12 per mg of protein. Molecular weight values of 61,000 to 66,000 were obtained by sedimentation equilibrium ultracentrifugation and amino acid and carbohydrate analysis. The protein contains 35.5% carbohydrate which accounts for the elevated molecular weight values (100,000 to 130,000) obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. Hog nonintrinsic factor retains its monomeric form in the presence of vitamin B12 under conditions in which hog intrinsic factor aggregates. Hog intrinsic factor and hog nonintrinsic factor differ significantly from each other in their amino acid and carbohydrate composition. These differences, together with differences in other parameters, demonstrate that these two proteins are distinct and separate species.