Isolation of Gastric Vitamin B12-binding Proteins Using Affinity Chromatography: I. PURIFICATION AND PROPERTIES OF HUMAN INTRINSIC FACTOR

Abstract

Abstract Human intrinsic factor has been isolated from gastric juice. The protein was purified 853-fold with a yield of 85% utilizing affinity chromatography on vitamin B12-Sepharose as the sole purification technique. The final preparation was homogeneous based on polyacrylamide gel electrophoresis and sedimentation equilibrium ultracentrifugation. The isolated protein (20 µg) corrected vitamin B12 malabsorption when given to a patient with pernicious anemia in a standard Schilling test. Human intrinsic factor binds 30.1 µg of vitamin B12 per mg of protein and contains a single vitamin B12-binding site with an association constant for vitamin B12 of 1.5 x 1010 m-1. The molecular weight determined by sedimentation equilibrium ultracentrifugation was 45,200 to 47,700 while that determined by amino acid and carbohydrate analyses was 44,200. The protein contains 15.0% carbohydrate which accounts for the elevated molecular weight values (59,000 to 66,000) obtained using sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. In the presence of vitamin B12, human intrinsic factor aggregates to form dimers and higher molecular weight oligomers. When vitamin B12 binds to human intrinsic factor, the spectral maximum for vitamin B12 shifts from 361 nm to 362 nm and the absolute absorbance at 361 nm increases by 32%.