Isolation of Vitamin B,,-binding Proteins Using Affinity Chromatography II. PURIFICATION AND PROPERTIES OF A HUMAN GRANULOCYTE VITAMIN B,z-BINDING PROTEIN*

A vitamin Blz-binding protein has been isolated from human granulocytes derived from patients with chronic granulocytic leukemia. Utilizing affinity chromatography as the sole purification technique, the protein was purified 9,860-fold with a yield of over 90% and was homogeneous based on polyacrylamide disc gel electrophoresis, sedimentation equilibrium ultracentrifugation, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein binds 34.9 pg of vitamin BIG per mg of protein and has a single vitamin Blz-binding site. The molecular weight determined by sedimentation equilibrium ultracentrifugation was 56,000, whereas that determined by amino acid and carbohydrate analysis was 58,200. The protein contains 33 % carbohydrate which accounts for the elevated molecular weight values (121,000 to 138,000) obtained using gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis.