Calcium binding proteins: purification of hepatocalcin-55 from bovine liver

1. Bovine liver was homogenized and the proteins were fractionated by two DEAE-Sephadex steps and a gel filtration. 2. All 150 fractions collected from the DEAE-Sephadex column were electrophoresed and combined to give ten groups of proteins with different SDS-electrophoresis patterns. 3. Fractions G and H, showing fast migrating proteins, were transblotted to a polyvinylidenedifluoride membrane. By incubation with 45CaCl2 and subsequent autoradiography one protein revealed strong calcium binding. 4. This protein, named hepatocalcin-55, was obtained in a pure form from the gel filtration through Sephadex G-75. The molecular weight (55,000) was determined by gel filtration. Since SDS electrophoresis shows one protein band at Mr = 27,000 the native hepatocalcin must be a dimer. Its isoelectric point was found to be at 4.9. 5. Gamma-carboxyglutamate could not be detected in alkaline hydrolyzates of the protein under study. No carbohydrates were found in the hepatocalcin.