Isolation and characterization of alpha-fetoprotein from the mouse hepatoma BW7756.

Purification of alpha-fetoprotein from mouse hepatoma BW7756 extracts was performed using ammonium sulfate precipitations, gel filtration, ion-exchange chromatography and isoelectric focusing. These procedures produced a 5.6% yield of alpha-fetoprotein with 96% purity. Polyacrylamide slab gel electrophoresis, extended agarose electrophoresis and immunoelectrophoresis demonstrated that mouse hepatoma alpha-fetoprotein migrated at pH 8.6 as a rapid alpha1, or postalbumin globulin. Crossed antibody electrophoresis of the agarose zone containing alpha-fetoprotein failed to demonstrate microheterogeneity. Molecular weight analysis of the mouse hepatoma alpha-fetoprotein on a calibrated Sephadex G-200 column yielded a value of 72 000-73 000 for the native protein. Sodium dodecyl sulfate gel electrophoresis subsequently demonstrated a single polypeptide chain with a molecular weight of 72 000. Amino acid analysis showed the alpha-fetoprotein to be an acidic protein dominated by hydrophobic residues. The total carbohydrate content was 5.5%, and 3 mol of sialic acid were detected per mol of alpha-fetoprotein. Although neutral sugars were the principal class present, galactosamine was the most abundant single sugar detected.