Isolation and characterization of murine lactoferrin.

Lactoferrin, a non-heme, iron-binding glycoprotein, was isolated from mouse milk. The purification procedure involved an initial centrifugation to remove much of the casein and to separate the fat from the whey, followed by ammonium sulfate precipitation and chromatography on carboxymethyl-Sephadex. The lactoferrin preparation obtained was highly purified as judged by polyacrylamide gel electrophoresis under both non-denaturing and denaturing conditions, and the presence of lysine as the only NH2-terminal amino acid. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated a single component having a molecular weight of 78 000 +/- 2800. Sedimentation equilibrium ultracentrifugation studies suggested that in addition to monomers of molecular weight 75 000 +/- 1000, other oligomeric forms of the protein were also present. Amino acid and amino sugar analyses revealed that there were 5 methionine, 10 histidine, 10 tryptophan and 5 glucosamine residues per mol of protein. Isoelectric focusing of lactoferrin in a polyacrylamide gel stabilized pH gradient indicated a single component having an isoelectric point of about 9; however, depending on the preparation examined, a range of 8.7-9.6 was noted. A single precipitin line was observed with rabbit anti-mouse lactoferrin when examined by immunodiffusion and immunoelectrophoresis. No immunological cross-reaction was observed between mouse lactoferrin and mouse transferrin.