Iron-dependent binding of bovine milk α-casein with holo-lactoferrin, but not holo-transferrin

Abstract

Bovine milk α-casein has been identified as an iron- and heme-binding protein. However, the physiological role of its iron-binding remains to be elucidated in more detail. α-Casein was immobilized on CNBr-activated Sepharose 4B beads, and the α-casein agarose beads efficiently bound hemin as well as ferrous ammonium sulfate (Fe(2+)) as compared with control beads. Additionally, α-casein-beads bound bovine holo-lactoferrin (Lf), but not holo-transferrin. Lf caused the release of Fe(2+) which had bound to the α-casein-agarose beads beforehand. These results suggest that bovine α-casein iron-dependently binds holo-bovine Lf more strongly than Fe(2+), and that strong binding between them may play a physiological role in regulating iron homeostasis in the bovine mammary gland.

Binding Analysis of Ferritin with Heme Using α-Casein and Biotinylated-Hemin: Detection of Heme-Binding Capacity of Dpr Derived from Heme Synthesis-Deficient <i>Streptococcus mutans</i>

“…Antibodies to commercial horse spleen ferritin were purified by affinity chromatography, and a part of the purified antibodies was biotinylated as described previously [22]. Bovine α-casein-Sepharose 4B was prepared by coupling 50 mg of α-casein (≥70% α S -casein, 90% purity by electrophoresis, Sigma-Aldrich Corp.) to 10 ml of CNBr-activated Sepharose 4B (GE Healthcare, Piscataway, NJ, U.S.A.) according to the manufacturer's instructions as previously described [19]. Hemin (ferriprotoporphyrin IX chloride) and biotinylated hemin were prepared as previously described [13].…”

“…A P-value below 0.001 was considered statistically significant. α-Casein has been shown to be an iron-and heme-binding protein in an analysis of its binding with ferritin [19,22]. Commercial horse spleen ferritin was mostly comprised of L subunits, and purified horse spleen ferritin contained both H and L subunits with an L/H subunit ratio of four [13,15].…”

“…The α-casein beads can be used for detection of proteins binding iron or heme [19], and hemin beads and biotinylated hemin can be used for detection of proteins with the hemebinding ability. Although the amino acid homology between S. mutans Dpr and P. gingivalis Dps or horse L subunit is low (<20%) [1,4,24,25], these iron-binding proteins may have developed as a divergent heme-binding family.…”