title: Heme-mediated binding of α-casein to ferritin: evidence for preferential α-casein binding to ferrous iron.
abstract: Bovine milk α-casein was identified as a ferritin-binding protein, and ferritin is known to be a heme-binding protein. In this study, we found that the binding of α-casein to bovine spleen ferritin in vitro was blocked by hemin, but not by iron-free hemin (protoporphyrin IX) or zinc-protoporphyrin IX, suggesting that the presence of iron in heme play a key role in this interaction. Indeed, the binding of α-casein to ferritin and biotinylated hemin was inhibited by adding excess ferrous ammonium sulfate (FAS). To further elucidate the binding mechanism of α-casein to biotinylated hemin, Ferrozine and nitrilotriacetic acid (NTA) were used as ferrous and ferric iron chelators, respectively. FAS-mediated inhibition of α-casein to biotinylated hemin was neutralized with Ferrozine, but not NTA, while FAS- as well as ferric chloride-mediated inhibition in their interaction was neutralized by NTA. The following ions also inhibited α-casein-biotinylated hemin binding in order of potency of inhibition: FAS (Fe(2+)) << ferric chloride (Fe(3+)) < copper sulfate (Cu(2+)) < zinc sulfate (Zn(2+)) < manganese chloride (Mn(2+)) < calcium chloride (Ca(2+)) < magnesium sulfate (Mg(2+)). These results suggests that the binding of α-casein to ferritin is heme-mediated through direct binding of α-casein to iron in the heme on the surface of ferritin molecule, and that α-casein preferentially binds Fe(2+) compared with any other metal ions, including Fe(3+).
authors: Laboratory of Veterinary Biochemistry, School of Veterinary Medicine, Kitasato University, Aomori, Japan.
authors: Aya
authors: A
authors: Usami
authors: Masakazu
authors: M
authors: Tanaka
authors: Yasunaga
authors: Y
authors: Yoshikawa
authors: Kiyotaka
authors: K
authors: Watanabe
authors: Hiromichi
authors: H
authors: Ohtsuka
authors: Koichi
authors: K
authors: Orino
mesh_terms: D000818:Animals; D002364:Caseins / Q000378:metabolism*; D002417:Cattle; D005293:Ferritins / Q000737:chemistry / Q000378:metabolism*; D005297:Ferrozine / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D011485:Protein Binding; D013154:Spleen / Q000737:chemistry
chemical_list: D002364:Caseins; D008670:Metals; D005297:Ferrozine; D006418:Heme; D005293:Ferritins; D007501:Iron