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Heme-mediated binding of α-casein to ferritin: evidence for preferential α-casein binding to ferrous iron

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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 (Fe2+) ≪ ferric chloride (Fe3+) < copper sulfate (Cu2+) < zinc sulfate (Zn2+) < manganese chloride (Mn2+) < calcium chloride (Ca2+) < magnesium sulfate (Mg2+). 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 Fe2+ compared with any other metal ions, including Fe3+.

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Acknowledgments

This study was supported in part by a Grant-in-Aid for Scientific Research (No. S1003) from the School of Veterinary Medicine, Kitasato University, Japan.

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Authors and Affiliations

  1. Laboratory of Veterinary Biochemistry, School of Veterinary Medicine, Kitasato University, Aomori, 034-8628, Japan

    Aya Usami, Masakazu Tanaka, Yasunaga Yoshikawa, Kiyotaka Watanabe & Koichi Orino

  2. Laboratory of Large Animal Internal Medicine, School of Veterinary Medicine, Kitasato University, Aomori, 034-8628, Japan

    Hiromichi Ohtsuka

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  1. Aya Usami
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  2. Masakazu Tanaka
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  3. Yasunaga Yoshikawa
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  5. Hiromichi Ohtsuka
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  6. Koichi Orino
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Correspondence to Koichi Orino.

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Usami, A., Tanaka, M., Yoshikawa, Y. et al. Heme-mediated binding of α-casein to ferritin: evidence for preferential α-casein binding to ferrous iron. Biometals 24, 1217–1224 (2011). https://doi.org/10.1007/s10534-011-9470-1

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