Laboratory of Veterinary Biochemistry, School of Veterinary Medicine, Kitasato University, Aomori, Japan.
Biometals. 2011 Dec;24(6):1217-24. doi: 10.1007/s10534-011-9470-1. Epub 2011 Jul 6.
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+).
牛乳α-酪蛋白被鉴定为一种铁蛋白结合蛋白，而铁蛋白是众所周知的血红素结合蛋白。在这项研究中，我们发现α-酪蛋白在体外与牛脾铁蛋白的结合被血红素所阻断，但不是无铁血红素（原卟啉 IX）或锌原卟啉 IX 所阻断，这表明血红素中铁的存在在这种相互作用中起着关键作用。事实上，α-酪蛋白与铁蛋白和生物素化血红素的结合被添加过量的硫酸亚铁铵（FAS）所抑制。为了进一步阐明α-酪蛋白与生物素化血红素的结合机制，分别使用 Ferrozine 和氮三乙酸（NTA）作为亚铁和三价铁螯合剂。FAS 介导的α-酪蛋白对生物素化血红素的抑制作用被 Ferrozine 中和，但不是 NTA，而 FAS 以及氯化铁介导的它们相互作用的抑制作用被 NTA 中和。以下离子也按抑制能力的强弱顺序抑制α-酪蛋白-生物素化血红素结合：FAS（Fe(2+)）<氯化铁（Fe(3+)）<硫酸铜（Cu(2+)）<硫酸锌（Zn(2+)）<氯化锰（Mn(2+)）<氯化钙（Ca(2+)）<硫酸镁（Mg(2+)）。这些结果表明，α-酪蛋白与铁蛋白的结合是通过α-酪蛋白直接与铁蛋白分子表面血红素中的铁结合介导的，并且α-酪蛋白优先与 Fe(2+)结合，而不是任何其他金属离子，包括 Fe(3+)。
