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

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Bacterial and mammalian ferritins are known to bind heme. The use of α-casein and biotinylated hemin could be applicable to detection of protein-bound heme and of proteins with heme-binding capacity, respectively. Although commercial horse spleen ferritin and purified horse spleen ferritin (L:H subunit ratio=4) bound to α-casein-coated plate, and this binding could be inhibited by hemin, recombinant iron-binding protein (rDpr), derived from heme-deficient Streptococcus mutans and expressed in Esherichia coli, did not bind to α-casein-coated plate. Both horse spleen ferritins bound to α-casein-immobilized beads. Commercial horse spleen ferritin and rDpr showed direct binding to hemin-agarose beads. After pre-incubation of commercial horse spleen ferritin or rDpr with biotinylated hemin, they showed indirect binding to avidin-immobilized beads through biotinylated hemin. These results demonstrate that α-casein is useful for detection of heme-binding ferritin, and both hemin-agarose and the combination of biotinylated hemin and avidin-beads are useful for the detection of heme-binding capacity of ferritin. In addition, this study also revealed that Dpr, decameric iron-binding protein, from heme-deficient cells binds heme.