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.

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 an 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 Escherichia coli, did not bind to an 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 preincubation 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 that both hemin-agarose and the combination of biotinylated hemin and avidin-beads are useful for detection of the heme-binding capacity of ferritin. In addition, this study also revealed that Dpr, a decameric iron-binding protein, from heme-deficient cells binds heme.