title: 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.
abstract: 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.
authors.affiliation: Laboratory of Veterinary Biochemistry, School of Veterinary Medicine, Kitasato University, Aomori 034-8628, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Mieno
authors.forename: Yuji
authors.initials: Y
authors.lastname: Yamamoto
authors.forename: Yasunaga
authors.initials: Y
authors.lastname: Yoshikawa
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Watanabe
authors.forename: Takao
authors.initials: T
authors.lastname: Mukai
authors.forename: Koichi
authors.initials: K
authors.lastname: Orino
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D019921:Biotinylation; D002364:Caseins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli; D005293:Ferritins / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism*; D006736:Horses; D011485:Protein Binding; D013295:Streptococcus mutans / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D002364:Caseins; D004268:DNA-Binding Proteins; C414750:Dpr protein, Streptococcus; D006418:Heme; D006427:Hemin; D005293:Ferritins