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

作者：Mieno Ayako; Yamamoto Yuji; Yoshikawa Yasunaga; Watanabe Kiyotaka; Mukai Takao; Orino Koichi^*

来源：Journal of Veterinary Medical Science, 2013, 75(8): 1101-1105.

DOI：10.1292/jvms.13-0095

• alpha-casein
• biotinylated hemin
• Dpr
• ferritin
• heme

Bacterial and mammalian ferritins are known to bind heme. The use of a-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 a-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 a-casein-coated plate. Both horse spleen ferritins bound to a-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 a-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.

• 出版日期2013-8