Binding Analysis of Horse and Bovine Ferritins using Beads Immobilized with Alpha-Casein: Detection of Ferritin Binding or Nonbinding with Heme using Alpha-Casein-Immobilized Beads

Abstract

Ferritin, iron storage protein, binds heme on its surface although it’s physiological role has not been elucidated yet. Firstly, this study analyzed the bindings of bovine and horse spleen ferritins to beads immobilized with α-casein (CasB) with the heme-mediated ferritin-binding capacity of α-casein (Cas). These spleen ferritins were added to solutions containing CasB, and the supernatant and beads obtained by centrifugation was subjected to polyacrylamide gel electrophoresis (SDS-PAGE or Native-PAGE) to detect ferritin. In case of horse spleen ferritin, CasB partly bound ferritin, and CasB-bound ferritin was detected only in the pelleted beads before the second addition of CasB to the first supernatant obtained before washing the beads, while the amount of ferritin detected in the first and second supernatants was similar. After addition of bovine spleen ferritin to CasB, some ferritin was also detected in the pelleted beads as CasB-binding protein, and the other ferritin was also detected in the supernatant as ferritin with no heme. On the other hand, the serum ferritin in fetal bovine serum showed little binding in the binding with CasB. These results suggest the following: CasB selectively detects heme-binding ferritin in tissue and serum ferritin, circulating ferritin contains little heme, and ferritin is intracellularly compartmented in heme catabolism as well as protection against its oxidative stress.