Apolipoprotein B binds ferritin by hemin-mediated binding: evidence of direct binding of apolipoprotein B and ferritin to hemin

Abstract

Apolipoprotein B (apoB) is known to be a ferritin-binding protein. Here we show that apoB binds to ferritin through hemin-mediated binding. Human apoB bound to bovine spleen, horse spleen, and canine liver ferritins, but did not bind to bovine apoferritin, even after incorporation of iron into it. Incubation of apoferritin with hemin resulted in apoB binding with apoferritin at the same level as with holoferritin. In contrast, hemin inhibited binding of apoB to ferritin. Bovine spleen apoferritin bound biotinylated hemin, and hemin inhibited the binding between the apoferritin and biotinylated hemin, suggesting that ferritin binds hemin directly. ApoB and LDL containing apoB bound biotinylated hemin, and their bindings were also inhibited by hemin, but not protoporphyrin IX. These data demonstrate that binding of apoB to ferritin is mediated through ferritin's binding to hemin, and also that apoB binds hemin directly.

Binding of Mammalian and Avian Ferritins with Biotinylated Hemin: Demonstration of Preferential Binding of the H Subunit to Heme

“…Human apolipoprotein B has been revealed to bind to horse spleen, bovine spleen and canine liver holoferritins through heme-mediated binding, suggesting that these ferritins bind hemin because heme is oxidized during these preparations [27]. Biotinylated hemin is available for the detection and purification of hemoproteins [8,27].…”

“…Biotinylated hemin is available for the detection and purification of hemoproteins [8,27]. Bovine spleen apoferritin has been found to bind biotinylated hemin, and the binding of spleen apoferritin with biotinylated hemin is inhibited by hemin but not by PPIX [27]. However, Crichton et al [6] reported that horse spleen apoferritin binds hemin to demetallate PPIX and then remetallates it using the iron released from hemin; demetallation and remetallation occur at between pH 6.5 Although there is no evidence for whether demetallation of heme/hemin by ferritin occurs under physiological conditions or in the experimental conditions in this study, purified mammalian tissue ferritins seem to keep heme (probably hemin) on the surface of the ferritin molecule even after harsh purification procedures, such as heat treatment (75ºC, 15 min) and acid treatment (pH 4.8), as well as gel filtration [13,27].…”

“…Apoferritins were prepared from each of the above ferritins using a reducing reagent as described previously [18]. Biotinylated hemin was used to examine the binding of holoferritin and its apoferritin with heme as described previously [9,27]. Both the mammalian holoferritins and apoferritins used in this study showed binding with biotinylated hemin; the binding activity was significantly higher for biotinylated hemin to apoferritin than to holoferritin, except in the case of canine liver ferritin, for which they had the same binding activity (Fig.…”

“…Hemin was biotinylated according to the manufacturer's instructions with soluble EZ-Link Biotin-PEO 2 -amine at a molar ratio of 20:1 (EZ-Link Biotin-PEO 2 -amine to hemin) with EDC as the cross-linking reagent, as previously described [27]. The hemin concentration was determined using its molar extinction coefficient at 385 nm ( 385 nm = 58,400 M -1 cm -1 ) [10].…”

“…Interestingly, horse spleen apoferritin not only binds hemin but also demetallates hemin and remetallates released iron to protoporphyrin IX (PPIX) based on spectroscopic data as a function of pH [6]. In addition, we have demonstrated that human apolipoprotein B binds ferritin through heme-mediated binding on the surface of the ferritin molecule using biotinylated hemin, which can safely detect hemoprotein without the use of radioisotopes, disposal of which is difficult [9,27], suggesting that mammalian holoferritins retain heme/hemin for a long time, even after harsh purification steps such as heat treatment (70°C for 20 min) and acid treatment (pH 4.8) [13,27]. Iron and hemin cause oxidative stress by reactive-oxygen species (ROS) production [19,25].…”