A Study on the Presence of Ferritin-binding Proteins in Fetal Horse Plasma

Abstract

In mammal circulation, ferritin-binding proteins (FBPs) are thought to be involved in clearance of circulating ferritin after complex formation with it through receptor-mediated uptake. However, there is no report on fetal FBP in fetal circulation. Although iron concentrations of fetal horse plasma were higher than those of adult horse plasma, plasma ferritin concentrations and ferritin-binding activities were found to be significantly lower in fetus than in adult. FBPs were purified from fetal or adult horse plasma on horse spleen ferritin-Sepharose 4B affinity column. Partially affinity-purified fetal horse plasma FBPs were mainly separated into 65 and 41 kDa bands in addition to minor bands with higher molecular masses ranged from 102 to 140 kDa on SDS-PAGE under reducing condition. The adult horse plasma FBPs were separated into 74, 54 and 28 kDa bands, and the 74 and 54 kDa bands reacted with antibodies specific for horse IgM and IgG heavy chains, respectively, by immunoblotting analyses. On the other hand, no antibodies to horse immunoglobulin classes detected any bands in fetal horse plasma FBPs. The affinity-purified adult and fetal horse plasma FBPs did not contain fibrinogen as a plasma specific FBP, probably due to its lower affinity to the ligand ferritin. These results demonstrate the presence of FBPs which are different from adult horse plasma FBPs including anti-ferritin autoantibodies in fetal plasma.

Keywords: adult horse; autoantibody; ferritin; ferritin-binding protein; fetal horse.

Fig. 1.

Iron (A) and ferritin (B) concentrations, and ferritin-binding activity (C) in adult and fetal horse plasma. Iron and ferritin concentrations in adult (closed bar) and fetal (open bar) horse plasma were determined by ferrozine reagent and sandwich ELISA as described in “Materials and Methods”. In ferritin-binding activity, a 100 μl of 10,000-fold diluted horse plasma with PBS was added to each well of microtiter plate and kept overnight at 4°C followed by addition of 100 μl of horse spleen ferritin (5 μg ml^–1). The ferritin bound to the well was detected with ALP-labeled anti-horse spleen ferritin antibody. Data indicate mean ± SD of respective six horse plasma samples. *p<0.01, compared with adult horse.

Fig. 2.

Separation by SDS-PAGE (A) and immunoblotting (B) of partially purified adult and fetal horse plasma FBPs. A) Partially purified adult (5 μg, lane 1) and fetal (5 μg, lane 2) horse plasma FBPs were run against commercial horse fibrinogen (F, 3 μg) and marker proteins (M, 1 μg each): phosphorylase b (92.5 kDa), serum albumin (66.3 kDa), ovalbumin (45.0 kDa), lactate dehydrogenase (36.0 kDa) and adenylate kinase (21.7 kDa). B) Partially purified adult horse plasma FBPs (lane 1, 2 μg) and fetal horse plasma FBPs (lane 2, 2 μg) were detected with ALP-conjugated polyclonal antibodies specific for horse IgM, IgG and IgA heavy chains. Arrows indicate bands detected with the specific antibodies used.