Detection of protein subunits of ferritin in situ in cells by immunofluorescence

Abstract

Antisera raised in rabbits against protein subunits of rat liver ferritin contain antibodies (IgG) that precipitate subunits and other antibodies (IgG) that precipitate undissociated ferritin and apoferritin. Removal of the latter antibodies from IgG fractions of immune sera by repeated addition of ferritin leaves antibodies that specifically precipitate subunits. Using these subunit-specific antibodies, we have demonstrated the presence of subunit-positive sites in cells by immunofluorescence. Subunit-positive sites were best demonstrated in rats that had been loaded with iron. These sites were diffusely spread through the cytoplasm of hepatocytes and Kupffer cells in rat livers and of macrophages and fibroblasts in rat livers and hearts. With antibodies to ferritin, the presence of ferritin was demonstrated in the same kinds of cells. Although, by immunofluorescence, the intracellular localization of ferritin-positive and subunit-positive sites was similar, ferritin-positive immunofluorescence was most intense in Prussian blue-positive cytoplasmic granules in which high concentrations of ferritin were presumed to be present on the basis of electron microscopic studies. Our findings can be interpreted to indicate either the presence of unassociated subunits or of fragments of subunits in the cytoplasm of the several kinds of cells studied. It is also possible that partly assembled molecules of ferritin or apoferritin in these cells reacted with subunit-specific antibodies. Subunit-specific antibodies may be useful for localizing sites of assembly of ferritin molecules at the level of cell fine structure and for elucidation of the biosynthesis of ferritin.