Horse spleen hemosiderin: II. Further characterization

Abstract

Horse spleen hemosiderin granules were fractionated into an iron-free soluble fraction and an iron-free residue fraction upon reductive removal of the iron and dialysis. These fractions were analyzed for iron, nitrogen, phosphorous, and sulfur. The widely held view that hemosiderin arises from aggregation of ferritin molecules rendered insoluble by an excessive iron load was tested on the grounds that apoferritin should be re-solubilized on removal of the iron from hemosiderin. Apoferritin was in this way successfully isolated in crystalline form, and was characterized antigenically and electrophoretically. It was demonstrated by a semiquantitative immunochemical technique in hemosiderin from a series of horses. The amount of apoferritin, however, was small in relation to the total protein, most of which remained as an insoluble residue after removal of the iron. In addition, a soluble protein other than apoferritin was demonstrated, and was distinguished from the latter by its solubility, dye-binding, and electrophoretic properties. Studies were carried out to elucidate the nature of the insoluble iron-free residue of hemosiderin. Analysis for N-terminal amino acids indicated heterogeneity in its protein composition. Dye-binding studies indicated significant differences from apoferritin. including a strong basophilia. Various methods of extraction of the residue revealed a pigment which behaved like heme. There was a significant amount of lipid, including cholesterol. The results of these analyses are consistent with a hypothesis suggested in the literature on the basis of electron micrographs of hemosiderin showing double membranes and possible crystae: that hemosiderin represents an iron-loaded subcellular organelle, perhaps a mitochondrion.