The apparent heterogeneity of erythropoietin

Abstract

Concentrates of urinary erythropoietin, from two patients with paroxysmal nocturnal hemoglobinuria, were electrofractionated exhaustively. Erythropoietin (ESF 1) was collected (biweekly for 8 weeks) from the anode section between Amicon membranes with molecular weight (MW) cutoffs at 20,000 to 30,000. An erythropoietin generating factor(s) (EGF)s was collected from the cathode section between Amicon membranes with MW cutoffs at 30,000 to 50,000. A relatively large erythropoiesis stimulating fraction (ESF 2) remained in the center section for several more weeks. The electrofractionation process was continued, however, and after 12 weeks all activity moved to the anode (ESF 1) and cathode sections (EGF). A previous report indicated the EGF fraction to contain proerythropoietin and an enzyme that converts proerythropoietin to erythropoietin, a reaction that is potentiated by a normal serum cofactor (9). Preparative acrylamide gel electrophoresis of urinary erythropoiesis regulatory factors was used to indicate the need of a component at about 32,000 MW (possibly also the normal serum cofactor) for the conversion, which is prevented by dithiothreitol and/or N-ethylmaleimide by apparently acting on the conversion factor. Erythropoietin appeared to be a monomer near to 23,000 MW, and proerythropoietin a dimer near to 45,000 MW. A complex of the monomer and dimer was above 50,000 MW. The apparent heterogeneity of erythropoietin can be explained by the erythropoietin monomer, the proerythropoietin dimer, and complexes of both (ESF 2).