The carbohydrate composition of human erythropoietin (epo) was determined by micro-GLC. Enzymic removal of most of the sugars results in aggregation of glycosidase-treated epo, loss of biological activity when assayed in mice, and retention of activity when assayed in marrow cell cultures or by RIA. Endoglycosidase F causes the removal of most of the carbohydrates indicating that the oligosaccharides are asparagine linked. The lack of O-linked sugar is confirmed by the absence of N-acetylgalactosamine. These findings indicate that the oligosaccharide portion of epo, although required for action in vivo, is not required for interaction with the target cells of the blood-forming system.

Erythropoietin (epo) is the glycoprotein hormone that induces normal red cell differentiation. Reaction of native or denatured epo with either [3H]iodoacetic acid or N-ethyl-2-[3H]maleimide did not result in the incorporation of any significant amount of radioactivity. Radiolabeling took place only if the protein were denatured before reduction and alkylation. When reduction was carried out in the presence of 6 M guanidine HCl, about 3.7 mol N-ethyl-2[3H]maleimide were covalently linked per mol epo. These results show that there are no free, accessible sulfhydryl groups in epo; there are two internal disulfide bonds. When epo was reduced in the presence of 6 M guanidine HCl and then reoxidized and the guanidine removed, about 85% of the biological activity was regenerated. The biological activity was lost irreversibly if the sulfhydryl groups were alkylated. Limited proteolysis of [3H]epo (labeled at sialic acid residues of the oligosaccharide chains) showed that it consists of two rather trypsin-resistant domains, each having a mol wt of about 16,000, connected by a small region of protein that is trypsin sensitive. The two large fragments contain most of the label. Biological activity and immunoreactivity are lost after limited tryptic proteolysis. Complexing epo with a neutralizing antibody protects its activity from proteolysis.