ABSTRACT
Urinary and recombinant human erythropoietin differ with respect to ease of iodination, inactivation by iodination, second derivative and circular dichroic spectra, rate of inactivation by trypsin and glycosylation pattern. All of these differences are compatible with a significant difference in conformation of these two forms of erythropoietin.
Subject(s)
Erythropoietin/chemistry , Erythropoietin/urine , Protein Conformation , Recombinant Proteins/chemistry , Animals , Circular Dichroism , Erythropoietin/genetics , Humans , Hydrolysis , Iodine Radioisotopes/chemistry , Iodine Radioisotopes/metabolism , Molecular Weight , Rats , Recombinant Proteins/metabolism , Spectrophotometry, Ultraviolet , Trypsin/chemistryABSTRACT
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.
Subject(s)
Erythropoietin , Chemical Phenomena , Chemistry , Ethylmaleimide , Humans , Hydrolysis , Molecular Weight , Oxidation-Reduction , Tritium , TrypsinABSTRACT
The frequencies of rat and mouse bone marrow cells capable of binding erythropoietin were studied by both direct fluorescence and indirect immunofluorescence. We found that between 1-2% of the cells bound erythropoietin, that the binding was specific, and that the number of cells that bound erythropoietin was, in part, a function of the erythropoietic state of the donor animal. A statistical method for evaluating the data obtained is included.
Subject(s)
Bone Marrow Cells , Erythropoietin/metabolism , Animals , Bone Marrow/metabolism , Cell Count , Fluorescent Antibody Technique , Mice , Microscopy, Fluorescence , RatsABSTRACT
Human erythropoietin, derived from urine of patients with aplastic anemia, has been purified to apparent homogeneity. The seven-step procedure, which included ion exchange chromatography, ethanol precipitation, gel filtration, and adsorption chromatography, yielded a preparation with a potency of 70,400 units/mg of protein in 21% yield. This represents a purification factor of 930. The purified hormone has a single electrophoretic component in polyacrylamide gels at pH 9, in the presence of sodium dodecylsulfate at pH 7, and in the presence of Triton X-100 at pH 6. Two fractions of the same potency and molecular size, by sodium dodecyl sulfate gel electrophoresis, but differing slightly in mobility at pH 9, were obtained at the last step of fractionation. The nature of the difference between these two components is not yet understood.
Subject(s)
Erythropoietin/isolation & purification , Anemia, Aplastic/urine , Electrophoresis, Polyacrylamide Gel , Erythropoietin/urine , Humans , Hydrogen-Ion Concentration , Molecular WeightSubject(s)
Erythropoietin/metabolism , Neuraminic Acids , Alcohol Oxidoreductases , Animals , Biological Assay , Bone Marrow/metabolism , Dose-Response Relationship, Drug , Galactose , Galactosidases , Iron Radioisotopes , Lactose/pharmacology , Male , Neuraminidase , Orosomucoid/pharmacology , Periodic Acid , Rats , Temperature , TrypsinSubject(s)
Erythropoietin/blood , Animals , Electrophoresis, Disc , Iodine Isotopes , Molecular Weight , Sheep , Sodium Dodecyl Sulfate , UltracentrifugationABSTRACT
Erythropoietin, the hormone that is the primary regulator of erythrocyte production, has been purified from anemic-sheep plasma, with a purification factor of more than 1 million. Gel electrophoresis shows that the major contaminant is desialated erythropoietin.
