estrogens are approximately the same. 4) When administered alone? these estrogens produce no significant change in either carbonic anhydrase titers or Gz 11 ratios of the endometrium. 5 ) The linear relationship with negative slope which is obtained between the logarithmic dose of the estrogens and the endometrial carbonic anhydrase content of the progesterone-treated Clauberg rabbit suggests the usefulness of the carbonic anhydrase method as an assay procedure for anti-progestational activity. 6 ) Comparison of the log-dose-response curves of progesterone with and without estradiol indicates that estrogen may depress reactivity of the endometrium to progesterone rather than neutralize or inactivate progesterone in the body.\Ye have recently isolated a heat stable erythropoietic factor, erythropoietin. and have shown it to be an acidic glycoprotein( 1). Because several workers have suggested that erythropoietin is not a protein (2,3,4) it was of interest to investigate the possibility that this glycoprotein subserves a carrier function. Since dialysis of erythropoietin does not reduce its activity( 5 ) , any 'carried factor' would be firmly bound. Therefore, erythropoietin was submitted to acid hydrolysis and the degradation products studied biologically and qualitatively by chromatography.Materials and methods. Purified erythropoietin was isolated from acidified, boiled plasma filtrates of anemic rabbits by DEAEcellulose ion-exchange chromatography as previously described (1 ) . It was dissolved in distilled water, 3 mg/ml. Five 1 ml aliquots were hydrolyzed in a water bath at 80' C for 1 hour with 5 ml H2S01 in final concentrations *Study supported by Contract with School of Aviation Medicine, U. S. Air Force, and from Eli L a y co. of 0.005 N, 0.01 N, 0.04 N, 0.08 N and 0.10 S respectively. A 1 ml aliquot brought to 5 ml with distilled water was carried through the procedure as a control. The hydrolysates were separately dialyzed in the cold against repeated changes of distilled water for 5 days. The dialysates, concentrated in vacuo, and protein dialysands were lyophilized and stored. Separations of 1 mg of the hydrolyzed protein were carried out on Whatman 3 MM paper a t 5 ma using veronal buffer pH 8.6, ionic strength 0.075. The strips were stained with bromphenol blue. Seven hour descending chromatograms of the dialysates in an ethyl acetate:pyridine:H20 (10:4:3) solvent system ( 6 ) were prepared. A mixture of neuraminic (sialic) acid, d-glucosamine, d-mannose, d-glucose, d-galactose and l-f ucose served as a reference material. After development and drying, the chromatograms were sprayed with aniline hydrogen phthalate reagent (7) and then heated at 110' C for 15 min. One half mg, in 0.9% NaCl, respectively of dialyzed 0.01 N hydrolysate of erythropoietin and in tact erythropoietin were injected subcutaneat JOHNS HOPKINS UNIVERSITY on June 9, 2015 ebm.sagepub.com Downloaded from
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