Isolation of testosterone-binding globulin from bovine serum by affinity chromatography and its molecular characterization.
The testosterone-binding globulin (TeBG) from bovine serum was purified by affinity chromatography and hydroxylapatite chromatography. The affinity column used was prepared by coupling 17 alpha-carboxyethynyl-17-hydroxy-4-androsten-3-one to aminoethyl-Sepharose. The compound was replaceable by 17alpha-carboxyethynyl-17-hydroxy-5alpha-androstan-3-one, but not by testosterone 17-hemisuccinate, estradiol 17-hemisuccinate, or testosterone 3-(O-carboxymethyl)oxime. The TeBG isolated was homogeneous on analytical polyacrylamide gel electrophoresis and equilibrium centrifugation. The protein was a glycoprotein having a molecular weight of 89,500 and a carbohydrate content of 17%. The association constant (M-1) at 4 degrees C was 1.1 X 10(8) and the number of binding sites per molecule was 0.8. Treatment with guanidine-HCl dissociated the protein into subunits having a molecular weight of 28,400 (about one-third of that of the original molecule). SDS-gel electrophoresis showed that two of the three subunits were slightly larger than the other. The dissociation into subunits could also be accomplished by GEDTA treatment with concomitant loss of testosterone-binding activity. The activity and molecular size were reversibly restored by incubation with excess Ca2+.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002241:Carbohydrates / Q000032:analysis; D002413:Cations, Divalent; D002417:Cattle; D002846:Chromatography, Affinity; D004492:Edetic Acid; D046911:Macromolecular Substances; D008970:Molecular Weight; D011487:Protein Conformation; D012738:Sex Hormone-Binding Globulin / Q000097:blood* / Q000302:isolation & purification; D013739:Testosterone / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D002413:Cations, Divalent; D046911:Macromolecular Substances; D012738:Sex Hormone-Binding Globulin; D013739:Testosterone; D004492:Edetic Acid