Purification of the sex steroid binding protein from human serum.
The sex steroid binding protein from human pregnancy serum was purified to homogeneity by affinity chromatography and preparative polyacrylamide gel electrophoresis. The selective adsorbants were prepared by coupling [3H]-5alpha-dihydrotestosterone 17beta-hemisuccinate to 3,3'-diaminodipropylamine-agarose, poly(Lys-DLAla)-agarose, and albumin-agarose. The most effective adsorbant purifying for the binding protein was 5alpha-dihydrotestosterone 17beta-hemisuccinyl-3,3'-diaminodipropylamine-agarose. A preparative procedure with 5alpha-dihydrotestosterone 17beta-hemisuccinyl-3,3'-diaminodipropylamine-agarose yielded active material which was further purified by preparative polyacrylamide electrophoresis at pH 9.5. Homogeneity was shown by analytical disc gel electrophoresis at three different pH units. A single radioactive band corresponding to the stained band was shown by incubating with [1,2-3H]-5alpha-dihydrotestosterone prior to electrophoresis. The radioactive peak corresponding to the pure sex steroid binding protein could not be detected when a 100-fold excess of 17beta-estradiol was present in the incubation prior to electrophoresis demonstrating the specific sex steroid binding properties of this protein. The migration of this peak was identical with that obtained when diluted serum was electrophoresed under the same conditions in the presence of [1,2-3H]-5alpha-dihydrotestosterone indicating that no significant changes in the molecular characteristics of the binding protein occurred during the purification procedure. The presence of carbohydrate in the pure protein was shown by the periodic acid-Schiff reagent procedure. Selective adsorbants containing 17beta-estradiol linked at the 3 position were ineffective in retaining sex steroid binding protein activity.
D001665:Binding Sites; D001798:Blood Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D013196:Dihydrotestosterone / Q000378:metabolism*; D004355:Drug Stability; D004589:Electrophoresis, Disc; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D006801:Humans; D007525:Isoelectric Focusing; D011247:Pregnancy; D011485:Protein Binding; D011956:Receptors, Cell Surface; D013997:Time Factors
D001798:Blood Proteins; D011956:Receptors, Cell Surface; D013196:Dihydrotestosterone; D004958:Estradiol