The use of the biotinyl estradiol-avidin system for the purification of "nontransformed" estrogen receptor by biohormonal affinity chromatography.
Several biotinyl estradiol derivatives have been prepared by coupling estradiol 7 alpha-carboxylic acid to biotin via different linear linkers. All these compounds exhibit a high affinity for the estrogen receptor as determined by competitive binding assays against [3H]estradiol. These compounds also displaced the dye 4-hydroxyazobenzene-2'-carboxylic acid from the biotin-binding sites of avidin free or immobilized on agarose. It was demonstrated that only the derivatives bearing a long spacer chain (greater than 42 A greater than) between estradiol and biotin were able to bind receptor and avidin simultaneously, suggesting some steric hindrance. The biotin-avidin system has been investigated for the purification of the cytosoluble "nontransformed" estrogen receptor stabilized by sodium molybdate. The method relies on: 1) high biohormonal affinity of receptor for biotinyl estradiol derivative; 2) the specific selection by avidin-agarose column of biotinyl estradiol-receptor complexes; and 3) the biohormonal elution step by an excess of radioactive estradiol. Starting from unfractionated cytosol containing molybdate-stabilized nontransformed 8S estrogen receptor with estradiol 7 alpha-(CH2)10-CO-NH-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)3-NH-biotin, preliminary experiments using avidin-agarose chromatography and then a specific elution step by exchange with free [3H]estradiol, allowed a 500-1,500-fold purification. Further purification of estrogen receptor was obtained by ion exchange chromatography through a DEAE-Sephacel column and led to a congruent to 20% pure protein, assuming one binding site/65,000-Da unit. The hydrodynamic parameters of the purified receptor were essentially identical to those of molybdate-stabilized nontransformed receptor present in crude cytosol. The advantages of this double biotinyl steroid derivative-avidin chromatographic technique over more conventional affinity procedures are discussed and make it applicable to the purification of minute amounts of steroid receptors in a wide variety of tissues.
D000818:Animals; D001360:Avidin / Q000378:metabolism*; D001667:Binding, Competitive; D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism; D005260:Female; D010047:Ovalbumin / Q000031:analogs & derivatives*; D011960:Receptors, Estrogen / Q000302:isolation & purification*; D014599:Uterus / Q000032:analysis
D011960:Receptors, Estrogen; D001360:Avidin; D004958:Estradiol; D001710:Biotin; D010047:Ovalbumin