Synthesis and evaluation of immobilized androgens for affinity chromatography in the purification of nuclear androgen receptor.
Seven biospecific adsorbents containing immobilized androgens were synthesized: dihydrotestosterone-17 beta-succinyl agarose, and both the unsubstituted and the 17 beta-acetyl derivatives of dihydrotestosterone-7 alpha-undecanoyl agarose, testosterone-7 alpha-undecanoyl agarose, and 19-nortestosterone-7 alpha-undecanoyl agarose. The retention capacities for nuclear androgen receptor were generally between 40-80% with little variation in reproducibility; the amount of binding was greatest with dihydrotestosterone-17 beta-succinyl agarose and dihydrotestosterone-17 beta-acetoxy-7 alpha-undecanoyl agarose. Rapid flow rates were obtained with all gels, and no tendency for decomposition was observed over a period of 1 year. Factors that affected retention included the concentration of immobilized androgen, length of the linker arm, occupation of receptor sites, interval of contact with the gel, and temperature of incubation. Chemical dissociation of androgens from androgen receptor complexes with 0.2 mM mersalyl increased the retention of receptor by dihydrotestosterone-17 beta-succinyl agarose. Two elutants showed promise for the dissociation of gel-bound receptor: 1) 0.2 mM mersalyl in the presence of 1.5 mg/ml of ovalbumin; 2) 10% (v/v) dimethylformamide:water containing 30 microM [1,2-3H] dihydrotestosterone and 0.5 M sodium thiocyanate.
D000728:Androgens / Q000138:chemical synthesis*; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D002847:Chromatography, Agarose; D007700:Kinetics; D008297:Male; D008634:Mersalyl; D011467:Prostate / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011944:Receptors, Androgen / Q000302:isolation & purification*; D011987:Receptors, Steroid / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives / Q000138:chemical synthesis; D013696:Temperature; D013739:Testosterone / Q000031:analogs & derivatives / Q000138:chemical synthesis; D013997:Time Factors
D000728:Androgens; D011944:Receptors, Androgen; D011987:Receptors, Steroid; D013739:Testosterone; D008634:Mersalyl; D012685:Sepharose