Purificación de la proteína transportadora de esteroides sexuales (SHBG) con un métodode doble cromatografía de afinidad

Purification of sex hormone bindingglobulin(SHBG) with two affinity chromatography methods. The Sex Hormone Binding Globulin (SHBG) transports steroids sex hormones, dihydrotestosterone (DHT), testosterone (T) and estradiol (E2) in the blood and regulates their access to target tissues. The obtention of a preparation of high purity of this protein is necessary for the development of analytical methods. The human serum albumin (HSA) is the principal contaminant to remove because its concentration is 150 times higher that SHBG (60 g/L vs 400 mg/ L) and also binds esteroid sex hormones and thus, can not be completely excluded by affinity chromatography purification. In this paper, the results of SHBG purification from human serum by affinity chromatography through a matrix made by coupling [3 H]-5 a dihydrotestosterone 17 b hemisuccinate to Sepharose, Gel chromatography (Sephadex G-200) and affinity chromatography through a matrix prepared coupling human albumin antibodies to CnBr Sepharose to eliminate the HSA contaminant. The process was monitored by an analytical method whit high sensibility and specify to HSA. The last step of immunoaffinity chromatography was introduced to eliminate the contaminantion with HSA. The analytical gel electrophoresis showed two bands corresponding to the molecular weight of SHBG (52 KDa and 48 KDa). The cumulative yield of protein was 25.8 % and a specific activity of 2116 nM of DHT bound per mg of protein. By analytical electrophoresis and the analytical method ELISA-HSA, employed we did not detect HSA.