Purification of estradiol-17 beta dehydrogenase from human placenta by affinity chromatography |
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Authors: | C Chin J C Warren |
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Affiliation: | Departments of Obstetrics-Gynecology and Biological Chemistry Washington University School of Medicine St. Louis, Missouri 63110 USA |
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Abstract: | Estriol 16-hemisuccinate has been synthesized and covalently attached to Sepharose through 1,5-diaminopentane. A crude preparation of estradiol-17β dehydrogenase from human placenta was adsorbed on the gel. After extensive washing, the enzyme was eluted by hydroxylamine in 0.1 potassium phosphate buffer (20–50% glycerol), pH 7, at room temperature. An apparently homogeneous enzyme with a specific activity of 7.2 U/mg (82% recovery) was obtained. It is stable for weeks in the eluting buffer. The hydroxylamine can be removed by passing the enzyme solution over a Sephadex G-100 column or by dialyzing it against 0.1 potassium phosphate buffer containing 20% glycerol. This one-step process makes purification of the enzyme simple and easy. |
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