Crystallization of human placental estradiol 17beta-dehydrogenase. A new method for crystallizing labile enzymes.
Estradiol 17beta-dehydrogenase from human placenta has been crystallized by a new technique, herein referred to as electrophoretic diffusion. This is the first crystallization of an enzyme from human placenta as well as the first crystallization of any steroid-converting enzyme of human source. A solution of the enzyme (specific activity 7.1 units/mg) in 1.5 ml of Tris-barbituric acid buffer, pH 7.0, containing 20% glycerol as stabilizer, was placed in an electrophoresis tube and the tube was closed at both ends with a dialysis membrane which permits the passage of substances of molecular weight less than 18,000. The tube was placed in a gel electrophoresis apparatus and the reservoirs filled with the Tris-barbituric acid buffer. A potential of 100 V was applied for 12 hours, then raised to 200 V for another 12 hours, and finally to 300 V until opalescence appeared at the bottom of the tube. Activity measurements showed that more than 90% of the enzyme had concentrated in the bottom 0.15-ml portion of the solution. When this section of the solution was removed and kept overnight at 4 degrees, gross and microscopic examination revealed a heavy crop of crystals which possessed a specific activity of 7.2 units/mg. The specific activity remained constant throughout three recrystallizations. The crystalline enzyme displayed a single band by analytical and sodium dodecyl sulfate-polyacrylamide gel analysis. Crystals of enzyme of high specific activity could also be obtained from an enzyme sample initially possessing a specific activity of only 4.5 units/mg. The new technique should be appliable for the crystallization of other labile enzymes and receptor proteins which have so far resisted crystallization by conventional methods.
D003460:Crystallization; D003956:Dialysis; D004960:Estradiol Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000302:isolation & purification*; D008433:Mathematics; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy
D006913:Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases