Crystallization of human placental estradiol 17beta-dehydrogenase. A new method for crystallizing labile enzymes.
Crystallization of human placental estradiol 17beta-dehydrogenase. A new method for crystallizing labile enzymes.
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人胎盘雌二醇 17β-脱氢酶的结晶。
DOI：
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发表时间：
1976
影响因子：
4.8
通讯作者：
J. C. Warren
中科院分区：
文献类型：
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作者：
C. Chin;J. B. Dence;J. C. Warren
用一种新的技术，本文称之为电泳扩散法，从人胎盘中结晶出了雌二醇17 β-脱氢酶。这是人类胎盘中酶的第一次结晶，也是人类来源的任何类固醇转化酶的第一次结晶。将酶（比活性7.1单位/毫克）在1.5毫升Tris-巴比妥酸缓冲液（pH7.0，含20%甘油作为稳定剂）中的溶液置于电泳管中，用透析膜封闭电泳管的两端，透析膜允许分子量小于18，000的物质通过。将管置于凝胶电泳装置中，并将储液器用Tris-巴比妥酸缓冲液填充。施加100 V的电位12小时，然后升高至200 V再持续12小时，最后升高至300 V直到在管底部出现乳光。活性测量表明，超过90%的酶已集中在底部的0.15毫升部分的溶液。当取出这部分溶液并在4 ℃下保持过夜时，肉眼和显微镜检查显示大量晶体，其比活度为7.2单位/mg。比活性在三次重结晶中保持恒定。通过分析和十二烷基硫酸钠-聚丙烯酰胺凝胶分析，晶体酶显示单一条带。高比活性的酶晶体也可以从最初仅具有4.5单位/mg比活性的酶样品中获得。新技术应该适用于结晶的其他不稳定的酶和受体蛋白，迄今为止，抵制结晶的常规方法。
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.