Bovine Thrombin PURIFICATION AND CERTAIN PROPERTIES
Bovine Thrombin PURIFICATION AND CERTAIN PROPERTIES
复制标题
牛凝血酶的纯化和某些特性
DOI：
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发表时间：
1967
期刊：
影响因子：
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通讯作者：
Waugh Df
中科院分区：
文献类型：
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作者：
B. Dj;Waugh Df
摘要：检查是由生物活化获得的粗凝血酶制剂的分离，即帕克戴维斯凝血酶，局部。研究了羧酸树脂柱、磷酸纤维素柱、二乙胺乙基纤维素柱和Sephadex G-100柱。仅使用羧酸树脂或磷酸纤维素获得的原料和所有产品都是不纯的，并且含有非酶抑制剂。一个简单的程序给出了分离稳定的凝血酶高，恒定的比活性。首先，在pH为7，Γ/2 = 0.1（磷酸盐）和23°的条件下，用deae -纤维素从溶液中去除抑制剂和一些非凝血酶蛋白。接下来，在相同的条件下使用纤维素磷酸盐柱吸附凝血酶并传递一些非凝血酶蛋白。在pH为7，Γ/2 = 0.15的条件下洗去额外的无活性蛋白后，在Γ/2 = 1.0的条件下洗脱凝血酶，得到凝血酶原液。此时的最低产率为原活性的91.1±8.4%。在Sephadex G-100上凝胶过滤原液凝血酶，可将原液凝血酶作为无活性蛋白平均额外分离24%的吸光度，并给出单个凝血酶峰。与已知蛋白比较，峰位置对应的分子量为36000±1600（95%置信限）。峰的后0.6具有恒定的比活性，对应于每吸光度单位1,160±130 NIH单位或每mg 2,260±250 NIH单位。在离子强度1.0，pH 7和4°时，原液凝血酶制剂的活性损失最大程度为每周0.1%。在Γ/2 = 0.1， pH 7和23°时，稀释的凝血酶（20秒凝血时间）每天失去1.6%的活性。结果表明，这不是由于抑制剂、外来酶或5 × 10-4 m KCN的存在，这些都是为了防止细菌生长而必须添加的。其机理为零级变性。通过硫酸铵沉淀或渗透溶剂去除来浓缩凝血酶的尝试经常导致产生无活性聚集体。在pH值为7时，从0.15 m氯化钙中析出的丙酮已成功地使浓度达到每毫升10个吸光度单位。
Abstract An examination is made of the fractionation of a crude thrombin preparation obtained by bioactivation, namely, Parke Davis thrombin, topical. Columns of carboxylic resin, cellulose phosphate, diethylaminoethyl cellulose, and Sephadex G-100 were studied. The starting material and all products obtained by using only carboxylic resin or cellulose phosphate are shown to be impure and to contain a nonenzymatic inhibitor. A simple procedure is given for isolating a stable thrombin of high, constant specific activity. The procedure involves removal from solution, first, of inhibitor and some nonthrombin protein by DEAE-cellulose at pH 7, Γ/2 = 0.1 (phosphate), and 23°. A cellulose phosphate column is next used under the same conditions to adsorb thrombin and pass some nonthrombin protein. After additional inactive protein is washed off at pH 7 and Γ/2 = 0.15, thrombin is eluted at Γ/2 = 1.0 to give stock thrombin. The procedure at this point gives a minimum yield of 91.1 ± 8.4% of the original activity. Gel filtration of stock thrombin on Sephadex G-100 separates an average additional 24% of the absorbance of stock thrombin as an inactive protein and gives a single thrombin peak. By comparison with known proteins the peak position corresponds to a molecular weight of 36,000 ± 1,600 (95% confidence limits). The last 0.6 of the peak has constant specific activity which corresponds to 1,160 ± 130 NIH units per absorbance unit or 2,260 ± 250 NIH units per mg. At ionic strength 1.0, pH 7, and 4°, stock thrombin preparations lose activity to the maximum extent of 0.1% per week. At Γ/2 = 0.1, pH 7, and 23°, diluted stock thrombin (20-sec clotting time) loses 1.6% of its activity per day. This is shown not to be due to an inhibitor, an extraneous enzyme, or the presence of 5 x 10-4 m KCN, which had to be added to prevent bacterial growth. The mechanism appears to be a zero order denaturation. Attempts to concentrate stock thrombin by ammonium sulfate precipitation or osmotic solvent removal have frequently led to the production of inactive aggregates. Acetone precipitation from 0.15 m calcium chloride at pH 7 has been successful in giving concentrations up to 10 absorbance units per ml.