Kolodzeĭskaia M V, Chernyshenko T M
Palladin Institute of Biochemirtsy, NAS of Ukraine, Kyiv.
Ukr Biokhim Zh (1999). 2002 Sep-Oct;74(5):27-33.
The work deals with estimation of thrombin preparation having such features as: sedimentation activity 3000-3200 NIH un. per 1 mg of protein and 97% of active centres. The enzyme isolated has been estimated according to the amidolytic activity on synthetic substrates S-2160 and BAPNA being equal 5200 and 185 milli un/mg of protein, respectively. According to the electrophoresis in PAAG in the presence of Ds-Na the preparation is homogenous, its molecular mass is 36000. The fibrinogen sedimentation time dependence on the isolated thrombin concentration has been estimated as well as the comparative analysis with the thrombin of the firm "Sigma" with the previously calibrated activity using the international standartion (coded P4) has been conducted. The absence of proportionality between the substrate sedimentation time and the preparation concentration has been determined. It has been revealed, that if the experimental findings are presented in the units 1/t against the thrombin units NIH the right lines are received within the limits used. The defreezing and secondary freezing of the preparation preserved under -20 degrees C have been showed as rendering an essential effect on thrombin activity. In order of the enzyme stabilizing at preserving the thrombin isolated has been concentrated applying the amycon membranes (MWCo: 30,000). While applying the thrombin water-saline solution in the conditions selected the preparation has showed itself practically stable during a year without utilizing any admixtures. The essential effect on thrombin has been found from the side of 1% glycin, 0.5% PEG, 1% saccharose and so on. The thrombin isolated high functional homogeneity, its stability permit to recommend the preparation as an operative standard.
沉降活性为每1毫克蛋白质3000 - 3200 NIH单位，活性中心为97%。所分离的酶根据其对合成底物S - 2160和BAPNA的酰胺水解活性进行评估，分别为5200和185毫单位/毫克蛋白质。根据在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）中的结果，该制剂是均一的，其分子量为36000。已评估了纤维蛋白原沉降时间对分离出的凝血酶浓度的依赖性，并与Sigma公司具有先前使用国际标准（编码P4）校准活性的凝血酶进行了比较分析。已确定底物沉降时间与制剂浓度之间不存在比例关系。结果表明，如果以1/t单位表示实验结果相对于凝血酶的NIH单位，则在所用范围内可得到直线关系。已表明在 - 20℃下保存的制剂解冻和二次冷冻对凝血酶活性有显著影响。为了在保存分离出的凝血酶时使其稳定，使用了超滤膜（截留分子量：30000）进行浓缩。在所选择的条件下使用凝血酶水溶液时，该制剂在一年中未使用任何添加剂的情况下表现出实际稳定性。已发现1%甘氨酸、0.5%聚乙二醇、1%蔗糖等对凝血酶有显著影响。所分离的凝血酶具有高度的功能同质性及其稳定性，使其可被推荐作为操作标准。
