Dodd I, Fears R, Robinson J H
Thromb Haemost. 1986 Feb 28;55(1):94-7.
Purified 2-chain recombinant tissue-type plasminogen activator (t-PA) was reduced under mild conditions - 10 mM dithiothreitol/5 degrees C/1.5 h - and the two chains were separated by chromatography on lysine Sepharose. The t-PA B chain was fully active as determined by its activity towards the chromogenic substrate S-2288 (H-D-ile-pro-arg p-nitroanilide). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing or non-reducing conditions revealed a single polypeptide at Mr = 35,000 or 29,000 respectively. In addition, under non-reducing conditions a fibrinolytic band at apparent Mr = 29,000 was present after fibrin zymography. The N-terminal sequence was confirmed as ile-lys-gly. The t-PA B chain had a specific amidolytic activity, using S-2288, of 170,000 to 210,000 SU/mg protein. (This compares to a specific activity of the native 2-chain t-PA of 170,000 SU/mg). It resembles urokinase-type plasminogen activator in its inability to be stimulated by fibrin and its dose response on human fibrin plates. However, t-PA B-chain was stimulated to almost the same extent as t-PA by poly-D-lysine. The isoelectric points, at pH 5.6 and 5.7, fall outside the range generally quoted for t-PA preparations (pH 7.8-8.8).
纯化的双链重组组织型纤溶酶原激活剂（t-PA）在温和条件下（10 mM二硫苏糖醇/5℃/1.5小时）进行还原，两条链通过赖氨酸琼脂糖凝胶柱层析分离。通过其对发色底物S-2288（H-D-异亮氨酸-脯氨酸-精氨酸对硝基苯胺）的活性测定，t-PA B链具有完全活性。在还原或非还原条件下进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析，分别显示在Mr = 35,000或29,000处有一条单一多肽带。此外，在非还原条件下，纤维蛋白酶谱分析后在表观Mr = 29,000处出现一条纤维蛋白溶解带。N端序列经确认是异亮氨酸-赖氨酸-甘氨酸。t-PA B链使用S-2288时的比酰胺水解活性为170,000至210,000 SU/mg蛋白质。（相比之下，天然双链t-PA的比活性为170,000 SU/mg）。它在不能被纤维蛋白刺激以及在人纤维蛋白平板上的剂量反应方面类似于尿激酶型纤溶酶原激活剂。然而，聚-D-赖氨酸对t-PA B链的刺激程度与对t-PA的刺激程度几乎相同。其等电点在pH 5.6和5.7，不在通常报道的t-PA制剂的等电点范围内（pH 7.8 - 8.8）。
