Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, 38400-902 Uberlândia-MG, Brazil.
Toxicon. 2010 Jun 15;55(7):1365-77. doi: 10.1016/j.toxicon.2010.02.014. Epub 2010 Feb 23.
A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 degrees C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aalpha-chain, apparently not degrading the Bbeta and gamma-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 degrees C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.
从矛头蝮蛇蛇毒中分离到一种丝氨酸蛋白酶，采用 DEAE-Sephacel、Sephadex G-75 和苯甲脒琼脂糖柱层析进行纯化。该纯化酶命名为 Bhalternin，在分析聚丙烯酰胺凝胶电泳（SDS-PAGE）上呈现单一蛋白质条带，在还原和非还原条件下的分子量分别为 31500 和 27000。通过 RT-PCR 获得其全长 cDNA，编码 236 个氨基酸残基的成熟蛋白。其推导的氨基酸序列的多重比对显示与其他蛇毒丝氨酸蛋白酶具有结构相似性。Bhalternin 对牛纤维蛋白原和白蛋白作为底物具有蛋白水解活性。当 Bhalternin 和牛纤维蛋白原在 37°C 下以 1:100（w/w）的比例孵育时，该酶优先切割 Aalpha 链，显然不降解 Bbeta 和 gamma 链。稳定性测试表明，纤维蛋白原水解活性的最适温度和 pH 区间分别为 30-40°C 和 7.0-8.0。此外，苯甲脒对 Bhalternin 纤维蛋白原水解活性的抑制作用表明它是一种丝氨酸蛋白酶。该酶可引起小鼠心脏、肝脏、肺和肌肉的形态改变，并发现其在体内给药时可引起体外凝血和纤维蛋白原降解，表明其为类凝血酶酶。因此，Bhaltenin 可能作为治疗和预防血栓形成障碍的治疗剂具有一定的应用前景。
