一种新型海洋纤溶酶的研究
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摘要
血栓性疾病是西方富裕国家人类死亡的首要原因。它主要危害心、脑、肺的血管系统,造成成年人死亡或残疾。在美国,每年大约有60万人发生肺栓塞,其中约20万人死亡;每年约有100万人发生急性心肌梗塞,其中20多万人需要住院进行治疗。在我国,每年大约有200万人死于栓塞性心脑血管疾病,每年需要进行溶栓治疗的病人超过300万。并且,这类疾病的发病率也有逐年上升趋势。血栓性疾病的主要治疗手段是溶栓,迄今溶栓剂已发展了三代,但都有一定的副作用。有鉴于此,我们从海洋无脊椎动物单环刺螠中寻找和分离了一种新型纤溶酶。现将主要研究结果分述如下:
1.单环刺螠纤溶酶在单环刺螠的血液及内脏组织中含量丰富,其大量制备工艺为:体腔液及内脏混合物经匀浆、PBS抽提、(NH_4)_2SO_4分级盐析、超滤、Q.Sepharose Fast Flow和Sephadex G-75、Sephacry S-100层析分离,最终获得纯酶。经过优化的本工艺可以高效率地提取这种新型海洋纤溶酶,并达到了电泳纯,在SDS-PAGE电泳图谱上显示一条带。
2.经SDS-PAGE和Native-PAGE聚丙烯酰胺凝胶电泳分析,单环刺螠纤溶酶分子量约为10,380 Da。与已经发现的陆地生物来源的纤溶酶以及海洋生物来源的纤溶酶相比,分子量相对较小。而苯酚-硫酸法测定糖含量表明,单环刺螠纤溶酶含糖量极低,所以可能不是糖蛋白。
3.与蚓激酶类似,单环刺螠纤溶酶既可直接水解纤维蛋白,又具有激酶活性,通过激活纤溶酶原并使之转化为纤溶酶,从而间接地水解纤维蛋白。因而该酶具有很高的纤维蛋白亲合性和酶活力。以尿激酶为对照,测得酶的比活力为5256 U/mg。
4.单环刺螠纤溶酶体外抗凝实验结果表明,不同浓度的酶均具有显著的抗凝作用,并且酶浓度越高,抗凝效果也越好。在酶浓度相同的情况下,单环刺螠纤溶酶的抗凝效果要优于蚓激酶。而且,单环刺螠纤溶酶作用相对温和,对细胞损
Thrombotic disease is the leading cause of human death in western affluent countries, which mainly damages the blood systems of heart, brain, and lung, resulting in human death and disability in adult population. In the United States, pulmonary embolism is a prevalent condition with approximately 600,000 patients affected, and there were as many as 200,000 deaths each year;approximately 1 million people per year experience acute myocardial infarction, and more than 200,000 people have been enrolled into clinical trials examining different reperfusion strategies. In China, 2000,000 people die of cardio-cerebrovascular disease each year, and more than 3000,000 people carry out thrombolytic therapy. The patients with thrombotic disease are increasingly increased so far.
Thrombolytic therapy is the most important pathway in treatment of thrombotic disease. There are all three generations of thrombolytic agent, but more or less side effects are present in each of them. For this reason, a novel fibrinolytic enzyme is investigated and isolated from a marine invertebrate, Urechis unicinctus, in this paper. The main achievements in our investigations are now showed as follows.
1.The fibrinolytic enzyme abounds in the haemocoelomic fluid and gut tissues of Urechis unicinctus, which isolation and purification include several steps. Mixture of haemocoelomic fluid and gut tissues was minced and extracted with PBS, then isolated with (NH_4)_2SO_4 precipitation and ultrafiltration, and purified by using of Q.Sepharose Fast Flow, Sephadex G-75, and Sephacry S-100 respectively, and finally obtained the pure enzyme. By use of all these procedures, the fibrinolytic enzyme of
1.单环刺螠纤溶酶在单环刺螠的血液及内脏组织中含量丰富,其大量制备工艺为:体腔液及内脏混合物经匀浆、PBS抽提、(NH_4)_2SO_4分级盐析、超滤、Q.Sepharose Fast Flow和Sephadex G-75、Sephacry S-100层析分离,最终获得纯酶。经过优化的本工艺可以高效率地提取这种新型海洋纤溶酶,并达到了电泳纯,在SDS-PAGE电泳图谱上显示一条带。
2.经SDS-PAGE和Native-PAGE聚丙烯酰胺凝胶电泳分析,单环刺螠纤溶酶分子量约为10,380 Da。与已经发现的陆地生物来源的纤溶酶以及海洋生物来源的纤溶酶相比,分子量相对较小。而苯酚-硫酸法测定糖含量表明,单环刺螠纤溶酶含糖量极低,所以可能不是糖蛋白。
3.与蚓激酶类似,单环刺螠纤溶酶既可直接水解纤维蛋白,又具有激酶活性,通过激活纤溶酶原并使之转化为纤溶酶,从而间接地水解纤维蛋白。因而该酶具有很高的纤维蛋白亲合性和酶活力。以尿激酶为对照,测得酶的比活力为5256 U/mg。
4.单环刺螠纤溶酶体外抗凝实验结果表明,不同浓度的酶均具有显著的抗凝作用,并且酶浓度越高,抗凝效果也越好。在酶浓度相同的情况下,单环刺螠纤溶酶的抗凝效果要优于蚓激酶。而且,单环刺螠纤溶酶作用相对温和,对细胞损
Thrombotic disease is the leading cause of human death in western affluent countries, which mainly damages the blood systems of heart, brain, and lung, resulting in human death and disability in adult population. In the United States, pulmonary embolism is a prevalent condition with approximately 600,000 patients affected, and there were as many as 200,000 deaths each year;approximately 1 million people per year experience acute myocardial infarction, and more than 200,000 people have been enrolled into clinical trials examining different reperfusion strategies. In China, 2000,000 people die of cardio-cerebrovascular disease each year, and more than 3000,000 people carry out thrombolytic therapy. The patients with thrombotic disease are increasingly increased so far.
Thrombolytic therapy is the most important pathway in treatment of thrombotic disease. There are all three generations of thrombolytic agent, but more or less side effects are present in each of them. For this reason, a novel fibrinolytic enzyme is investigated and isolated from a marine invertebrate, Urechis unicinctus, in this paper. The main achievements in our investigations are now showed as follows.
1.The fibrinolytic enzyme abounds in the haemocoelomic fluid and gut tissues of Urechis unicinctus, which isolation and purification include several steps. Mixture of haemocoelomic fluid and gut tissues was minced and extracted with PBS, then isolated with (NH_4)_2SO_4 precipitation and ultrafiltration, and purified by using of Q.Sepharose Fast Flow, Sephadex G-75, and Sephacry S-100 respectively, and finally obtained the pure enzyme. By use of all these procedures, the fibrinolytic enzyme of
引文
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