单环刺螠不同部位中纤溶酶的分离纯化及其活性差异

对单环刺螠不同部位中的纤溶酶(UFE)进行分离纯化,并比较其活性差异.选择单环刺螠体壁肌、内脏、体腔液作为UFE的提取来源,通过离心分离、SephacrylS 100凝胶柱层析、Q-Sepharose fast flow阴离子交换层析等工艺获得UFE单一组分,采用Folin-酚试剂法测定其酶活性,并研究UFE对AT-Ⅲ,HC-Ⅱ,凝血因子和钙离子的影响,验证不同部位UFE的活性差异.结果表明:从秋季单环刺螠体腔液中提取的总蛋白质量为39.8 mg,UFE的比活力为4 695.94 mkat·g^-1,纯化倍数为13.8,明显优于内脏和体壁肌的提取所得;在抗凝作用实验中,加入体腔液中所得UFE后,标准血浆、乏AT-Ⅲ血浆和乏HC-Ⅱ血浆凝血酶原时间、凝血酶时间均极显著延长,加入不同浓度氯化钙后,能显著延长凝血时间,同时,能够极显著降低凝血因子Ⅴ,Ⅶ,Ⅷ,Ⅸ,Ⅹ,Ⅻ的活性及大鼠血液中的钙离子浓度;不同部位来源的UFE活性有较大差异,与内脏及体壁肌相比,体腔液为UFE更优提取部位.

Purification and Activity Difference of Fibrinolytic EnzymeFrom Different Parts of Urechis unicinctus

The activity difference of Urechis unicinctus fibrinolytic enzyme(UFE)was compared after separation and purification from different parts.The body wall muscle, viscera and body cavity fluid of Urechis unicinctus were selected as the extraction source of UFE, and the single component of UFE was obtained by centrifugation separation, SephacrylS 100 gel column and Q-Sepharose fast flow anion exchange chromatography. The viability of UFE was measured by Folin-phenol reagent method, and the effects of UFE on AT-Ⅲ, HC-Ⅱ, coagulation factors and calcium ions were studied to verify the difference of UFE activity from different parts. The results show that the total protein weight extracted from the body cavity in autumn was 39.8 mg. The specific activity of UFE reached 4 695.94 mkat·g^-1 and the purification ratio was 13.8 times, which was significantly better than that obtained from the visceral and body wall muscles. In the anticoagulation experiment, the levels of prothrombin time and thrombin time in standard plasma, deficient AT-Ⅲ and HC-Ⅱ deficiency prolonged significantly after body cavity fluid of UFE added, and the clotting time also prolonged significantly after adding different concentrations of calcium chloride. At the same time, it could significantly reduce the activity of coagulation factor Ⅴ, Ⅶ, Ⅷ, Ⅸ, Ⅹ, Ⅻ and the concentration of calcium in rat blood. The activity of UFE in different parts were different, and the body cavity fluid is better as UFE extraction site compared with visceral and body wall muscles.