抗凝组分Acoagulatin的纯化鉴定及其抗凝特性

Purification, identification of acoagulatin, an anticoagulation factor from the venom of Chinese Agkistrodon, and observation of its anticoagulation effect

目的 从蝮蛇毒中分离纯化一种组分acoagulatin并进行鉴定,观察其抗凝作用。方法 经DEAE-Sepharose Fast Flow和CM-sepharose Fast Flow离子交换柱层析,从蝮蛇毒中分离、纯化得到acoagulatin,分别采用Biosep-sec-s 2000型HPLC分子筛柱层析、还原性SDS-PAGE(5％浓缩胶,12％分离胶)电泳进行纯度和相对分子质量测定,将不同浓度acoagulatin与抗凝兔血混合,测定凝血时间(TT)、凝血酶原时间(PT)、部分凝血活酶时间(APTT)。结果 Acoagulatin的相对分子质量为31400,由两个亚基组成,相对分子质量分别为14400和17000,能显著地延长PT和APTT,对TT没有影响。结论 采用DEAE-Sepharose Fast Flow和CM-sepharose Fast Flow两种离子交换柱层析分离蛇毒,能分离出纯度高的组分acoagulatin,该组分具有抗凝活性。

Objective To isolate and identify an anticoagulantion factor, acoagulatin, from the venom of Chinese Agkistrodon and to observe its anticoagulation effect. Method The venom of Chinese Agkistrodon was isolated and purified using ion-exchange chromatography on DEAE-Sepharose Fast Flow and CM-sepharose Fast Flow. High-performance liquid chromatography (HPLC) was performed for determination of the purity of acoagulatin, and SDS-PAGE electrophoresis (with 5% concentrated gel ofpH 6.8, 12% separation gel of pH 8.8, Tris-aminoacetic acid buffer of pH 8.3 as the electrode buffer) for determining the relative molecular mass. For observation of the anticoagulation effect, 20 μ1 acoagulatin solution at the concentration of 0.30, 0.20 and 0.15 μg/μ1, respectively, was mixed with 100 μ1 rabbit anticoagulated plasma and the thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT) were determined. Results Acoagulatin was found to consist of two subunits with relative molecular mass of 14 400 and 17 000 respectively, resulting in the total relative molecular mass of 31 400 as determined by SDS-PAGE. HPLC demonstrated good homogeneity of this protein, which significantly prolonged the PT and APTT without affecting TT. Conclusion DEAE-Sepharose Fast Flow and CM-sepharose Fast Flow ion exchange column chromatographies are effective to isolate acoagulatin of high purity, which possesses anticoagulation effect.