摘要
用DEAE-SephadexA-25、Sephadex-G-100和CM-SephadexC-50三步柱层析分离法,从白唇竹叶青(Trimeresurus albolabris)蛇毒中分离纯化出具有5′-核苷酸酶活性的组分。SDS-聚丙烯酰胺凝胶电泳测定其分子量为48.03kDa,HPLC柱层析图谱为单一峰。该组分是一个糖蛋白,以一磷酸腺苷(AMP)为底物时,其酶活力为330.33μg Pi/(min·mg);而以二磷酸腺苷(ADP)为底物时,其酶活力为123.56μg Pi/(min·mg)。金属离子Zn2+、Fe3+和Cu2+对5′-核苷酸酶活性有显著的抑制作用,EDTA可完全抑制其酶活性。该酶的最适pH为9,最适温度为50℃。该组分还具有抑制由ADP诱导的血小板聚集的生物功能。
A 5＇-nucleotidase was isolated and purified from the snake venom of T. albolabris using three steps of chromatography including DEAE-SephadexA-25, Sephadex-G-100 and CM-Sephadex C-50. Using SDS-PAGE and HPLC column chromatography the purified 5＇-nucleotidase proved to be homogenous. It was a glycoprotein with a molecular weight of 48.03kDa. The enzymatic activities of the purified 5＇-nucleotidase were 330.331ag Pi/min mg and 123.561μg Pi/min mg when using AMP （adenosine monophosphate） and ADP （adenosine diphosphate） as substrates, respectively. Metal ions, including Zn^2＋, Fe^3＋ and Cu^2＋, could inhibit 5＇-nucleotidase activity, as did EDTA. Its optimum pH was nine and its optimum temperature was 50℃ It has a potent inhibitory effect on rabbit platelet aggregation induced by ADP.
基金
重庆市自然科学基金重点项目(CSTC2006BA5032)
重庆市教委基金项目(KJ080824)
关键词
白唇竹叶青蛇毒
5'-核苷酸酶
分离纯化
Trimeresurus albolabris
Snake venom
5＇-nucleotidase
Isolation and purification
