【作者】 潘剑茹；

【导师】 王中来； 饶平凡；

【作者基本信息】 福州大学 ， 生物化学与分子生物学， 2002， 硕士

【摘要】 本论文研究的是福建产蕲蛇粗毒尝试着用嗜硫色谱进行分离后，其洗脱峰在室温下放置一个晚上后，发生降解现象的原因，并对发生降解的蛋白组分进行纯化和表征。 蕲蛇粗毒用嗜硫色谱分离所得的洗脱峰，在室温下放置一个晚上后，经SDS-PAGE检测发现其组分中分子量44kDa左右的蛋白质发生了降解，新生成分子量29kDa左右的蛋白质。 为弄清这一现象的产生原因，将嗜硫色谱洗脱峰通过POROS20HQ离子交换色谱进一步分离，获得5个分子量非常接近的组分。PAGE电泳结果表明，其中第五个组分(HQ-5)会发生降解。经SDS-PAGE电泳和CLC鉴定，为纯组分。由此可断定嗜硫色谱洗脱峰的降解现象是由HQ-5的自降解引起的。 HQ-5经SDS-PAGE电泳测得分子量为47.6kDa，含有链内二硫键，加DTT处理后，分子量为50.8kDa。HQ-5完全降解的最快条件为：在70℃和pH9.0条件下保温80分钟。此组分具有碱性蛋白酶活性，最佳反应pH约为9.5，不耐高温，EDTA能抑制其蛋白酶活性，是一种金属蛋白酶。此外该组分还具有抗凝血活性，且能耐受较高温度。该组分是一种糖蛋白，中性糖含量为3.5%。 将嗜硫色谱的洗脱峰在40℃水浴中保温2小时后，再通过TSK G3000SW分离得到新的蛋白组分(TSK-2)。该组分为单链蛋白质，分子量为26.8kDa，具有抗凝血活性，是嗜硫色谱洗脱峰降解后新产生的蛋白组分。其一级序列为RTEFQRYMEIVV。 与从蕲蛇蛇毒中分离出的其它组分相比，HQ-5及其降解产生的组分(TSK-2)为新的抗凝血组分。更多还原

【Abstract】 This article researched for the reason of the proteolytic phenomenon of the proteins of Agkistrodon acutus snake venom adsorbed in thiophilic adsorption chromatography and purify and characterize the proteolytic proteins.The proteins in the eluant of the thiophilic adsorption chromatography of MW 44kDa were converted to a new protein of MW29kDa when they were put under room temperature over night.To reveal the reason of above phenomenon, we purified the eluant by POROS 20HQ ion-exchange chromatography and got five proteins. One of them(HQ-S) was proved to can be proteolytic. When it was verified to be pure by SDS-PAGE, PAGE and CLC, it could be concluded that HQ-5’s autoproteolysis just was the cause of the proteolytic phenomenon of eluant of the thiophilic adsorption chromatographyHQ-5 was identified as a glycoprotein containing 3.5% neutral carbohydrates. Its molecular weight was determined to be 47.6kDa by SDS-PAGE and about SO.SkDa under reduced conditions. It is a metalloprotease, having a basic proteolytic activity with the optimum pH of 9.5 and being inhibited by EDTA . Its anti-coagulation activity was characterized. HQ-5 could be converted to a new protein autoproteolytically when it was incubated hi the basic solution or higher temperature.The new protein (TSK-2) was isolated and purified to homogeneity by TSK G3000SW chromatography from the elutriant of thiophilic adsorption, which was incubated in the condition of 40℃ for 2 hours. Its molecular weight was determined to be 26.8kDa by SDS-PAGE and the same under reduced conditions. Its anti-coagulation activity was characterized. The N-terminal sequence of TSK-2 is RTEFQRYMEIW.It is shown that the obtained proteins(HQ-5 & TSK-2) can be identified as novel components of anti-coagulation enzyme family in the venom of Agkistrodon acutus by the analysis of the N-terminal sequence of TSK-2.更多还原