湖南尖吻蝮(D. acutus)蛇毒出血毒素的纯化、性质和溶液构象研究
文献类型：学位论文
| 作者 | 陶勇 |
| 学位类别 | 硕士 |
| 答辩日期 | 1991 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 冉永禄 |
| 关键词 | 湖南 尖吻蝮 出血毒素 金属蛋白酶 溶液构象 圆二色性 |
| 中文摘要 | 通过Sephadex G-75凝胶过滤，QAE-Sephadex A-50和CM_Sephadex C-25离子交换的步骤，我们从湖南产尖吻蝮(Dienagkistrodon acutus)蛇毒中纯化出两个出血毒素，分别称之为DaHT-1和DaHT-2。在聚丙烯酰胺凝胶电泳(PAGE)和SDS-聚丙烯酰胺凝胶电泳中均呈单一蛋白带，显示两个出血毒素皆为电泳纯。DaHT-1和DaHT-2的分子量相同，都为23,500道尔顿，具有相似的氨基酸组成，其中酸性氨基酸(Asx和GLx)分别占23%和24%。经等电聚焦(IEF)测得它们的等电点分别为5.6和5.2。两个出血毒素具有较强的出血活性(MHD分别为0.5和0.8μg)，都具蛋白水解酶活力，无精氨酸水解酶和PLA~2活性，但蛋白水解酶活性与出血活性并非正相关。DaHT-1，DaHT-2的最适温度分别为35 ℃，40 ℃；最适pH为6-9。对热不稳定，温度变高于60 ℃，活性完全丧失。在中性和碱性条件下稳定，在酸性条件下不稳定，pH<3，出血活性丧失。EDTA完全抑制，半胱氨酸部分抑制它们的出血活性，表明两个出血毒素都是依赖金属离子的蛋白酶，且二硫键对其活性是必需的。金属离子的分析表明每摩尔毒蛋白大约含0.5摩尔的Zn，1摩尔的Ca，较多的Na，K。Mg，不含Co。两者是糖蛋白，含糖总量分别为11%和7%。用远紫外CD谱探讨DaHT-1和DaHT-2的溶液构象所得DaHT-1的α-螺旋，β-折叠和无规卷曲分别为36.9%，27.6%和31.4%；DaHT-2的α-螺旋，β-折迭和无规卷曲分别为23.4%，37.3%和45.3%。随着pH的增大或减少，DaHT-1和DaHT-2的峰位蓝移，在酸性条件下的变化比在碱性条件下大，计算表明：α-螺旋减少，无规卷曲增多，β-折迭基本未变。温度的影响和pH相似，50 ℃时峰位蓝移，α-螺旋减少，无规卷曲增多。EDTA对其影响很大，0.02M EDTA便导致两个出血毒素呈极度的无序状态。 |
| 英文摘要 | By Sephadex G-75, QAE-Sephadex A-50 and CM-Sephadex C-25 chromatography, two hemorrhagic toxins (DaHT-1 and DaHT-2) were isolatated from the venom of D. acutus (from Hunan). they were single polypeptide chain and homogeneous judged by PAGE and SDS-PAGE. These hemorrhagins have similar mol. wts of about 23,500 and their isoelectric point were pH 5.6 and 5.2 for DaHT-1 and DaHT-2, respectively. DaHT-1 and DaHT-2 were glycoprotein contained 11% and 7% carbohydrate, respectively. DaHT-1 was composed of about 217 amino acid residues while DaHT-2 was 203 amino acid residues which contained high Asp and Glu. Both were basic stable but labile to acid and heat. The optimal pH of DaHT-1 and DaHT-2 were same, pH7.0 and 5 ℃ and 40 ℃ were the optimal temperature for DaHT-1 and DaHT-2, respectively. They were strong hemorrhagic toxin with proteolytic activity. EDTA and cysteine could inhibit both hemorrhagic and proteolytic avtivity but Trypsin had no effect, suggesting that the toxins are metalloproteins and difulfide bridge had contribution to the confromation of activities. Analyses for metals indicated that zinc and caium atoms were present in both toxins with roughly 0.5mol zinc and 1 mol caium to 1 mol protein. Circular dichroism (CD) was used to examine the comformation of DaHT-1 and DaHT-2. In the far CD spectrum, it showed a wide negative peak at 217 nm for DaHT-1 and a negative peak at 208 nm with a shoulder peak at 217 nm for DaHT-2. Although there is some indication of a high β-sheet content, it is possible that the aromatic region contributed to this complex spectrum for DaHT-2. The secondary structure contents were calculated by the method of Chen and Yang and for DaHT-1 were found to be 36.9% α-helix, 35.5% β-sheet and 27.6% random-coil. For DaHT-2, 23.4%α-helix, 31.3% β-sheet and 45.3% random-coil. Effect of pH temperature and EDTA on CD spectrum of the hemorrhagin could be found that the conformation of DaHT-1 and DaHT-2 have some changes. As determined by the method of Chen and Yang, the content of α-helis decreased while that random-coil increased. |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6223]  |
| 专题 | 昆明动物研究所_其他 |
| 推荐引用方式 GB/T 7714 | 陶勇. 湖南尖吻蝮(D. acutus)蛇毒出血毒素的纯化、性质和溶液构象研究[D]. 北京. 中国科学院研究生院. 1991. |
入库方式： OAI收割
来源：昆明动物研究所
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