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研究生:顏德民
研究生(外文):Teh-ming Yen
論文名稱:利用分析級超高速離心機研究蛇毒毒蛋白之分子結合作用
論文名稱(外文):The Molecular Association of Snake Venom Protein as Studied by Analytical Ultracentrifugation
指導教授:吳文桂
指導教授(外文):Wen-guey Wu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:70
中文關鍵詞:分析級超高速離心機磷脂水解酵素A2寡聚體四級結構沈降速率法
外文關鍵詞:analytical ultracentrifugationphospholipase A2oligomerquaternary structuresedimentation velocitycalcium
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近年的研究顯示磷脂水解酵素A2 (PLA2),具有功能的多樣性。而純化自毒蛇Naja nigricollis毒液中的鹼性PLA2(N-PLA2),同時具有水解磷脂質、溶血、抗凝聚、心臟毒性以及肌肉毒性等藥理作用。由以往對於蛇毒PLA2的觀察之中,被認為在缺乏鈣離子情況下,會產生寡聚體(oligomer)的四級結構。而在本篇論文第一部份裡,運用了分析級超高速離心機,於水溶液條件下,來對鹼性N-PLA2蛋白自體結合(self-association)模式和鈣離子濃度的關連性進行研究,直接證實了鈣離子確實擁有著調控蛇毒N-PLA2四級結構的能力。此外,由過去多項不同的研究及已得晶體結構中,蛇毒PLA2分子被推測具有兩個鈣離子結合位置。在本文中,對於其他二價陽離子所造成N-PLA2的不同四級結構變化,進行了系列的實驗,並進而得知二價陽離子結合於N-PLA2的本質結合位置(essential site),極為可能扮演著調控N-PLA2四級結構的功能。

此論文中第二部份,在探討心臟毒素與肝素之間的結合模式。心臟毒素是眼鏡蛇毒蛋白的主要成分,當這種多正電荷的毒蛋白接近標的細胞時,細胞表面的醣類分子可能扮演了重要的調控角色。過去,我們實驗室曾分析過醣胺素與心臟毒素的作用並提出兩者結合的特異性。而在策略上,則運用了酵素分解肝素使之成為短鍊片段,並透過不同的官能基再加以純化區分,用以模擬細胞表面高含量的硫酸乙醯肝素上高度硫酸化區域,並藉此希望瞭解不同長度或特定官能基的醣鏈,對蛇毒蛋白的相互作用情形。由蛇毒蛋白A3或Tr與肝素的異體結合(hetero-association)模式實驗中,在方法上,使用了吸收圖譜的沈降速率法,來追蹤蛋白分子流體力學數值的改變,由此得到巨分子在溶液中分子量的變化情形,進而瞭解蛋白與肝素間可能的結合模式。期望藉著心臟毒素與不同長度或特定官能基肝素間的結合模式,能帶來兩分子在水溶液下的相互作用資訊。
Ⅰ.緒論…………………………………………………………1
1.1蛇毒磷脂水解酵素………………………………………1
1.1.1蛇毒…………………………………………………1
1.1.2磷脂水解酵素A2……………………………………2
1.1.3鈣離子在磷脂水解酵素A2的結合位置和功能……3
1.2心臟毒素…………………………………………………4
1.2.1心臟毒素……………………………………………4
1.2.2心臟毒素的生理特性和可能機制…………………5
1.3醣胺素……………………………………………………6
1.3.1關於醣胺素…………………………………………6
1.3.2醣胺素與蛋白的相互作用…………………………7
1.4研究目的…………………………………………………9
圖表…………………………………………………………….12

Ⅱ.實驗材料及方法……………………………………………22
2.1實驗材料…………………………………………………22
2.1.1蛇毒磷脂水解酵素A2及心臟毒素的製備…………22
2.1.2不同片段醣類分子的製備…………………………23
2.1.3蛋白質與醣類分子濃度之測定…………………………23
2.2分析級超高速離心機……………………………………24
2.2.1實驗裝置……………………………………………24
2.2.2沉降速率方法與原理………………………………25
2.2.3沉降平衡方法與原理………..................26
2.2.4連續尺寸分布分析法的應用及原理………………27
2.3酵素活性方法所得PLA2與不同二價陽離子的關連性…28
圖表…………………………………………………………….31

Ⅲ.實驗結果……………………………………………………38
3.1磷脂水解酵素A2的自體結合作用………………………38
3.1.1鈣離子對N-PLA2四級結構的影響…………………38
3.1.2不同二價陽離子對N-PLA2四級結構的影響…………..41
3.2肝素與心臟毒素的結合模式……………………………42
3.2.1不同長度肝素片段與心臟毒素A3的結合模式………..42
3.2.2完全硫酸化肝素與心臟毒素A3的結合模式………44
3.2.3檸檬酸對心臟毒素-肝素複合體結合模式的影響.44
圖表…………………………………………………………….46

Ⅳ.討論…………………………………………………………59
圖表…………………………………………………………….61
Ⅴ.參考資料……………………………………………………63
Ⅵ.附錄…………………………………………………………69
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