台灣眼鏡蛇以地區分為東岸和西岸眼鏡蛇，東岸眼鏡蛇體型較大且蛇腹全黑，西岸體型較小且蛇腹為白色或灰色，先前研究發現，東西岸眼鏡蛇心臟毒素也有地區上的分別，西岸白腹眼鏡蛇毒液中含有較多的心臟毒素A2和A4，但缺少A6，這一發現在東岸黑腹眼鏡蛇毒液則剛好相反。
陰離子檸檬酸是毒液中的主要成分，大約有50 mM的檸檬酸在眼鏡蛇毒液中，檸檬酸對毒性的作用除了對毒液中毒素的陽離子依賴的抑制作用以外，其他作用了解甚少。先前的研究發現，檸檬酸離子結合到心臟毒素三指環II區末端的Lys-23和Lys-31位置穩定了心臟毒素A3二聚體結構，並且在功能重要的環I和II區疏水接觸，根據序列比對觀察，發現A3和A6都有Lys-23和Lys-31的保留序列，但A2和A4沒有Lys-31的序列，取而代之的是Thr-31，因此我們想知道在檸檬酸緩衝液下，肝素對於黑腹、白腹心臟毒素的交互作用中，是否讓黑腹心臟毒素會比白腹心臟毒素在細胞膜滯留中扮演更重要的角色。
我們的研究結果表明，在表面電漿共振的實驗中顯示在檸檬酸緩衝液的情況下，黑腹心臟毒素在肝素的滯留會比白腹心臟毒素強大約六倍，結果說明Lys-23和Lys-31對於肝素的滯留確實有影響，另外，我們從高效液相層析儀分析滯留於肝素的黑、白腹心臟毒素，發現所有心臟毒素中，A3有最明顯的滯留現象，進一步從十二烷基硫酸鈉聚丙烯醯胺凝膠電泳觀察，發現加入交聯劑反應之後，心臟毒素A3-A6有檸檬酸異二聚體的現象，此現象比心臟毒素A2-A3和A3-A4更加明顯；在細胞實驗中顯示，白腹心臟毒素對於黑腹心臟毒素有更強的細胞毒性，而加入檸檬酸組又比沒有加檸檬酸組有更高的細胞致死率，另一方面，隨著檸檬酸濃度的增加細胞死亡率也跟著提高；結論，檸檬酸會明顯誘導黑腹心臟毒素二聚體的形成，且檸檬酸和心臟毒素作用後造成細胞死亡的更多，顯示細胞死亡可能和二聚體結構有關係，也可以發現黑、白腹心臟毒素細胞致死機制完全不同。

Taiwan Cobra (Naja atra) is distributed based on eastern and western regions. Eastern cobra (bCTXs) is larger in size and fully black on its belly, whereas western cobra (wCTXs) is smaller and belly is white or gray. In the previous study from our lab, we found that east and west cobra cardiotoxin also have regional difference. West cobra venom contains more of cardiotoxin (CTX) A2 and A4, but lacks A6.
Anionic citrate is one of a major component of cobra venom and around 50 mM citrate is present in Taiwan cobra venom. In cobra venom, effect of citrate on toxicity is poorly understood than inhibitory effect on cation-dependence action from venom toxins. In previous study, a citrate ion bound to Lys-23 and Lys-31 near tip of loop II stabilizes CTX-A3 homodimer and hydrophobic contact of functionally important loop I and II regions. Sequence alignment revels that both Lys-23 and Lys-31 are conserved residues in A3 and A6 (bCTXs) but not for A2 and A4 (wCTXs). The interaction of glycosaminoglycan and CTX in citrate might play an important role in cell membrane retention for bCTXs than wCTXs.
Our results shows that bCTXs is about six times stronger than wCTXs in retention of heparin surface when added citrate in surface plasmon resonance(SPR), indicating that Lys-23 and Lys-31 have an impact for retention. From SDS-PAGE, we observed that adding cross-linking reagent and citrate induced heterodimer in CTX A3-A6 which was more evident than CTX A2-A3 and CTX A4-A3 heterodimer.
Further retention of heparin surface in bCTXs and wCTXs are analyzed from reverse phase high performance liquid chromatography and we found that A3 had most significant retention in all CTXs. In cell viability assay, wCTXs is more cytotoxicity than bCTXs and citrate group have higher cell death than non-citrate group. On the other hand, increase in citrate concentration leads to improve in cell death. These results suggest that citrate is involved significantly to induce the formation of dimerization in bCTXs which can lead to more cell death. The dimerization in cardiotoxins might suggest relationships with cell death. We also found wCTXs and bCTXs cell death mechanism is completely different.

目錄
第一章 緒論
1-1 台灣眼鏡蛇…………………………………………………………….......1
1-2 眼鏡蛇毒簡介……………………………………………………………...1
1-3 黑、白腹台灣眼鏡蛇毒之差異……………………………………….…..2
1-4 心臟毒素…………………………………………………………………...3
1-5 肝素………………………………………………………………………...4
1-6 表面電漿共振……………………………………………….……………..4
1-7 蛇毒中檸檬酸角色與能量代謝途徑……………………………….……..5
1-8 心臟毒素A3二聚體結構與肝素、檸檬酸形成複合物…………………7
1-9 研究目的…………………………………………………………….……..7
第二章 材料與方法
2-1 材料準備………………………………………………………….………12
2-2 實驗過程……………………………………………………………….…12
2-2-1 純化白腹眼鏡蛇之心臟毒素………………………………….………12
2-2-2 純化黑腹眼鏡蛇之心臟毒素…………………………………...…..…12
2-2-3 逆相高效液相層析 ……………………………………………..…….13
2-2-4 心臟毒素濃度定量……………………………………………...……..14
2-2-5 表面電漿共振 肝素晶片…………………………………………...…14
2-2-6 交聯試劑…………………………………………………………….....15
2-2-7 細胞存活率檢測…………………………………………………..…...16
第三章 結果
3-1 純化白腹眼鏡蛇之心臟毒素 ……………………………………….…..17
3-2 純化黑腹眼鏡蛇之心臟毒素 …………………………………...………17
3-3 表面電漿共振 肝素晶片……………………………………………...…17
3-4 黑、白腹心臟毒素-肝素複合體在檸檬酸緩衝液下的影響…………....18
3-5 肝素管柱分析有較強親和性之心臟毒素……….. ………..……...…….19
3-6 交聯試劑觀察心臟毒素之二聚體和三聚體結構之形成.………………19
3-7 有無檸檬酸條件下，黑、白腹心臟毒素細胞毒性…………………….…20
第四章 討論
4-1 黑、白腹心臟毒素毒性差異…………………………………….………..31
4-2 蛇毒中檸檬酸的角色與檸檬酸對於細胞致死之影響………….………31
4-3 黑、白腹眼鏡蛇地域上差異…………………………..……………….…32
4-4 黑、白腹眼鏡蛇神經毒素差異…………………………………...………33
4-5 心臟毒素和肝素之強弱親和性分析………………………….…………33
4-6 影響肝素表面滯留原因………………………….………………………33
4-7 不同緩衝液之影響於黑、白腹心臟毒素……………………………...…34
4-8 抑制劑抑制檸檬酸進入細胞質之細胞膜轉運蛋白..………………...…34
第五章 結論……………………………………………………………………..…38
第六章 參考文獻……………………………………………………………..……39

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