自台灣眼鏡蛇毒(Naja naja atra) 純化的心臟毒素(cardiotoxins)，在文獻報導中指出具有六種isoforms，本論文以cardiotoxin 3 (CTX3)作為實驗材料。CTX3是由60個胺基酸所組成的polypeptide-chain，並有四對雙硫鍵構成的三個指環結構(Three-finger loops)。CTX3會造成紅血球的溶血反應，並在人工脂質體上聚集成多聚體而形成類似通道的孔洞使脂質體內含物釋出。因此本論文將探討何種因素影響CTX3在細胞膜上的作用。首先以Methyl-β-cyclodextrin (MβCD)將紅血球細胞上的膽固醇予以剔除後觀察到CTX3對於紅血球細胞的破膜能力有明顯上升。而脂質體中加入膽固醇，發現CTX3結合到脂質體的量與破膜能力都有顯著的下降。對CTX3的離胺酸進行Guanidination (Gu-CTX3)化學修飾後再對與氮端氨基進行Trinitrobenzoylation (TNP-Gu-CTX3)修飾，對於脂質體無論有無膽固醇，其結合量與破膜能力都無太大改變。這些蛋白質的結合量會隨著環境的鹽類濃度增加而減少；而破膜能力也和上述結果一致，會隨著鹽類濃度增加而減少。但CTX3不同於Gu-CTX3和TNP- Gu-CTX3，其破膜能力與聚集情形在1 M鹽類離子環境下幾乎完全消失。FTIR spectra結果顯示在有或無膽固醇的脂質體上，此三種蛋白質的構型都有所不同。綜合上述結果，CTX3在膜上的構型會受到膽固醇以及化學修飾離胺酸而影響其活性。

Taiwan cobra (Naja naja atra) cardiotoxins, are composed of 60 amino acids and structurally stabilized by four disulfide bonds to form three loops structure. Cardiotoxins induced hemolysis of red blood cells and form a channel-like structure in inducing phospholipid vesicles leakage. The aim of the present study is to explore the events affect the membrane-damaging activity of cardiotoxin 3 (CTX3). Depletion of cholesterol in membrane of red blood cells using methyl-β-cyclodextrin (MβCD) enhanced the hemolytic activity of CTX3. Expectedly, CTX3 showed a low binding capability and lower membrane-damaging activity toward phospholipid vesicles containing cholesterol. Guanidinated of lysine residues and selective trinitrobenzoylation (TNP) at N-terminus of guanidinated CTX3 (Gu-CTX3) insignificantly altered binding capability and membrane-damaging activity of CTX3 regardless of phospholipid vesicles containing cholesterol. The binding of CTX3, Gu-CTX3 and TNP-Gu-CTX3 with phospholipid vesicles was reduced by increasing NaCl concentration. Consistent with these observations, membrane-damaging activity of CTX3, Gu-CTX3 and TNP-Gu-CTX3 were also decreased with increasing NaCl concentration. Unlike that of Gu-CTX3 and TNP-Gu-CTX3, membrane-damaging activity and membrane-induced oligomerization of CTX3 was completely abolished by 1 M NaCl. Fourier transform infrared spectroscopy indicated that CTX3, Gu-CTX3 and TNP-Gu-CTX3 adopted different conformation on binding with phospholipid vesicles and phospholipid/cholesterol vesicles. Taken together, our data indicate that membrane-bound conformation of CTX3 is affected by cholesterol and guanidination of lysine residues.

目錄 1
摘要 2
縮寫表 4
前言 5
實驗材料 12
研究方法與材料 13
結果 22
討論 31
參考文獻 37
圖 44

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