臺灣博碩士論文加值系統

粒線體釋放之細胞色素c (cytochrome c)會引發細胞凋亡(apotosis)內源路徑，而此釋放行為與細胞色素c和心磷脂(cardiolipin)之間交互作用有關；在電子傳遞鏈(electron transport chain)中氧化態細胞色素c及還原態細胞色素c對複合體(complex III and complex IV)的交互作用有所不同，因此了解氧化及還原態細胞色素c與心磷脂之作用機制將有助於解析細胞凋亡路徑。
我們運用最佳化的羧酸基和醇基混合的自組裝單分子層(self-assembled monolayer)靜電吸附細胞色素c，並利用電化學技術分析經心磷脂吸附後之細胞色素c氧化還原反應(redox reaction)，循環伏安圖(cyclic voltammogram)及交流伏安圖(AC voltammogram)指出氧化態細胞色素c與心磷脂作用後均比還原態細胞色素c造成較大的電位負方向偏移，並且氧化態細胞色素c與心磷脂作用後形式電位(19.5 mV和15 mV)和峰值(24.1 mV和9.2 mV)電位差值均比後者還原態細胞色素c大。我們進一步使用石英晶體微天平─耗散監測(Quartz Crystal Microbalance with Dissipation monitoring)系統對細胞色素c和心磷脂之間交互作用進行探討，結果顯示氧化態細胞色素c與心磷脂的結合量(經模擬後以質量表示)大於還原態的兩倍，氧化態細胞色素c與心磷脂的能量耗散也顯示比還原態大；還原態細胞色素c與心磷脂結合後則表現出較大的彈性係數(shear modulus)為µ = 4.325 x 10^3 Pa，經由耗散對頻率圖(D-f plot)中的斜率得知，細胞色素c於第一層吸附作用時單位質量氧化態細胞色素c比還原態造成較大的能量耗散，兩者於開始吸附後的第一分鐘亦有不同行為表現，綜合上述結果，我們推知氧化態細胞色素c（相對於還原態）和心磷脂交互作用後可能呈現較鬆散的結構，例如解折疊(extended)狀態。此結果和近期文獻中所報導的結論呼應，證實石英晶體微天平─耗散監測系統具有依時間解析(time-resolved)氧化態及還原態細胞色素c對於心磷脂之間交互作用的能力，此結果亦助於釐清細胞凋亡的病理學機制。

Cytochrome c (cyt c), a protein inherent with a redox-centered heme in ferric/ferrous states, locates in the mitochondrial intermembrane space in eukaryotic cells. The oxidation state of heme regulates structure of cyt c and its affinity to electron transport complexes, in which the ferric cyt c tends to bind to complex III and the ferrous cyt c associates with complex IV. Approximate 15 % of the positively charged cyt c associates with the mitochondrial cardiolipin (CL) in homeostatic conditions and function as a peroxidase to promote CL oxidation which is referred as an early step towards apoptosis. Although ferric and ferrous cyt c have been shown to form complexes with CL, the exact mechanism leading to apoptosis in connection to oxidation state of cyt c remains unresolved.
We performed electrochemical techniques to interrogate effect of redox switch (cyt c) upon the interaction between cyt c and CL. Cyt c was functionalized on a carboxylic- and hydroxyl-groups terminated self-assembled monolayer on gold electrode by electrostatic and hydrophobic attraction. Cyclic voltammogram reveals that the formal potentials acquired from both ferric and ferrous cyt c shifted to less positive potential (by 19.5 and 15 mV) upon the association with CL. The potential separation of redox waves also increased upon the cyt c-CL association. The results correlated with the conclusion obtained in AC voltammetric experiments wherein the feeric cyt c exhibited a potential shift (24.1 mV), remarkably greater than 9.2 mV of ferrous cyt c, inferring a substantial structural change of ferric cyt c upon the association with CL. A molecular modeling also suggested a more perpendicular orientation of heme-plane (in the adsorbed ferrous cyt c) to the electrode surface.
The cyt c-CL interaction was further interrogated using Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). The mass the ferric cyt c adsorbed onto a phosphatidylcholine (PC)/CL (4:1 in molar ratio) lipid bilayer was 2.4-fold greater than the ferrous one which, however, represented greater shear modulus (by a difference ofµ = 1.44 x 103 Pa) for the binding of cyt c to CL. The D-f plot revealed different slopes given by ferrous and ferric cyt c, presumably ascribed to distinct adsorption kinetics and different affinity to CL. Moreover, we used the D-f plot and the time-resolved with ∆D and ∆f to assume that the effect of the interaction between ferric/ferrous cyt c with CL. Finally we hope to understand deeply the mechanism of the interaction between ferric/ferrous cyt c with CL, which is a cause to pro-apoptosis signal released, that can use into regulation and clinical applications in the future.

謝誌
中文摘要
Abstract
目錄
圖目錄
表目錄
第一章緒論 (Introduction)
1.1細胞色素c (cytochrome c, cyt c)
1.2心磷脂(cardiolipin, CL)
1.3電子傳遞鏈(electron transport chain, ETC)
1.4細胞凋亡(apotosis)與相關疾病
1.5細胞色素c與心磷脂之相互作用
1.6氧化態及還原態細胞色素c
1.7動機
第二章實驗材料與方法
2.1藥品試劑
2.2實驗儀器與材料
2.3電極製備
2.4微脂體 (liposome) 製備
2.5還原態細胞色素c製備
2.6 Bradford蛋白質定量法
2.7紫外可見吸收光譜確認還原態細胞色素c
2.8電化學感測
2.8.1感測氧化/還原態細胞色素c
2.8.2感測心磷脂CL (18:1) 與氧化/還原態細胞色素c的相互作用力
2.9二氧化矽感測晶片實驗前處理
2.10心磷脂CL (18:1)吸附於二氧化矽感測晶片
2.11使用螢光染劑 (10-N-Nonyl acridine orange) 確認心磷脂CL (18:1) 於二氧化矽感測晶片
第三章結果與討論
3.1測定氧化/還原態細胞色素c
3.2自組裝單分子層靜電吸附細胞色素c ─電化學法之鑑定
3.3自組裝單分子層羧酸、醇官能基比例最佳化
3.4自組裝單分子層羧酸官能基長度選擇最佳化
3.5交流伏安法分析氧化態及還原態細胞色素c ─心磷脂交互作用
3.6循環伏安法分析氧化態及還原態細胞色素c ─心磷脂交互作用
3.7石英晶體微天平─耗散監測氧化態及還原態細胞色素c ─心磷脂
3.7.1石英晶體微天平─耗散監測氧化態及還原態細胞色素c ─心磷脂 (質量)
3.7.2石英晶體微天平─耗散監測氧化態及還原態細胞色素c─心磷脂(D-f 圖)
3.8以10-N-nonyl acridine orange (NAO)螢光檢測心磷脂
3.9蛋白質定量還原態細胞色素c
第四章結論
第五章參考文獻

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