電化學及石英晶體微天平─耗散監測分析法探討氧化/還原態細胞色素c和心磷脂交互作用之研究

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题名:	電化學及石英晶體微天平─耗散監測分析法探討氧化/還原態細胞色素c和心磷脂交互作用之研究
其它题名:	Interaction between Ferric/Ferrous Cytochrome c and Cardiolipin: An Electrochemical and Quartz Crystal Microbalance with Dissipation Study
作者:	孫心慈 Sun, Sin-Cih
贡献者:	莊旻傑 Chuang, Min-Chieh 化學系
关键词:	細胞色素c;心磷脂;電化學;石英晶體微天平─耗散監測;循環伏安法;交流伏安法 Cytochrome c;Cardiolipin;Electrochemistry;Quartz Crystal Microbalance with Dissipation;Cyclic voltammetry;AC voltammetry
日期:	2015
上传时间:	2015-12-15T07:43:03Z (UTC)
摘要:	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.
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