細菌視紫質為一存在於嗜鹽古生菌Halobacterium salinarum細胞膜上的跨膜蛋白，其受光激發後會進行光迴圈反應，在細胞膜內外側產生氫離子濃度梯度，具備質子幫浦的功能，而此古生菌得利用此化學勢位合成生物生存所需的三磷酸腺苷。先前不乏以電化學方法對細菌視紫質進行質子幫浦的研究，但分析方法除了涵蓋複雜的假設外，亦隨樣品製程方式不同而有差異。本論文利用瞬態可見光吸收光譜技術以及電化學裝置觀察pH 6.3－8.1之間，紫膜溶液中細菌視紫質基態回復的速率以及質子幫浦行為，並建立一個概念簡單的動力學模型描述以連續光激發細菌視紫質所觀察到的電流訊號。
由於細菌視紫質其光誘發質子傳遞的特性，電化學裝置可偵測到溶液中瞬間質子濃度變化。在連續光源激發下，紫膜溶液產生的質子濃度差可由一系列的瞬態氫離子濃度差擬合。當細菌視紫質被光激發後須完成光迴圈反應才能回到基態再次受激發，因此隨著時間有效細菌視紫質濃度逐漸減少，瞬態氫離子濃度差之振幅也隨之遞減。吾人使用瞬態可見光吸收光譜觀察細菌視紫質光迴圈完成之速率並搭配實驗條件的評估，判斷細菌視紫質隨時間遞減的程度。接著以累加一系列瞬態氫離子濃度差的方式，得到氫離子隨時間的總濃度差，最後將氫離子總濃度差微分後可得光電流。吾人利用上述概念所建構之動力學模型適解pH 6.3－8.1範圍內的光電流訊號，不僅得到與實驗相似的波形，其質子幫浦動力學參數也與文獻中Kuo及Chu利用脈衝光激發之電流模型所獲得結果一致。故吾人利用本實驗以細菌視紫質為例，證明利用一連串瞬態事件的組合可用於分析連續激發下的現象。

The proton pump activity of bacteriorhodopsin in aqueous solution upon excitation with modulated continuous light was monitored electrochemically and analyzed by superimposing a series of transient proton translocation events Hi+(t). An evolution function , including a decay and a stationary offset, was introduced to weight the contribution of the individual transient events evolving with time in the envelope of the steady-state event. The evolution of the total proton concentration can be treated as an ensemble of weighted sequential transient events, , and the temporal profile of the photocurrent is derived by differentiating the proton concentration with respect to time, . The temporal profiles of the bacteriorhodopsin photocurrent in pH range 6.3–8.1 were analyzed using a well-defined kinetics model and restricted mathematical formulization, and fitted temporal behaviors agreed with the observations. This successful proof-of-concept study on analyzing a steady-state phenomenon using an ensemble of sequential transient events can be generalized to quantify other phenomena upon continuous stimulation, such as estimation of light-driven ion pump activities of the photosynthetic proteins upon illumination.

第一章 緒論
1.1 前言
1.2 文獻回顧與實驗動機
1.2.1 嗜鹽古細菌、紫膜及細菌視紫質的發現與研究
1.2.2 實驗動機與目的
第二章 細菌視紫質與紫膜的性質
2.1 細菌視紫質結構
2.2 紫膜的組成及結構
2.3 紫膜的光譜特性
2.4 細菌視紫質的光迴圈特性
2.5 質子幫浦
2.5.1 細菌視紫質的質子幫浦通道
2.5.2 紫膜質子幫浦行為與周圍脂質關係
2.6 紫膜的光迴圈、質子幫浦行為與環境pH值的相依性
2.7 紫膜的光致電流特性
2.7.1 脈衝雷射激發
2.7.2 連續光激發
2.8 光致電流理論模型
第三章 實驗技術原理簡介、儀器架設與實驗步驟
3.1 實驗技術原理簡介
3.1.1 穩態可見光吸收光譜原理
3.1.2 瞬態吸收光譜原理
3.1.3 光致電流偵測系統原理
3.2 儀器架設
3.2.1 穩態紫外光/可見光吸收光譜儀
3.2.2 單波長可見光瞬態吸收光譜儀
3.2.3 光致電流偵測系統
3.2.4 電子快門響應時間
3.3 樣品製備
3.3.1 嗜鹽菌的培養方法
3.3.2 紫膜純化處理
3.3.3 實驗樣品的製備
3.3.4 實驗藥品
3.4 儀器參數設定
3.4.1 紫外光可見光光譜儀
3.4.2 單波長瞬態吸收光譜儀
3.4.3 光致電流系統
第四章 實驗結果與討論
4.1 紫膜的瞬態吸收光譜
4.2 紫膜的光致電流
4.2.1 雜訊處理
4.2.2 連續光源下之光致電流動力學模型
4.2.2.1 單一事件中質子濃度的時間側寫
4.2.2.2 有效濃度遞減函數
4.2.2.3 質子總濃度差在調制連續光激發下的時間側寫
4.2.2.4 光電流在調制連續光激發下的時間側寫
4.2.2.5 紫膜的光致電流與pH相依性
4.2.2.6 光致電流動力學速率常數分析
第五章 結論
附錄
1.1 傅立葉轉換
1.2 離散傅立葉變換
1.3 快速傅立葉變換
2.1 簡化速率常數k1
3.1 紫膜的光致電流與激發光能量相依性
4.1 紫膜溶液在不同pH環境中的紫外光/可見光吸收光譜

[1] Vsevolodov, N. N., Biomolecular Electronics: An Introduction Via Photosensitive Proteins. Birkhauser Boston: 1998; p 3.
[2] Stoeckenius, W.; Kunau, W. H. J. Cell Biol. 1967, 34, 365–393.
[3] Lanyi, J. K., Bacteriorhodopsin, Chemistry of. In Wiley Encyclopedia of Chemical Biology, Begley, T. P., Ed. John Wiley & Sons, Inc.: 2007.
[4] Lanyi, J. K. Biochim. Biolphys. Acta 2006, 1757, 1012–1018.
[5] Stoeckenius, W.; Rowen, R. J. Cell Biol. 1967, 34, 365–393.
[6] Oesterhelt, D.; Stoeckenius, W. Nat. New. Biol. 1971, 233, 149–152.
[7] Oesterhelt, D.; Stoeckenius, W. Proc. Nat. Acad. Sci. U.S.A. 1973, 70, 2853–2857.
[8] Stoeckenius, W.; Bogomolni, R. A. Annu. Rev. Biochem. 1982, 51, 587–616.
[9] Smith, S. O.; Pardoen, J. A.; Mulder, P. P. J.; Curry, B.; Lugtenburg, J.; Mathies, R. A. Biochemistry 1983, 22, 6141–6148.
[10] Sharkov, A. V.; Pakulev, A. V.; Chekalin, S. V.; Matveetz, Y. A. Biochim. Biophys. Acta 1985, 808, 94–102.
[11] Gerwert, K.; Souvignier, G.; Hess, B. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 9774–9778.
[12] Lanyi, J. K. Annu. Rev. Physiol. 2004, 66, 665–688.
[13] Garczarek, F.; Gerwert, K. Nature 2006, 439, 109–112.
[14] Mathies, R.; Brito Cruz, C.; Pollard, W.; Shank, C. Science 1988, 240, 777–779.
[15] Neutze, R.; Pebay-Peyroula, E.; Edman, K.; Royant, A.; Navarro, J.; Landau, E. M. Biochim. Biophys. Acta 2002, 1565, 144–167.
[16] Mathies, R.; Brito Cruz, C.; Pollard, W.; Shunk, C. Science 1988, 240, 777–779.
[17] Rothschild, K. J.; Zagaeski, M.; Cantore, W. A. Biochem. Biophys. Res. Commun. 1981, 103, 483–489.
[18] Degroot, H. J. M.; Harbison, G. S.; Herzfeld, J.; Griffin, R. G. Biochemistry 1989, 28, 3346–3353.
[19] Henderson, R.; Unwin, P. N. T. Nature 1975, 257, 28–32.
[20] Blaurock, A. E. J. Mol. Biol. 1975, 93, 139–158.
[21] Khorana, H. G.; Gerber, G. E.; Herlihy, W. C.; Gray, C. P.; Anderegg, R. J.; Nihei, K.; Biemann, K. Proc. Natl. Acad. Sci. USA 1979, 76, 5046–5050.
[22] Chen, Z. P.; Birge, R. R. Trends Biotechnol. 1993, 11, 292–300.
[23] Vsevolodov, N. N.; Ivanitsky, G. R. Biofizika 1985, 30, 883–887.
[24] Brauchle, C.; Hampp, N.; Oesterhelt, D. Adv. Mater. 1991, 3, 420–428.
[25] Zhao,S.; Cunha, C.; Zhang, F.; Liu, Q.; Gloss, B.; Deisseroth, K.; Augustine, G.J.; Feng, G. Brain Cell Biol. 2008, 36, 141–154.
[26] Zhang, F.; Wang, L.P.; Boyden, E. S.; Deisseroth, K. Nat. Methods 2006, 3, 785–792.
[27] Boyden, E. S.; Zhang, F.; Bamberg, E.; Nagel, G.; Deisseroth, K. Nat. Neurosci. 2005, 8, 1263–1268.
[28] Deisseroth, K. Nat. Methods 2011, 8, 26–29.
[29] Miyasaka, T.; Koyama, K. Thin Solid Films 1992, 210, 146–149.
[30] Koyama, K.; Yamaguchi, N.; Miyasaka, T. Science 1994, 265, 762–765.
[31] Keszthelyi, L. Biochim. Biophys. Acta 1980, 598, 429–436.
[32] Keszthelyi, L.; Ormos, P. FEBS Lett. 1980, 109, 189–193.
[33] Wang, J. P.; Yoo, S. K.; Song, L.; El-Sayed, M. A. J. Phys. Chem. B 1997, 101, 3420–3423.
[34] Wang, J. P.; Song, L.; Yoo, S. K.; El-Sayed, M. A. J. Phys. Chem. B 1997, 101, 10599–10604.
[35] Chu, L. K.; Yen, C. W.; El-Sayed, M. A. Biosens. Bioelectro. 2010, 26, 620–626.
[36] Trissl, H. W.; Montal, M. Nature 1977, 266, 655–657.
[37] Grzesiek, S.; Dencher, N. A. FEBS Lett. 1986, 208, 337–342.
[38] Heberle J., Riesle J., Thiedemann G., Oesterhelt D., Dencher N.A. Nature 1994, 370, 379–382.
[39] Scherrer, P.; Alexiev, U.; Marti, T.; Khorana, H. G.; Heyn, M. P. Biochemistry 1994, 33, 13684–13692.
[40] Drachev, L. A.; Kaulen, A. D.; Skulachev, V. P. FEBS Lett. 1978, 87, 161–167.
[41] Robertson, B.; Lukashev, E. P. Biophys. J. 1995, 68, 1507–1517.
[42] Miyasaka, T.; Koyama, K.; Itoh, I. Science 1992, 255, 342–344.
[43] Braun, D.; Dencher, N. A.; Fahr, A.; Laindau, M.; Heyn, M. P. Biophys. J. 1988, 53, 617–621.
[44] Keszthelyi, L.; Ormos., P. Biophys. Chem. 1983, 18, 397–405.
[45] Hong, F. T. Prog. Surf. Sci.1999, 62, 1–237.

[46] Hong, F. T. Biosystems 1986, 19, 223–236.
[47] Miyazaki, S.; Matsumoto, M.; Brier, S. B.; Higaki, T.; Yamada, T.; Okamoto, T.; Ueno, H.; Toyabe, S.; Muneyuki, E. Eur. Biophys. J. Biophys. Lett. 2013, 42, 257–265.
[48] Hong, F. T. Photochem. Photobiol. 1976, 24, 155–189.
[49] Hong, F. T. Mater. Sci. Eng. C 1997, 4, 267–268.
[50] Birge, R. R. Annu. Rev. Biophys. Bioeng. 1981, 10, 315–354.
[51] Leucke, H.; Schobert, B.; Richter, H. T.; Cartailler, J. P.; Lanyi, J. K. J.Mol. Biol. 1999, 291, 899–911.
[52] Blaurock, A. E.; Stoeckenius, W. Nat. New. Biol. 1971, 233, 152–155.
[53] Henderson, R. J. Mol. Biol. 1975, 93, 123–138.
[54] Mukhopadhyay, A. K.; Bose, S.; Hendler, R. W. Biochemistry 1994, 33, 10889–10895.
[55] Becher, B.; Tokunaga, F.; Ebrey, T. G. Biochemistry 1978, 17, 2293–2300.
[56] Kalisky, O.; Feitelson, J.; Ottolenghi, M. Biochemistry 1981, 20, 205–209.
[57] Rehorek, M.; Heyn, M. P. Biochemistry 1979, 18, 4977–4983.
[58] Birge, R. R.; Schulten, K.; Karplus, M. Chem. Phys. Lett. 1975, 31, 451–454.
[59] Balashov, S. P.; Imasheva, E. S.; Govindjee, R.; Ebrey, T. G. Biophys. J. 1996, 70, 473–481.
[60] Lozier, R. H.; Bogomolni, R. A.; Stoecknius, W. Biophys. J. 1975, 15, 955–962.
[61] Stoeckenius, W.; Lozier, R. H. J. Supramol. Struct. 1974, 2, 769–74.
[62] Scherrer, P.; Mathew, M. K.; Sperling, W.; Stoeckenius, W. Biochemistry 1989, 28, 829–834.
[63] Bennett, J. A.; Birge, R. R. J. Chem. Phys. 1980, 73, 4234–4246.
[64] Lanyi, J. K. J. Struct. Biol. 1998, 124, 164–178.
[65] Gergely, C.; Zimányi, L.; Váró, G. J. Phys. Chem. B, 1997, 101, 9390–9395.
[66] Harbison, G. S.; Mulder, P. P. J.; Pardoen, H.; Lugtenburg, J.; Herzfeld, J.; Griffin, R. G. J. Am. Chem. Soc. 1985, 107, 4810–4816.
[67] Wada, M.; Sakurai, M.; Inoue, Y.; Tamura, Y.; Watanabe, Y. J. Am. Chem. Soc. 1994, 116, 1537–1545.
[68] Schobert, B.; Cupp-Vickery, J.; Hornak, V.; Smith, S. O.; Lanyi, J. L. J. Mol. Biol. 2002, 321, 715–726.
[69] Beppu, Y.; Kakitani, T. Photochem. Photobiol. 1994, 59, 660–669.
[70] Birge, R. R.; Gillespie, N. B.; Izaguirre, E.W.; Kusnetzow, A.; Lawrence, A. F.; Singh, D.; Song, Q. W.; Schmidt, E.; Stuart, J. A.; Seetharaman, S.; Wise, K. J. J. Phys. Chem. B 1999, 103, 10746–10766.
[71] Richter, H. T.; Needleman, R.; Kandori, H.; Maeda, A.; Lanyi, J. K. Biochemistry 1996, 35, 15461–15466.
[72] Lanyi, L. K. Biochem. Biophys. Acta 1993, 1183, 241–261.
[73] Zimányi, L.; Váró, G., Chang, M.; Ni, B.; Needleman, R.; Lanyi. J. K. Biochemistry 1992, 31, 8535–8543.
[74] Morgan, J. E.; Vakkasoglu, A. S.; Lanyi, J. K.; Gennis, R. B.; Maeda, A. Biochemistry 2010, 49, 3273–3281.
[75] Lanyi, J. K. A Structure View of Proton Transfer by Bacteriorhodopsin. In Biophysical and Structural Aspects of Bioenergetics, Wikstrom, M., Ed. The Royal Society of Chemistry: 2005; pp 227–248.
[76] Hendler, R. W.; Dracheva, S. Biochemistry-Moscow 2001, 66, 1311–1314.
[77] Sternberg, B.; L'Hostis, C.; Whiteway, C. A.; Watts, A. Biochim. Biophys. Acta 1992, 1108, 21–30.
[78] Joshi, M. K.; Dracheva, S.; Mukhopadhyay, A. K.; Bose, S.; Hendler, R. W. Biochemistry 1998, 37, 14463–14470.
[79] Hendler, R. W.; Barnett, S. M.; Dracheva, S.; Bose, S.; Levin, I. W. Eur. J. Biochem. 2003, 270, 1920–1925.
[80] Heberle, J. Biochim. Biophys. Acta 2000, 1458, 135–147.
[81] Mowery, P. C.; Lozier, R. H.; Chae, Q.; Tseng, Y. W.; Tayor, M.; Stoeckenius, W. Biochemistry 1979, 18, 4100–4107.
[82] Subramaniam, S.; Marti, T.; Khorana, H. G. Proc. Nat. Acad. Sci. U. S. A. 1990, 87, 1013–1017.
[83] Balashov, S. P. Biochim. Biophys. Acta 2000, 1460, 75–94.
[84] Ludmann, K.; Gergely, C.;Váró, G. Biophys. J. 1998, 75, 3110–3119.
[85] Váró, G.; Lanyi, J. K. Biophys. J. 1989, 56, 1143–1151.
[86] Bressler, S.; Friedman, N.; Li, Q.; Ottolenghi, M.; Saha, C.; Sheves, M. Biochemistry 1999, 38, 2018–2025.
[87] Liu, S. Y.; Govindjee, R.; Ebrey, T. G. Biophys. J. 1995, 68, 1507–1517.
[88] Okajima, T. L.; Hong, F. T. Biophys. J. 1986, 50, 901–912.
[89] Simmeth, R.; Rayfield, G. W. Biophys. J. 1990, 57, 1099–1101.
[90] Lu, T.; Li, B. F.; Jiang, L; Rothe, U.; Bakowsky, U. J. Chem. Soc.-Faraday Trans. 1998, 94, 79–81.
[91] Tamogami, J.; Kikukawa, T.; Miyauchi, S.; Muneyuki, E.; Kamo, N. Photochem. Photobiol. 2009, 85, 578–589.
[92] Ormos, P.; Hristova, S.; Keszthelyi, L. Biochim. Biophys. Acta 1985, 809, 181–186.
[93] Liu, S. Y. Biophys. J. 1990, 57, 943–950.
[94] Kuo, C. L.; Chu, L. K. Bioelectrochem. 2014, 99, 1–7.
[95] Tkachenko, N. V., Steady State Absorption Spectroscopy. In Optical Spectroscopy: Methods and Instrumentations, Elservier Science: 2006; p 90.
[96] Tkachenko, N. V., Flashphotolysis. In Optical Spectroscopy: Methods and Instrumentations, Elservier Science: 2006; p 130.
[97] Oesterhelt, D.; Stoeckenius, W. Methods Enzym. 1974, 31, 667–678.
[98] USB Optical Bench Options
http:// www.oceanoptics.com/products/benchoptions_usb4.asp
[99] 才富國; 朱立岡. 嗜鹽古細菌H. marismortui之雙細菌視紫質系統的光化學反應–HmbRⅠ與HmbRⅡ. 國立清華大學, 新竹市, 2014.
[100] Rohr, M.; Gäertner, W.;Schweitzer, G.;Holzwarth, A. R.; Braslavsky, S. E. J. Phys. Chem. 1992, 96, 6055–6061.
[101] Birgham, E. O. The Fast Fourier Transform, Prentice Hall: 1974.