一、利用電子激發態分子探測釕金屬修飾蛋白質內的長距離電子傳遞及檢測十二烷基硫酸鈉的濃度二、七種台灣精油的化學組成及對大腸桿菌的抗菌效果

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(1)國立臺灣師範大學化學系博士論文. 指導教授：張一知博士. (I) Probing long range electron transfer in ruthenium modified proteins and evaluating the SDS concentration by using electronically excited molecules (II) Essential oils from Taiwan: chemical composition and antibacterial activity against Escherichia coli. 研究生：林柏成中華民國一百零五年十一月.

(2) 誌謝首先感謝張一知老師多年來的教導，沒有她也就沒有這本論文的出現，還有實驗室的學長姊弟妹及助理(依純,千瑋,益維,昀達,宏裕,一宏,茲嶸,恆毅,以文,彥宏, 忠達,坤陽,家惠,弼豊,哲宇,瑩翰,筱婷,家昇,家翎,郁凰,峻文,哲雄,靖雯,延展,積瑞, 琍敏,恩涵,俊良,昱凡,貞瑋,慧群)，謝謝大家這段期間的照顧。四位口試委員(洪偉修老師、葉怡均老師、清大化學蔡易州老師及陳建添老師)在口試中對我的論文提出了很多有用的意見，非常感謝他們的指教。再來感謝師大化學系的每位師長還有謝明惠 lab(繆佳曄學長,朱晏頤學姊, 林建男,邱蓉怡,陳思瑋,簡立慈,黃宗毅,邢凱捷,林俞君,余家齊)、簡敦誠 lab(何智豊學長,張永育學長,史諭樵學長,林豪俊,廖振傑,吳元仁,馬義翔,林建邦,吳啟誠, 楊雅媖,陳家蓉,王建泓)、李位仁 lab(汪子立學長,趙宏杰學長,江建緯學長,鄭惠文, 陳許志勇,周彥甫,溫淑如,陳虹伶)、林文偉 lab(徐祥恩,蔡乙鈴,陳科維,陳昱彰, 李玉婷,楊玫春,李家睿,許家寧,陳錡翰,張耕華)、林震煌 lab(林鉅逢,林建宏,賀怡珊, 鄭元凱,張嘉芸,陳冠甫,吳卉馨)、洪偉修 lab(張晉豪,莊培佑,林靖衛,黃彥翔, 陳誌濠)、王忠茂 lab(黃祥盈,韓岳樺,邱柏豪)、陳家俊 lab(王迪彥學長,李政宏)、黃文彰 lab(游岳寧)、王禎翰 lab(毛永祥,張軒誌)、陳焜銘 lab(管軒浩,張惠茹, 葉倫輔)、姚清發 lab(王振權)、葉名倉 lab(白蕙棻學姊,林欣慧,李冠儀)、吳學亮 lab(陳俊志)、陳炳宇 lab(姜博仁,藍國峻,簡佑芩,辜大維)、系上助教(顏碧秀, 郭依婷,鄭雅純,龐玉珍,陳美玲,何秋慧,賴怡旬,林彥慧,邢泰莉,張靜芬,林懿雯)及技士(姜大鵬,戴興堂,陳夢麒)，還有許多人，恕我無法一一點名，謝謝大家在這段時間的幫忙與鼓勵。謝謝師大生科系的王玉麒老師及 lab(汪宗明,詹豐碩,李卓然)在抗菌實驗上的幫忙與指點，王震哲老師及 lab 幫忙製作植物標本。謝謝送貨員(林誠意)每天辛苦幫我們送大桶 solvent、工程師(張葉程,王興詩, 郭志秉)隨傳隨到幫我們維修儀器、業務(林秋仲,陳孟甫)一邊推銷一邊打屁。.

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(4) Abstract Ruthenium bipyridine-type compounds, [Ru((CH3)2bpy)2(im)2]2+ and [Ru((COO−)2bpy)2(im)2]2−, were synthesized to evaluate the protein electron transfer property by flash-quench method. After reacting with Ru(NH3)63+, [Ru((CH3)2bpy)2(im)2]2+, with electron donating substitutents, gives quenching yield of 25.1% and formation yield of [RuIII(LL)2(im)2]3+ species of 42.0%. While [Ru((COO−)2bpy)2(im)2]2−, with electron withdrawing substitutents, has 65.2% of quenching yield and 19.6% of formation yield of [RuIII(LL)2(im)2]3+ species. In those ruthenium modified cytochrome c, Ru((CH3)2bpy)2(im)(His33)-Fe2+-cyt c has the largest quantum yield of intramolecular electron transfer (17.6%) and the smallest for Ru((COO−)2bpy)2(im)(His33)-Fe2+-cyt c (11.9%). Although driving force favors for Ru((COO−)2bpy)2(im)(His33)-Fe2+-cyt c, cage effect and chemical reaction are other variable factors in the trend. Ru(bpy)2dppz2+, known for its light switch property, had been utilized to evaluate the concentration of SDS. As the concentration of SDS increases, the emission intensity of Ru(bpy)2dppz2+ increases. Unfortunately, at the attempt to lower the SDS concentration below 0.1%, Ru(bpy)2dppz2+ precipitates, therefore, ethidium bromide (EtdBr) was employed. In the low concentration of SDS (0−0.1%), the wavelength of absorption maximum red shifts and the emission intensity decreases. While the concentration of SDS is above 0.1%, the wavelength of absorption maximum blue shifts and the emission intensity is recovering. At above the CMC of SDS, the emission intensity remains unchanged and is higher than that without SDS. An assay for evaluating of SDS concentration by EtdBr has been proposed. Chemical compositions of seven essential oils from Taiwan had been analyzed by gas chromatography−mass spectroscopy. The eluates had been identified by matching the mass fragment patents to the NIST 08 database. Quantitatively analysis showed the major components are somewhat different from the same essential oils reported that are obtained from other origins. The antibacterial activity of the essential oils against Escherichia coli was evaluated by optical density method. Patchouli is a very effective inhibitor that completely inhibits the growth of E. coli at 0.05%. Clove basil and sweet marjoram are good inhibitors and their upper limits of minimum inhibitory concentration are 0.1%..

(5) 摘要本研究合成出釕金屬聯吡啶錯合物([Ru((CH3)2bpy)2(im)2]2+與 [Ru((COO−)2bpy)2(im)2]2−)，並將其修飾在細胞色素 c (cyt c)上，再藉由閃光淬熄法來探測蛋白質內的電子傳遞。修飾上推電子取代基的[Ru((CH3)2bpy)2(im)2]2+ 與 Ru(NH3)63+反應後，得到 25.1%的激發態淬熄率和 42.0%的三價釕金屬 ([RuIII(LL)2(im)2]3+)生成率；修飾上拉電子取代基的[Ru((COO−)2bpy)2(im)2]2−則有 65.2%激發態淬熄率和 19.6%的三價釕金屬生成率。同樣將釕金屬修飾蛋白質與 Ru(NH3)63+反應，發現 Ru((CH3)2bpy)2(im)(His33)-Fe2+-cyt c 的分子內電子傳遞之量子產率是 17.6%，而 Ru((COO−)2bpy)2(im)(His33)-Fe2+-cyt c 的分子內電子傳遞之量子產率則是 11.9%。儘管反應驅動力預測 Ru((COO−)2bpy)2(im)(His33)-Fe2+-cyt c 有較大的量子產率，但還要考慮另外兩個變數的影響(籠蔽效應跟化學反應)。利用 Ru(bpy)2dppz2+的光開關性質來檢測十二烷基硫酸鈉的濃度，隨著十二烷基硫酸鈉的濃度增加，Ru(bpy)2dppz2+的磷光強度也增加。然而，當十二烷基硫酸鈉的濃度小於 0.1%時，Ru(bpy)2dppz2+會沉澱析出，因此換用溴化乙錠。在低濃度的十二烷基硫酸鈉(0−0.1%)，溴化乙錠的吸收波長最大值會紅位移而螢光強度會降低；當十二烷基硫酸鈉的濃度超過 0.1%，溴化乙錠的吸收波長最大值會藍位移而螢光強度會增強；當十二烷基硫酸鈉的濃度超過臨界微胞濃度後，溴化乙錠的螢光強度維持不變。利用上述現象，溴化乙錠可以拿來檢測十二烷基硫酸鈉的濃度。利用氣相層析質譜儀分離鑑定七種台灣精油，再透過 NIST 08 資料庫的比對，可以清楚辨識主要的化學成分。藉由定量分析的實驗，可以得知精油的主要成分含量，比較文獻後發現，不同產地的精油其組成成分會有很大的差異。將七種精油分別加入大腸桿菌培養液中，經過 24 小時後，發現廣藿香的抑菌效果非常好，只要 0.05%的濃度就可以完全抑制大腸桿菌的生長；而丁香羅勒和甜馬鬱蘭的抑菌效果也不差，兩者的最低抑菌濃度都是 0.1%。.

(6) Keywords flash-quench, cytochrome c, bimolecular quenching reaction, cage effect, ethidium bromide, sodium dodecyl sulfate, critical micelle concentration, assay, antibacterial activity, chemical composition, Escherichia coli, essential oil, gas chromatography−mass spectrometry. 關鍵字閃光淬熄法，細胞色素 c，雙分子淬熄反應，籠蔽效應，溴化乙錠，十二烷基硫酸鈉，臨界微胞濃度，檢測，抗菌性，化學組成，大腸桿菌，精油，氣相層析質譜儀.

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(8) Table of contents List of Figures………………………………………………………………………...iv List of Tables………………………………………………………………………...xiii List of Schemes……………………………………………………………………...xiv. Chapter 1………………………………………………………………………………1 Abstract…………………………………………………………………………..2 Introduction………………………………………………………………………3 Experimental Section……………………………………………………………11 Results and Discussion………………………………………………………….18 Electronic absorption and emission spectra of ruthenium model compounds………………………………………………………………...18 Electrochemistry of ruthenium model compounds………………………..19 Radiative and non-radiative decay rate constant of ruthenium model compounds………………………………………………………………...20 Bimolecular quenching reaction of ruthenium model compounds………..22 Driving force dependence of the bimolecular quenching reaction………...24 Comparison of quenching rate constant and driving force of the bimolecular quenching reaction…………………………………………...26 Comparison of the excited state quenching yield and the formation yield of [RuIII(LL)2(im)2]3+ species……………………………………………...29 Photophysical properties of ruthenium modified Fe3+-cytochrome c……..33 Bimolecular quenching reaction of ruthenium modified Fe3+-cytochrome c…………………………………………………………35 Bimolecular quenching reaction of ruthenium modified Fe2+-cytochrome c…………………………………………………………38 i.

(9) Intramolecular electron transfer in ruthenium modified Fe2+-cytochrome c…………………………………………………………40 Driving force dependence of the intramolecular electron transfer reaction…………………………………………………………………….45 Conclusions……………………………………………………………………..49 References………………………………………………………………………51 Supporting Information…………………………………………………………54. Chapter 2……………………………………………………………………………..59 Abstract…………………………………………………………………………60 Introduction……………………………………………………………………..61 Experimental Section…………………………………………………………...65 Results and Discussion………………………………………………………….69 Interaction between Ru(bpy)2dppz2+ and surfactants……………………...69 UV−Visible absorption and luminescence spectra for Ru(bpy)2dppz2+ in SDS aqueous solution……………………………………………………...69 UV−Visible absorption and luminescence spectra for Ru(bpy)2dppz2+ in TX-100 and CTAB aqueous solution……………………………………...72 Summary of the interaction between Ru(bpy)2dppz2+ and surfactants……74 Interaction between EtdBr and surfactants………………………………...75 UV−Visible absorption and luminescence spectra for EtdBr in SDS aqueous solution…………………………………………………………...75 UV−Visible absorption and luminescence spectra for EtdBr in TX-100 aqueous solution…………………………………………….......................84 The effect of micelle formation for EtdBr in SDS and TX-100 aqueous solution…………………………………………………………………….88 ii.

(10) UV−Visible absorption and luminescence spectra for EtdBr in CTAB aqueous solution…………………………………………….......................89 Solvatochromic effect for EtdBr in SDS aqueous solution………………..91 Detail discussion with the emissive property of EtdBr in SDS aqueous solution…………………………………………………………………….93 Surfactant chain length effect on the photophysical properties of EtdBr……………………………………………………………………..102 Assay of estimating for the concentration of SDS in aqueous solution….111 Conclusions……………………………………………………………………113 References……………………………………………………………………..116 Supporting Information………………………………………………………..120. Chapter 3……………………………………………………………………………131 Abstract………………………………………………………………………..132 Introduction……………………………………………………………………133 Experimental Section………………………………………………………….135 Results and Discussion………………………………………………………...139 Method development……………………………………………………..139 Qualitative and quantitative analysis of essential oils……………………142 Major component in Taiwan species and comparison with various origins…………………………………………………………………….146 High content of component and its application on biology………………148 Antibacterial activity against E. coli……………………………………..149 Conclusions……………………………………………………………………153 References……………………………………………………………………..154 Supporting Information………………………………………………………..158 iii.

(11) List of Figures Chapter 1 Figure 1. Photosynthetic electron transport chain……………………………………..4 Figure 2. Electron transport chain of cellular respiration……………………………...6 Figure 3. Latimer diagram of [Ru(bpy)3]2+ complex………………………………….7 Figure 4. Structure of the ruthenium model compounds……………………………..12 Figure 5. Structure of the ruthenium modified cytochrome c………………………..12 Figure 6. Electronic absorption and emission spectra of ruthenium model compounds in 50 mM NaPi buffer solution………………………………………….19 Figure 7. Cyclic voltammogram of ruthenium model compounds in 50 mM NaPi buffer solution………………………………………………………………………..20 Figure 8. Emission spectra of [Ru((COO−)2bpy)2(im)2]2− with various concentration of Rua63+………………………………………………………………22 Figure 9. Driving force (−GQ) versus natural logarithm of quenching rate constant for the bimolecular quenching reaction between ruthenium model compound and Rua63+………………………………………………………………...25 Figure 10. Driving force dependence of electron transfer rate constants predicted by semi-classical Marcus theory………………………………………………………...27 Figure 11. Nanosecond transient absorption spectra of ruthenium model compounds without and with Rua63+ at the ground state bleach wavelength……………………..30 Figure 12. The absorption spectra of [Ru((CH3)2bpy)2(im)2]2+ with 5 mM Rua63+ before experiment, after emission measurement and after transient absorption measurement………………………………………………………………………….32 Figure 13. Electronic absorption spectra of ruthenium modified Fe3+-cyt c in 50 mM NaPi buffer solution……………………………………………………………..34. iv.

(12) Figure 14. Emission spectra of ruthenium modified Fe3+-cyt c in 50 mM NaPi buffer solution………………………………………………………………………..35 Figure 15. Nanosecond transient absorption spectra of ruthenium modified Fe3+-cyt c with Rua63+ at the ground state bleach wavelength……………………….37 Figure 16. Difference absorption spectrum of oxidized and reduced form of cyt c….39 Figure 17. Nanosecond transient absorption spectra of ruthenium modified Fe2+-cyt c with Rua63+ at 550 nm…………………………………………………….41 Figure 18. Nanosecond transient absorption spectra of ruthenium modified Fe2+-cyt c with Rua63+ at 390 nm…………………………………………………….42 Figure 19. Nanosecond transient absorption spectrum of Ru(bpy)2(im)(His33)-Fe2+-cyt c with Rua63+ and the difference absorption spectrum of oxidized and reduced cyt c………………………………………………………...43 Figure 20. Nanosecond transient absorption spectrum of Ru((CH3)2bpy)2(im)(His33)-Fe2+-cyt c with Rua63+ and the difference absorption spectrum of oxidized and reduced dm-cyt c………………………………………….44 Figure 21. Nanosecond transient absorption spectrum of Ru((COO−)2bpy)2(im)(His33)-Fe2+-cyt c with Rua63+ and the difference absorption spectrum of oxidized and reduced dc-cyt c…………………………………………..44 Figure 22. Driving force (−GET) versus natural logarithm of intramolecular electron transfer rate constant for the ruthenium modified Fe2+-cyt c……………….46 Figure 23. Driving force (−GET) versus quantum yields of intramolecular electron transfer for the ruthenium modified Fe2+-cyt c………………………………………47. Figure S1. Emission spectra of [Ru(bpy)2(im)2]2+ with various concentrations of Rua63+………………………………………………………………………………...54 v.

(13) Figure S2. Emission spectra of [Ru((CH3)2bpy)2(im)2]2+ with various concentrations of Rua63+……………………………………………………………...54 Figure S3. The Stern-Volmer plot of emission intensity of [Ru(bpy)2(im)2]2+ with Rua63+…………………………………………………………………………...........55 Figure S4. The Stern-Volmer plot of emission lifetime of [Ru(bpy)2(im)2]2+ with Rua63+…………………………………………………………………………...........55 Figure S5. The Stern-Volmer plot of emission intensity of [Ru((CH3)2bpy)2(im)2]2+ with Rua63+…………………………………………………………………………...56 Figure S6. The Stern-Volmer plot of emission lifetime of [Ru((CH3)2bpy)2(im)2]2+ with Rua63+…………………………………………………………………………...56 Figure S7. The Stern-Volmer plot of emission intensity of [Ru((COO−)2bpy)2(im)2]2− with Rua63+………………………………………………57 Figure S8. The Stern-Volmer plot of emission lifetime of [Ru((COO−)2bpy)2(im)2]2− with Rua63+………………………………………………57 Figure S9. The Stern-Volmer plot of emission lifetime of [Ru(phen)2(im)2]2+ with Rua63+………………………………………………………………………………58. Chapter 2 Figure 1. Structure of EtdBr and Ru(bpy)2dppz2+……………………………………63 Figure 2. Structures of the three kinds of surfactants………………………………...69 Figure 3. UV−Visible absorption spectra for Ru(bpy)2dppz2+ in 0% and 2% of SDS…………………………………………………………………………………...70 Figure 4. Phosphorescence spectra for Ru(bpy)2dppz2+ between 0−2% of SDS…….71 Figure 5. Emission intensity at 635 nm for Ru(bpy)2dppz2+ between 0−2% of SDS…………………………………………………………………………………...72 vi.

(14) Figure 6. UV−Visible absorption spectra for Ru(bpy)2dppz2+ in 0% and 2% of TX-100……………………………………………………………………………….73 Figure 7. UV−Visible absorption spectra for Ru(bpy)2dppz2+ in 0% and 2% of CTAB…………………………………………………………………………………74 Figure 8. UV−Visible absorption spectra for EtdBr in various concentrations of SDS…………………………………………………………………………………...76 Figure 9. UV−Visible absorption spectra for diluted EtdBr between 0−2% of SDS…………………………………………………………………………………...77 Figure 10. Fluorescence spectra for EtdBr in various concentrations of SDS……….78 Figure 11. Absorption maximum of n→* transition for EtdBr between 0−2% of SDS…………………………………………………………………………………...79 Figure 12. Job plot for EtdBr-SDS complex by monitoring the change of emission intensity at 615 nm…………………………………………………………80 Figure 13. Diagram of the interaction for (a) EtdBr-SDS complex and (b) EtdBr-SDS micelle…………………………………………………………………...81 Figure 14. Calculating the binding constant from the fitting curve for the plot of EtdBr emission intensity at 623 nm versus SDS concentration……………………...82 Figure 15. Emission intensity ratio at 623 nm for EtdBr between 0−2% of SDS……83 Figure 16. UV−Visible absorption spectra for EtdBr between 0−2% of TX-100……84 Figure 17. Fluorescence spectra for EtdBr between 0−2% of TX-100………………85 Figure 18. Emission intensity ratio at 628 nm for EtdBr between 0−2% of TX-100……………………………………………………………………………….85 Figure 19. Diagram of the interaction for EtdBr-TX-100 micelle…………………...87 Figure 20. Absorption maximum of n→* transition for EtdBr in SDS and TX-100 solution……………………………………………………………………………….88 Figure 21. Emission intensity ratio for EtdBr in SDS and TX-100 solution…………89 vii.

(15) Figure 22. UV−Visible absorption spectra for EtdBr in 0% and 2% of CTAB……...90 Figure 23. Fluorescence spectra for EtdBr in 0% and 2% of CTAB…………………90 Figure 24. UV−Visible absorption spectra of EtdBr in 0% (H2O), 0.1% and 2% SDS aqueous solution and two organic solvent DCM and DMSO………………......92 Figure 25. Resonance structures of ethidium cation…………………………………94 Figure 26. Emission intensity at 615 nm and absorption maximum of n→* transition for EtdBr between pH 0−14 in NaPi buffer solution……………………...95 Figure 27. Emission intensity at 610 nm and absorption maximum of n→* transition for EtdBr between pH 1−10 in CH3CN……………....................................95 Figure 28. UV−Visible absorption spectrum of ethidium cation with predicted TD-DFT transition……………………………………………………………………97 Figure 29. UV−Visible absorption spectrum of deprotonated ethidium cation (Etd-RH) with predicted TD-DFT transition…………………………………………97 Figure 30. UV−Visible absorption spectrum of deprotonated ethidium cation (Etd-LH) with predicted TD-DFT transition…………………………………………98 Figure 31. Luminescence decay of EtdBr in 0.1% of SDS…………………………..99 Figure 32. Emission intensity ratio at 623 nm and fluorescence lifetime ratio at 615 nm for EtdBr below 0.1% of SDS……………………………………………...100 Figure 33. Emission intensity ratio at 623 nm and fluorescence lifetime ratio at 615 nm for EtdBr below 1% of SDS………………………………………………..101 Figure 34. UV−Visible absorption spectra for EtdBr in 0%, 0.1% and 1% of sodium sulfate………………………………………………………………………102 Figure 35. Fluorescence spectra for EtdBr in 0%, 0.1% and 1% of sodium sulfate……………………………………………………………………………….103 Figure 36. UV−Visible absorption spectra for EtdBr in 0%, 0.1% and 1% of sodium methyl sulfate………………………………………………………………103 viii.

(16) Figure 37. Fluorescence spectra for EtdBr in 0%, 0.1% and 1% of sodium methyl sulfate……………………………………………………………………………….104 Figure 38. UV−Visible absorption spectra for EtdBr in 0%, 0.1%, 0.5% and 1% of sodium hexyl sulfate……………………………………………………………..105 Figure 39. Fluorescence spectra for EtdBr in 0%, 0.1%, 0.5% and 1% of sodium hexyl sulfate………………………………………………………………………...105 Figure 40. UV−Visible absorption spectra for EtdBr in 0%, 0.1%, 0.5% and 1% of sodium octyl sulfate……………………………………………………………...106 Figure 41. Fluorescence spectra for EtdBr in 0%, 0.1%, 0.5% and 1% of sodium octyl sulfate…………………………………………………………………………106 Figure 42. UV−Visible absorption spectra for EtdBr between 0−0.1% of sodium tetradecyl sulfate…………………………………………………………………….108 Figure 43. Fluorescence spectra for EtdBr between 0−0.1% of sodium tetradecyl sulfate……………………………………………………………………………….109 Figure 44. Emission intensity ratio at 615 nm for EtdBr between 0−0.1% of sodium tetradecyl sulfate……………………………………………………………110 Figure 45. Calculating the binding constant from the fitting curve for the plot of EtdBr emission intensity at 615 nm versus sodium tetradecyl sulfate concentration………………………………………………………………………..110 Figure 46. Absorption maximum of n→* transition and emission intensity ratio at 623 nm for EtdBr between 0−2% of SDS………………………………………..114 Figure 47. Absorption maximum of n→* transition and emission intensity ratio at 628 nm for EtdBr between 0−2% of TX-100…………………………………….114. ix.

(17) Figure S1. Phosphorescence spectra for Ru(bpy)2dppz2+ between 0−2% of TX-100……………………………………………………………………………...120 Figure S2. Phosphorescence spectra for Ru(bpy)2dppz2+ between 0−2% of CTAB………………………………………………………………………………..120 Figure S3. Fluorescence spectra for diluted EtdBr between 0−2% of SDS………...121 Figure S4. Emission intensity ratio at 620 nm for diluted EtdBr between 0−2% of SDS………………………………………………………………………………….122 Figure S5. UV−Visible absorption spectra for EtdBr in various pH values of NaPi buffer solution………………………………………………………………………122 Figure S6. Fluorescence spectra for EtdBr in various pH values of NaPi buffer solution……………………………………………………………………………...123 Figure S7. UV−Visible absorption spectra for EtdBr in basic condition of CH3CN……………………………………………………………………………...124 Figure S8. Fluorescence spectra for EtdBr in basic condition of CH3CN………….124 Figure S9. UV−Visible absorption spectra for EtdBr in acidic condition of CH3CN……………………………………………………………………………...125 Figure S10. Fluorescence spectra for EtdBr in acidic condition of CH3CN………..125 Figure S11. Luminescence decay of EtdBr in 0.001% of SDS……………………..126 Figure S12. Luminescence decay of EtdBr in 0.005% of SDS……………………..126 Figure S13. Luminescence decay of EtdBr in 0.01% of SDS………………………127 Figure S14. Luminescence decay of EtdBr in 0.05% of SDS………………………127 Figure S15. Luminescence decay of EtdBr in 0.2% of SDS………………………..128 Figure S16. Luminescence decay of EtdBr in 1% of SDS………………………….128 Figure S17. Luminescence decay of EtdBr in pure water…………………………..129. x.

(18) Chapter 3 Figure 1. GC−MS chromatogram of seven essential oils (linear temperature gradient)…………………………………………………………………………….139 Figure 2. GC−MS chromatogram for tea tree essential oil…………………………140 Figure 3. GC−MS chromatogram for rose geranium essential oil………………….141 Figure 4. GC−MS chromatogram of seven essential oils (step temperature gradient)…………………………………………………………………………….141 Figure 5. Growth curves of E. coli in LB medium in the absence and presence of patchouli essential oil……………………………………………………………….150 Figure 6. Inhibitory effect of E. coli growth by 0.01%, 0.05% and 0.1% of seven essential oils after 24 hours of incubation…………………………………………..152. Figure S1. GC−MS chromatogram for lemon verbena essential oil……………......158 Figure S2. GC−MS chromatogram for sweet marjoram essential oil………………159 Figure S3. GC−MS chromatogram for clove basil essential oil……………………159 Figure S4. GC−MS chromatogram for patchouli essential oil……………………...160 Figure S5. GC−MS chromatogram for rosemary essential oil……………………...160 Figure S6. The regression relationship between the concentration of geraniol and its integrated area of abundance in the GC−MS chromatogram……………………161 Figure S7. The regression relationship between the concentration of 1,8-cineole and its integrated area of abundance in the GC−MS chromatogram……………….161 Figure S8. The regression relationship between the concentration of -caryophyllene and its integrated area of abundance in the GC−MS chromatogram……………………………………………………………………….162. xi.

(19) Figure S9. Growth curves of E. coli in LB medium in the absence and presence of clove basil essential oil…………………………………………………………..162 Figure S10. Growth curves of E. coli in LB medium in the absence and presence of sweet marjoram essential oil……………………………………………………..163 Figure S11. Growth curves of E. coli in LB medium in the absence and presence of lemon verbena essential oil………………………………………………………163 Figure S12. Growth curves of E. coli in LB medium in the absence and presence of tea tree essential oil………………………………………………………………164 Figure S13. Growth curves of E. coli in LB medium in the absence and presence of rosemary essential oil…………………………………………………………….164 Figure S14. Growth curves of E. coli in LB medium in the absence and presence of rose geranium essential oil……………………………………………………….165 Figure S15. Inhibitory effect of E. coli growth by 0.01%, 0.02%, 0.05% and 0.1% of patchouli essential oil after 24 hours of incubation……………………………...165 Figure S16. Inhibitory effect of E. coli growth by 0.01%, 0.05% and 0.1% of clove basil essential oil after 24 hours of incubation……………………………….166 Figure S17. Inhibitory effect of E. coli growth by 0.01%, 0.05% and 0.1% of sweet marjoram essential oil after 24 hours of incubation………………………….166 Figure S18. Inhibitory effect of E. coli growth by 0.01%, 0.05% and 0.1% of lemon verbena essential oil after 24 hours of incubation…………………………...167 Figure S19. Inhibitory effect of E. coli growth by 0.01%, 0.05% and 0.1% of tea tree essential oil after 24 hours of incubation……………………………………….167 Figure S20. Inhibitory effect of E. coli growth by 0.01%, 0.05% and 0.1% of rosemary essential oil after 24 hours of incubation…………………………………168 Figure S21. Inhibitory effect of E. coli growth by 0.01%, 0.05% and 0.1% of rose geranium essential oil after 24 hours of incubation……………………………168 xii.

(20) List of Tables Chapter 1 Table 1. Absorption and emission maximum of ruthenium model compounds in 50 mM NaPi buffer solution………………………………………………………….19 Table 2. Photophysical properties of ruthenium model compounds in 50 mM NaPi buffer solution………………………………………………………………………..21 Table 3. Quenching efficiency between ruthenium model compounds and Rua63+….24 Table 4. Driving forces of the bimolecular quenching reaction between ruthenium model compounds and Rua63+………………………………………………………..25 Table 5. Bimolecular quenching reaction between ruthenium model compounds and Rua63+…………………………………………………………………………….31 Table 6. Bimolecular quenching reaction between ruthenium modified Fe3+-cyt c and Rua63+…………………………………………………………………………….36 Table 7. Bimolecular quenching reaction between ruthenium modified Fe2+-cyt c and Rua63+…………………………………………………………………………….38 Table 8. Intramolecular electron transfer rate constants for the ruthenium modified Fe2+-cyt c……………………………………………………………………………..42 Table 9. Intramolecular electron transfer within the ruthenium modified Fe2+-cyt c……………………………………………………………………………..46. Table S1. Supplement for the bimolecular quenching reaction between ruthenium modified Fe3+-cyt c and Rua63+………………………………………………………58 Table S2. Supplement for the bimolecular quenching reaction between ruthenium modified Fe2+-cyt c and Rua63+………………………………………………………58. xiii.

(21) Chapter 2 Table 1. Absorption and emission maximums of EtdBr in H2O, DCM and DMSO with the specific physical properties of solvents……………………………………..92 Table 2. Fluorescence lifetime and normalized emission intensity of EtdBr in various concentrations of SDS……………………………………………………...100. Chapter 3 Table 1. Volatile components of the seven essential oils and their relative abundance…………………………………………………………………………...143 Table 2. The major components and their structures of the seven essential oils……147 Table 3. Comparison for major components with various origins of tea tree essential oils………………………………………………………………………...148. List of Schemes Chapter 1 Scheme 1. Mechanism of the direct photoinduced electron transfer for Ru2+(bpy)2(dcbpy)-Lys-Fe3+-cyt c……………………………………………………..8 Scheme 2. Mechanism of the flash-quench method for Ru2+(bpy)2(im)(His33)-Fe2+-cyt c……………………………………………………...9 Scheme 3. Synthetic scheme of ruthenium model compounds………………………11. Chapter 2 Scheme 1. Assay of estimating for the concentration of SDS in aqueous solution…112 xiv.

(22) Chapter 1. Study of the yield of flash-quench method applied to probe the long range electron transfer in ruthenium modified protein. 1.

(23) Abstract The flash-quench method is developed to produce long-lived potent reactants. This method has been widely applied to protein and DNA electron transfer reactions. However, the current method is low in quantum yield and makes the data analysis difficult. The goal for this research is to increase the yield for the flash-quench product. Alternating the excited state electron density distribution can perturb the efficiency of electron transfer. Three ruthenium compounds were synthesized; [Ru(bpy)2(im)2]2+, [Ru((CH3)2bpy)2(im)2]2+, and [Ru((COO−)2bpy)2(im)2]2−. For [Ru((CH3)2bpy)2(im)2]2+, the excited state quenching yield is the smallest but the formation yield of [RuIII(LL)2(im)2]3+ species is the largest. [Ru((COO−)2bpy)2(im)2]2− has the opposite result. Driving force effect can not explain the trend of excited state quenching yield. Charge interaction and hydrophobic interaction play important roles in these results. Corresponding ruthenium carbonate complex is readily to react with the surface histidine of cytochrome c to produce the ruthenium modified cytochrome c. By using the oxidative quencher, Rua63+, intramolecular electron transfer was monitored by transient absorption spectra. Similar to the model compound, Ru((CH3)2bpy)2(im)(His33)-Fe2+-cyt c has the largest quantum yield of intramolecular electron transfer and the smallest for Ru((COO−)2bpy)2(im)(His33)-Fe2+-cyt c. Although driving force favors for Ru((COO−)2bpy)2(im)(His33)-Fe2+-cyt c, cage effect and chemical reaction on the methyl substitutent are other variable factors in the trend.. 2.

(24) Introduction Long range electron transfer is extremely important in biological system such as photosynthesis and cellular respiration. The photosynthesis of plants is by far the most important energy generation process in nature. To convert the transient energy of photon into stable chemical energy, three integrated membrane protein complexes involved are as following: photosystem II (PSII), cytochrome b6f (cyt b6f), and photosystem I (PSI).1-3 Electron transfer takes place between these proteins which is known as the light reaction. In the light reaction, light is converted to the energy in the form of proton gradient and NADPH as the following equation: 1 H 2 O  NADP   4photons  NADPH  H   O 2 2. E o  1.14 V. (1). Equation 1 is a concise final overall reaction, but there are numerous electron transfer steps involved. Some steps are just electron transfer and others are proton coupled electron transfer. The electron transport chain in photosynthetic reaction contains sequential electron transfer reactions, step-by-step over a long distance (Figure 1).. 3.

(25) Chloroplast stroma. e flow. ADP + Pi. H+ flow. NADP+ + H+. ATP H+. NADPH. ATP FNR. PSII. 2H+. Cyt b6f. PSI. QB. FA / FB. QA. FX. Pheo. FeS. A0. Cyt f. Tyrz. Fd. A1. PQH2. P680. synthase. P700. OEC PC. H+. 2H+ 2H2O. O2 + 4H+ Thylakoid lumen. Figure 1. Photosynthetic electron transport chain. OEC: oxygen evolving complex; TyrZ: tyrosine; P680: reaction center chlorophyll; Pheo: pheophytin; QA and QB: bound plastoquinone; PQH2: protonated and reduced plastoquinone; FeS: Rieske iron-sulfur center; PC: plastocyanin; P700: reaction center chlorophyll; A0: chlorophyll; A1: phylloquinone; FX and FA/FB: iron-sulfur center; Fd: ferredoxin; FNR: ferredoxin/NADP+ reductase. NADPH: nicotinamide adenine dinucleotide phosphate. ADP and ATP: adenosine diphosphate and adenosine triphosphate. Assuming that the absorbed wavelength is at 700 nm (~40 kcal/mol), four photons absorbed will provide 160 kcal/mol as in equation 1. However, after long range electron transfer, the energy storage is about 50 kcal/mol at most. There are many charge separation states lie in as the intermediates and it consumes some energy for each step. Therefore, only about one-third of the energy is stored. Nonetheless photosynthesis of plants is still the most efficiently species to store the solar energy by far. Solar energy is the handiest, cleanest, and most economical energy on earth. Simulating the mechanism of photosynthesis of plants by artificial photosynthesis. 4.

(26) reaction center and enhancing the reaction yield are the goals for many scientists nowadays.4-6 Cellular respiration supplies energy to all the activities of the living beings. In the beginning, glucose is converted to pyruvate in the process of glycolysis. Pyruvates undergo oxidative decarboxylation and enter the citric acid cycle. NADH is the product from citric acid cycle and it initializes the electron transfer to promote adenosine triphosphate (ATP) production. The electron transport chain in cellular respiration is shown in Figure 2. Four membrane-bound protein complexes involve and three of them are proton pumps.7-9 Again, many electron transfer steps take place among the protein complexes. Finally, oxygen is reduced to water molecule and ATP is formed to provide chemical energy within cells for metabolism. Step-by-step electron transfer is crucial for the long range electron transfer. For instance, the distance between reaction center of cytochrome c (cyt c) and cytochrome c oxidase is above 20 Å .10 How to efficiently transfer electron without wasting unnecessary energy across a long distance in the living system has aroused many researchers’ attention.. 5.

(27) Intermembrane space. e flow H+ flow. H+. H+. H+ I. H+. Cyt c. III. IV Cyt c1. CoQ. CuA FeS Cyt a. FeS II FeS. Cyt a3. FeS FAD. FMN. fumarate 2H2O. NAD+ + H2O succinate. O2 + 4H+. ATP synthase. NADH + H+ + 1/2O2 H+. H+. H+ H+ ADP + Pi ATP. Matrix. Figure 2. Electron transport chain of cellular respiration. Complex I: NADH dehydrogenase; Complex II: succinate dehydrogenase; Complex III: cytochrome bc1 complex; Complex IV: cytochrome c oxidase; FMN: flavin mononucleotide; FeS: iron-sulfur cluster; FAD: flavin adenine dinucleotide; CoQ: coenzyme Q, ubiquinone. Metalloproteins function as electron transport are usually small (molecular weight range from 10000~15000) and water soluble. Cyt c, a heme protein, is a typical electron transport protein and plays an important role in biological system.11 The iron in cyt c has two oxidation states; Fe2+ and Fe3+, both show distinct UV-Visible spectra which give a perfect spectroscopic handle for studying the redox chemistry of cyt c. Ruthenium bipyridine type complex has been utilized to understand the long range electron transfer in proteins.12-18 It is soluble in aqueous and organic solvent. Therefore, it is ready to apply to biological system. Ruthenium bipyridine type complex is the famous photosensitizer and has been investigated extensively.. 6.

(28) Ruthenium trisbipyridine, [Ru(bpy)3]2+, has a metal-to-ligand charge-transfer (MLCT) absorption band in the visible region. Electronically excited [Ru(bpy)3]2+ has a typical 3. MLCT emission profile in the visible region and the excited state energy (E00) of. [Ru(bpy)3]2+ is 2.10 eV. In the ground state, [Ru(bpy)3]2+ is a stable compound which requires -1.26 V to reduce or oxidize (Figure 3). However, at the excited state, *. [Ru(bpy)3]2+ is highly reactive. With proper redox partner presents, *[Ru(bpy)3]2+. behaves as a strong reductant or oxidant.19 In addition, the excited state lifetime of [Ru(bpy)3]2+ is about 600 ns which is long enough for photoinduced bimolecular reaction.. Figure 3. Modified Latimer diagram of [Ru(bpy)3]2+ complex (the present potential are standard reduction potential versus SCE). There are several methods to study electron transfer in ruthenium modified cytochrome c. Millett and Durham had singly labeled ruthenium bis(bipyridine) dicarboxybipyridine, [Ru(bpy)2(dcbpy)]2+, at lysine amino group on cyt c.20 Upon exciting to MLCT excited state, [Ru(bpy)2(dcbpy)]2+ behaves similar to that of well-known [Ru(bpy)3]2+ complex. With Fe3+-cyt c attached, *[Ru(bpy)2(dcbpy)]2+ acts as a strong reducing agent and transfers electron to Fe3+-cyt c due to the large 7.

(29) driving force (photoinduced reaction). The resulting product is metastable state and undergoes the thermodynamic favored electron recombination to the ground state. Detail mechanism is outlined in Scheme 1. Scheme 1. Mechanism of the direct photoinduced electron transfer for Ru2+(bpy)2(dcbpy)-Lys-Fe3+-cyt c (Ru2+Fe3+). Where kd is the excited state decay rate constant, kFET is the forward electron transfer rate constant and kBET is the back electron transfer rate constant.. *Ru2+. Fe3+ cyt c Fe3+ cyt c. *Ru2+. kFET. h. kd. cyt c. kBET. Ru3+. Ru2+. Fe2+ 3+ Ru. Fe2+ cyt c. Fe3+ cyt c. The above method is called direct photoinduced electron transfer which has been widely adopted to study electron transfer in many fields. However, the types of electron transfer reaction are limited by the excited state lifetime of the photosensitizer. If the electron transfer reaction is slower than the excited state decay, the method would fail. In the biological system, the distance between electron donor and acceptor is quite long so that the electron transfer reaction is generally slow. Therefore, direct photoinduced electron transfer by ruthenium bipyridine type complexes is inadequate for probing long range electron transfer. 8.

(30) Flash-quench method is developed to take the advantage of the robust excited state of ruthenium bipyridine type complexes. The lifetime of reactive species is dramatically prolonged. Chang and Gray had modified [Ru(bpy)2(im)]2+ at histidine amino group on cyt c surface.21 Histidine has an imidazole side chain which may serve as a ligand. Therefore, mixing coordinatively unsaturated ruthenium complex, [Ru(bpy)2(H2O)2]2+, and cyt c and adding excess imidazole to replace remaining aqua ligand result ruthenium modified protein, Ru(bpy)2(im)(His33)-cyt c. This protein modification has hence been widely adopted to explore the long range electron transfer in biomolecules. Although neither [Ru(bpy)2(im)(His33)]2+ nor cyt c has long-lived excited state, the electron transfer reaction is initiated and monitored according to Scheme 2. Scheme 2. Mechanism of the flash-quench method for Ru2+(bpy)2(im)(His33)-Fe2+-cyt c (Ru2+Fe2+). Where kd is the excited state decay rate constant, k1 is the quenching rate constant, kET is the electron transfer rate constant, k2 is the intermolecular charge recombination rate constant and Q is the oxidative quencher.. *Ru2+. Fe2+ cyt c. Q. Q. Fe2+ 3+ Ru cyt c. k1. h. kET Ru3+. kd. k2. Ru2+. Fe2+. Q. Q. cyt c. 9. Ru2+. Fe3+ cyt c. Fe2+ cyt c.

(31) In contrast to the direct photoinduced electron transfer, an oxidative quencher (Q) has been added. Photoinduced bimolecular reaction between the ruthenium excited state and oxidative quencher produces a strong oxidant, Ru3+. The consequent electron transfer between Ru3+ and Fe2+-cyt c proceeds. Even though the lifetime of ruthenium excited state is short, high concentration of oxidative quencher is sufficient to produce partial Ru3+ rapidly. The Ru3+ has much longer lifetime (~100 ms) compared to the original excited state (60 ns). At this stage, time window is much wider to study most of long range electron transfer reactions. On the other hand, back reaction between Ru3+ and reduced quencher, Q, is very slow (in seconds) due to the low concentration of both Ru3+ and Q. Flash-quench method is powerful for extending the lifetime of a strong oxidant/reductant. However, it also has a disadvantage which is low quantum yield for generating this long-lived oxidant/reductant. The quantum yield is related to the excited state quenching yield which depends on the quenching rate constant, the excited state lifetime of photosensitizer and the concentration of quencher (means it is concentration dependent). However the variable factors of the quenching rate constant are extensive and the added concentration of quencher is limited. Therefore, to increase the formation yield of Ru3+, prolonging the excited state lifetime is necessary. In this chapter, 2,2’-bipyridine (bpy) ligand and its substituent, such as 4,4’-dimethyl-2,2’-bipyridine (dm) and 4,4’-dicarboxyl-2,2’-bipyridine (dc) have been coordinated with ruthenium to form the complexes of [Ru(LL)2(im)2]2+ (LL = bpy, dm and dc; im = imidazole). Three ruthenium modified cytochrome c are synthesized. The effect of electron donating and withdrawing substituents on the bipyridine ligand to the bimolecular quenching reaction is discussed.. 10.

(32) Experimental Section Materials Imidazole (im), Na2HPO4, NaH2PO4·H2O and NaCl were purchased from Merck. Horse heart cytochrome c (cyt c) and Na2S2O4 were obtained form Aldrich. Ligands of 2,2’-bipyridine (bpy) was obtained from Fluka and 1,10-phenanthroline (phen) was purchased from Alfa Aesar. Ru(NH3)6Cl3 was purchased from Strem. All chemicals were used as received. Milli-Q-grade water (18.3 M·cm) was used to prepare all aqueous solution. Organic solvents for the synthesis reaction were reagent grade.. Synthesis of ruthenium model compounds Substituted bipyridine ligands, 4,4’-dimethyl-2,2’-bipyridine (dm) and 4,4’-dicarboxyl-2,2’-bipyridine (dc), were synthesized according to the reported procedures.22 Ruthenium model compounds, [Ru(LL)2(im)2]2+ (LL = bpy, dm, dc and phen; im = imidazole), were synthesized according to the reported methods which is briefly summarized in Scheme 3.23,24 The structure of the ruthenium model compounds are shown in Figure 4. Scheme 3. Synthetic scheme of ruthenium model compounds. Ru( LL)2Cl2. 10 eq Na2CO3 H2O. Ru( LL)2CO3. 10 eq im H2O. LL = bpy, dm. RuCl3. 2 eq LL & 5 eq LiCl DMF LL = dc, phen. Ru( LL)2Cl2. 2 eq AgNO3 & 10 eq im H2O. 11. Ru( LL)2(im)2. Ru( LL)2(im)2.

(33) Figure 4. Structure of the ruthenium model compounds (the dicarboxyl substituents on the dc ligands are deprotonated at pH 7 in NaPi buffer solution).. Figure 5. Structure of the ruthenium modified cytochrome c.. Synthesis of ruthenium modified cytochrome c Ruthenium complex was modified on cytochrome c according to the literature method and briefly summarized as the following procedures.25 The correspondent ruthenium carbonate complex (5 mM) was mixed with cyt c (100 mg in 25 mL, 0.32. 12.

(34) mM) in 85 mM NaPi buffer solution at 35ºC and shaken at 150 rpm in incubator. The reaction was traced by measuring the absorbance ratio between 290 nm and 410 nm every hour and stopped when the ratio reached 1.6. High concentration of imidazole (1 M) was added into the solution to replace aqua ligand on the ruthenium complex for 24 hours. The excess ruthenium complexes and imidazole were removed by G-25 gel filtration. Final purification of ruthenium modified cyt c was performed on FPLC by using strong cation exchange resin, Mono S column. The eluting solvents were 50 mM NaPi buffer and 50 mM NaPi buffer mixed with 250 mM NaCl. Each eluate bands was pooled together, and was concentrated and desalted by ultrafiltration. The structure of the ruthenium modified cytochrome c are shown in Figure 5.. General procedure and method Sodium phosphate buffer solution To maintain physiological condition for all experiments, a pH 7, sodium phosphate (NaPi) buffer was employed. For protein modification, high ionic strength, 85 mM NaPi buffer solution was prepared by using 4.54 g Na2HPO4 and 7.40 g NaH2PO4·H2O to a final l L solution. To reduce the non-specific bonding between highly charged dc ligand in protein modification, a higher ionic strength, 150 mM NaPi buffer solution was prepared (7.37 g Na2HPO4 and 13.72 g NaH2PO4·H2O in l L solution). For protein purification, low ionic strength, NaPi buffer solution (50 mM) was prepared by mixing 2.73 g of Na2HPO4 and 3.80 g of NaH2PO4·H2O to final l L solution.. G-25 gel filtration Gel filtration is a chromatographic method to separate molecules according to their sizes. The stationary phase consists fixed hole size resins. Small molecules can 13.

(35) diffuse into the porous gel matrix and elute out slowly while large molecules are prevented from diffusing into the porous gel matrix and bypass out quickly. When the molecular size difference is larger than factor of 10, it is called group separation, which is practiced to routine desalting and buffer exchange procedures. In this study, cyt c (native and modified) was separated from excess unreactive ruthenium complex and other small molecular weight reagents by using gel filtration. Sephadex G-25 (GE Healthcare), cross-linking dextran, was utilized as the gel. The hydrophilic resin reduces the non-specific bonding and provides high recoveries.. Fast protein liquid chromatography Fast protein liquid chromatography (FPLC) is a high performance system in protein purification. Despite its similarity to HPLC system; it is designed highly anticorrosive to operate in aqueous solution and the solvent pressure is less than 4 MPa so that the flow rate is relatively high. Pharmacia system with two P-500 pumps and LCC-500 Plus controller were exploited. The glass column, glass-cylinder pumps and plastic tubing allow immediate inspection of the system. Mono S, a strong cation exchanger column, was utilized to separate the native and modified cyt c. The column is prepacked with a hydrophilic resin (10 m MonoBeads with charged group of -CH2SO32-). The higher positive charged compounds bind to the resin strongly and elute out later. The net charge of cyt c is +9 while ruthenium modified cyt c has net charge of +11 (for bpy and dm) or +5 (for dc). The eluates are tracked by UV−Vis detector (UV-1575, Jasco) and collected by fraction collector (RediFrac, Pharmacia LKB).. Ultrafiltration Stirred cell (Series 8000, Model 8050, Amicon) with very small pore size. 14.

(36) membrane (PLBC, Ultracel, Millipore) was utilized to concentrate and desalt the eluates after FPLC. It requires high pressure nitrogen to force the sample solution to transport through the membrane in the airtight system. The gentle magnetic stirring provides homogeneous condition, prevents sample aggregation and minimizes sample denaturation. The nominal molecular weight limit of the regenerated cellulose membrane is 3000 Da for globular proteins. Therefore only NaPi buffer and ruthenium complexes passed out and cyt c (native and modified) will stay in.. Sample preparation For spectroscopic measurement, sample was dissolved in 50 mM NaPi buffer solution which was stored in a custom-designed quartz cuvette (1 cm path length). This cuvette was fitted with Schlenk ware with stopcock and vacuum inlet. Pump-and-fill with high purity nitrogen was operating for five cycles to ensure anaerobic condition. Reduced Fe2+-cyt c (native and modified) was prepared by adding excess Na2S2O4 to Fe3+-cyt c solution. The excess Na2S2O4 was removed by G-25 gel filtration. Reduced Fe2+-cyt c was pooled in the centrifugal filter unit (Amicon Ultra-4, Millipore, with regenerated cellulose membrane whose molecular weight cut-off is 3000 Da for globular proteins) and centrifuged at 7000 g for 10 minutes at 25ºC (Centrifuge 5430 R, eppendorf) to give proper concentration. Prior to kinetic measurements, absorption spectrum was measured to confirm the oxidation state of cyt c.. Instrument UV−Visible spectra were recorded on Agilent 8453 diode array spectrophotometer. Steady state luminescence spectra were obtained by using 15.

(37) Aminco-Bowman Series 2 Spectrofluorometer. The light source was 150 W xenon lamp and the emitting light was collected into photomultiplier tube which was perpendicular to the excitation light. The excitation wavelength was selected at the MLCT band. The scan rate was 1 nm/s and was repeated for four times. All the spectroscopic measurements were performed at room temperature. Photoluminescence decay was performed on the equipment which was designed by Pascher Instruments. The third harmonic of the Q-switched Nd:YAG laser (Spectra-physics, Quanta-Ray INDI series compact, 8 ns FWHM, 10 Hz) was utilized as the excitation wavelength (355 nm). Probe light for the transient absorption spectra was provided by a 75 W xenon short arc lamp (USHIO, UXL-75XE, power-supplied by LPS-220B, Photon Technology International). The probe light can be pulsed (Analog modules Inc, Laser diode driver model 779 series) to produce higher intensity white light. A monochromator (Acton research corporation, SpectraPro 2150i) with 1 mm slit was used to select the probe wavelength. The signal was amplified by the photomultiplier tube (Hamamatsu, model R928) and recorded by the digitizer (LeCroy 9350A, 500 MHz oscilloscope). Long pass filters were employed to remove scattered excitation light. The kinetic of the signal was fitted by the software of ns KinFit 1.3.6. (Pascher Instruments). Electrochemistry was performed on EG&G Princeton applied research Potentiostat/Galvanostat model 273. Ruthenium model compounds were dissolved in 50 mM NaPi buffer solution with concentration around 1~1.5 mM. The measurements were executed in a standard three-electrode, single compartment cell. The working electrode was glassy carbon (1 mm2). The counter and reference electrodes were platinum wire and homemade saturated calomel electrode (SCE); respectively. The sample solution was purged with high purity nitrogen prior to each measurement. Redox potential of ruthenium model compounds were measured by cyclic 16.

一、利用電子激發態分子探測釕金屬修飾蛋白質內的長距離電子傳遞及檢測十二烷基硫酸鈉的濃度 二、七種台灣精油的化學組成及對大腸桿菌的抗菌效果

一、利用電子激發態分子探測釕金屬修飾蛋白質內的長距離電子傳遞及檢測十二烷基硫酸鈉的濃度二、七種台灣精油的化學組成及對大腸桿菌的抗菌效果