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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/3958


    Title: 以螢光光譜觀測蛋白質吸附於疏水表面後之構型變化與吸附位向;Monitoring the Conformation and Orientation of Protein on a Hydrophobic Surface
    Authors: 李柏毅;Po-I Lee
    Contributors: 化學工程與材料工程研究所
    Keywords: 吸附;蛋白質;變性;protein;adsorption;denature
    Date: 2006-06-19
    Issue Date: 2009-09-21 12:27:01 (UTC+8)
    Publisher: 國立中央大學圖書館
    Abstract: 本研究的目的,在利用色胺酸的螢光光譜,來推測蛋白質在Octadecyltrimethoxysilane (ODS)疏水的表面吸附後之構形變化、吸附量及吸附位向。我們所選用的蛋白質,包括BAA、BSA及Lysozyme。首先,根據色胺酸螢光光譜的分析,我們發現當其所處環境為極性時,訊號強度會較所處環境為非極性時來得大。且在非極性環境時,最大波峰位置有藍移的現象發生。 在Lysozyme的吸附實驗中,我們發現當不含DL-Ditheothiol (DTT),吸附於ODS表面後,其構形改變不大。並推測Lysozyme先是以α-Domain的Trp123暴露區域為吸附位向,另外兩個暴露之色胺酸(β-Domain-Trp62、Trp63)再漸漸吸附至疏水表面。而當Lysozyme在含有DTT之情況下,吸附於ODS表面後,訊號強度明顯上升且波峰紅移,明顯表示其構形改變。應是因雙硫鍵被破壞,使得內部的Trp28及Trp108暴露出來。 BSA吸附的實驗中發現,吸附後之波峰位置由340nm先藍移至310nm最後漸漸紅移至325nm,我們認為BSA是以位於IB區域暴露之色胺酸(Trp158)吸附於表面,使得吸附後BSA第I與第II區域逐漸攤開轉向。吸附後色胺酸的螢光幾乎完全被淬滅。 在BAA吸附的實驗部份,我們發現BAA的訊號因吸附而下降,但λmax並無改變,因此構形並無隨時間而改變。並由淬滅率的計算推測,而其可能吸附位向為七個色胺酸的暴露面。 本研究利用色胺酸含量多於酪胺酸的Lysozyme探討時,其形變可由內部色胺酸之暴露而明顯的觀察到。又利用色胺酸含量遠少於酪胺酸的BSA探討,其初始吸附方位可很容易的由其明顯的藍位移及螢光淬滅程度而推測。最後,我們探討色胺酸及酪胺酸均富的BAA,也可由其螢光淬滅程度來推測其吸附位向。 In this study, the orientation and conformation change of protein hydrophobic adsorption were examined. There were three proteins been chosen, which were BAA, BSA and Lysozyme. The quartz surface was modified by octadecyltrimethoxysilane to form the hydrophobic surface. At first, according to the tryptophan fluorescence spectrum analysis, the intensity of tryptophan in polar environment was higher than nonpolar environment and the wavelength of lambda maximum was blue shift when tryptophan located in nonpolar environment. In the experiment of Lysozyme hydrophobic adsorption, it can be found that the structure of Lysozyme on ODS surface did not change obviously in phosphate buffer. The signal of tryptophan was quenched slowly as time increase, we could infer that Lysozyme adsorbed firstly by Try123 at α-Domain and then adsorbed gradually by Trp62 and Trp63 at β-Domain. However, when protein solution contained DL-Dithiothreitol, we could obtain that the intensity of lambda 340nm increased and the lambda maximum was red shift. These indicated that the protein structure was changed because of the disulfide bonds were broken which caused that Trp28 and Trp108 exposed outside the protein surface and oriented to polar environment. In the experiment of BSA, we found that when BSA adsorbed on ODS surface the wavelength was blue shift from 340nm to 310nm then gradually red shift to 325nm. We thought that BSA was adsorbed by Trp158 located at IB-domain firstly then forced the I domain and II domain of BSA changed the direction gradually, and the fluorescence of tryptophan was almost disappear after adsorption. Finally, in BAA hydrophobic adsorption experiment, we found that the intensity of 340nm was decrease due to tryptophan be quenched and the lambda maximum was no change which indicated that the native structure of BAA was still maintains on the ODS surface. Furthermore, by calculating the quench ratio of BAA, we could conjecture that the orientation was the plane of BAA which contained seven tryptophan. In consequence, the conformation change and orientation surmise of protein hydrophobic adsorption can be obtained via the fluorescence spectrum analysis directly.
    Appears in Collections:[化學工程與材料工程研究所] 博碩士論文

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