摘要 本研究利用表面電漿共振生物感測器(SPR)測出角度的變化量，進而得到金膜上蛋白質構型的改變，再利用單分子模型槽(L-B Trough)、圓二色光譜儀(CD)與螢光光譜儀(Fluorescence)得到蛋白質更精確的結構藉以輔佐SPR的結果；實驗上利用溶菌酶(Lysozyme)與核醣核酸酶(Ribonuclease A)這兩種蛋白質，利用有無添加還原劑(i.e. DTT)與不同濃度變性劑(i.e. Urea與GuHCl)的環境下看蛋白質構形的改變。 SPR實驗利用Self-Assembly Monolayer(SAM)及表面活化的方法把蛋白質固定在金膜上，再通入還原劑與不同濃度的變性劑；在沒有添加還原劑的環境下，SPR角度變化量有一個正的趨勢，認為在只有變性劑下的蛋白質結構並沒有完全被破壞，變性劑只是吸附在蛋白質的表面，因變性劑的折射係數(refractive index)較緩衝溶液來的大，因此較高濃度的變性劑下得到較高的角度變化量；在有還原劑的環境下，SPR則是一個負的趨勢，因為雙硫鍵被打斷後，加入變性劑能穩定不穩定的去摺疊狀態，造成蛋白質結構變較鬆散，使得折射係數降低，進而得到一個負的趨勢。而在L-B trough的實驗中，所得到在氣/液界面上每分子所佔據的面積與平衡的表面壓，也可說明以上的現象。最後在輔以CD及Fluorescence能更清楚的知道影響SPR訊號主要是以蛋白質三級結構為主。 Abstract Behaviors of proteins immobilized on a gold surface were investigated using a home-made biosensor utilizing surface plasmon resonance(SPR). In order to examine the possibility of the use of SPR for high sensitive, real-time and label-free monitoring of the protein conformation of unfolding and refolding process, the protein immobilized on a self-assembled monolayer (SAM) layer was analyzed. Specific structure was assigned to the changing surface plasmon resonance(SPR) signals of the immobilized proteins using Langmuir-Blodgett Trough(L-B Trough), Circular Dichroism(CD) and Fluorescence spectroscopies, which are established techniques for the conformational analysis of proteins. For the oxidized proteins, from L-B trough, CD and fluorescence infer that the hydrophobic region was not exposed and retain most of the ordered secondary and tertiary structure. The denaturants(i.e. urea and guanidine hydrochloride) were bound to the protein at specifically hydrophobic sites, therefore SPR angle shift was increasing with concentration of denaturants. The reducing agent affect the conformational changes of proteins in SPR signal, make a positive SPR angle shift. From L-B truogh experiments reveal a compact conformation and hydrophobic region exposed unobviously, therefore, protein film became compactly and increase the refractive index of protein layers. For the reduced proteins, the denaturants not only destroy the ordered secondary and tertiary structure but also stabilize the unfolded conformation, that would increase the thickness of the protein layers and decrease the refractive index of the protein layers, and therefore SPR angle shift was decreasing with the concentration of denaturants.
