利用旋光量測技術研究蛋白質二級結構的變化

Studies on the structure change of biopolymers by optical rotation measurement

郭豐瑋(Feng-Weei Kuo)

指導教授 ： 吳見明

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2006年)

https://doi.org/10.6843/NTHU.2006.00684

摘要

蛋白質通常具有旋光性，且蛋白質中的二級結構如α螺旋、β平板因其構型不同所貢獻的旋光量亦不同。已知蛋白質會受外界環境，如溫度、pH值或經UV照射後的影響而使得其結構發生變化，而此變化通常是迅速的。傳統用以觀察蛋白質的結構的方法，如X-ray晶格繞射、核磁共振(NMR)等皆難以應用於其變化過程的觀察。在本次的研究中，我們利用了量測旋光角的方法來對蛋白質的構型變化作即時性的觀察。本研究利用相位可調式波片來放大旋光訊號，並結合鎖相放大器及數位資料擷取卡來建造一高解析度(6.41×10-4度)與即時性量測的系統。BSA是一種常用於免疫分析的蛋白質，其構型一半以上皆由α螺旋構成，其餘為隨機的圈。在本實驗中，我們藉由加熱BSA來觀察其構型的變化過程。結果指出，我們加熱濃度分別為0.67g/100mL, 1.33g/100mL以及2.66g/100mL的BSA所得的相位訊號變化的斜率成正比，斜率分別為-0.012±0.009, -0.031±0.004以及-0.067±0.013。由於量測到的旋光訊號變化是起因於其α螺旋經加熱後破壞成隨機的圈，因此結果說明了隨濃度的增加被破壞的α螺旋亦增多，使得量測得的訊號成比例關係。而由測到的斜率為負值亦可了解α螺旋在632.8nm的旋光貢獻為右旋。

關鍵字

旋光 ； 蛋白質 ； 二級結構 ； 變質

並列摘要

The secondary structures, i.e., the α-helix, β-sheet, and triple-helix, of protein may be denatured at a certain condition, for example, the pH-value, concentration of solvent, and beyond certain temperature. The transformation of denaturation is not well understood now. The X-ray diffraction crystallography and the nuclear magnetic resonance (NMR) can be used to detect the structures of protein; however, these two methods can not be applied to monitor the denaturation process of proteins at the present time. In addition, the X-ray diffraction crystallography can be only applied to observe protein powder or protein crystallization; therefore, it can’t observe the structures of protein dissolved in liquid. On the other hand, Circular dichroism (CD) spectroscopy is usually used to calculate the proportion of different protein structures; however, it is not a real-time system because the spectrum needs to be scanned. For this reason to construct a real-time detection system is highly required.
In this study, we have built up a high resolution and real-time detection system to monitor the changes of secondary structure by utilizing a variable-retarder to amplify the rotation angle and a lock-in amplifier to enhance the signal-to-noise-ratio. We have verified that the structure changes and optical rotation changes of bovine serum albumin (BSA) were occurred via heating, the so-called thermal effect, where the thermal effect is directly related to denaturation of protein and therefore causes changes in optical rotation. We have demonstrated that by heating different concentrations of BSA, i.e., 0.67 % wt, 1.33 % wt, 2.66 % wt, the corresponding phase signal variations, as function of BSA concentration,　are -0.012±0.009, -0.031±0.004 and -0.067±0.013, respectively, where the phase signal is directly corresponding to optical rotation.

並列關鍵字

optical rotation ； protein ； secondary structure ； denaturation