利用外差式光學偏光儀研究三氟乙醇與甘油對膠原蛋白熱變性之影響

Using Heterodyne optical polarimeter to study the effect on thermal denaturation of Collagen with Trifluoroethanol and Glycerol

曾楷涵(Kai-Han Tseng)

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(2013年)

摘要

近年來，有許多的量測方法皆可用來研究蛋白質在加熱後其結構之變化，例如差分掃描法、圓偏振二色光譜法、X-ray 晶格繞射法等等，但這些方法皆無法即時量測出蛋白質水溶液在加熱後結構的快速變化。因此本研究使用具有放大旋光訊號效果約40倍的自製光學外差式偏光儀，以及精密的熱電致冷晶片溫度控制器，設計了四部份的實驗來詳細探討膠原蛋白溶液在加熱後的熱變性現象。
實驗結果指出，膠原蛋白溶液從室溫25oC加熱至55oC時，在40.2±0.2°C 會產生明顯的熱變性現象，當從55oC降溫回25oC時，約66%的結構再度摺疊回三股螺旋的形式。而膠原蛋白溶液之濃度、加熱系統之加熱速度雖然皆會些許的影響其變性溫度，但皆不是影響蛋白質實際變性溫度的主要因素。加入不同濃度的三氟乙醇(TFE)後，可以觀察到初始旋光數值的降低及變性溫度的提前，皆可證明TFE對膠原蛋白結構的破壞，且當濃度到達20%(v/v)後，TFE對膠原蛋白的作用似乎到達飽和。最後，當加入不同濃度的甘油以及不同濃度的TFE後，其結果可以觀察出在不同濃度的TFE下，甘油濃度提高，保護效果越顯著，而在相同濃度的甘油下，TFE濃度越高，變性溫度延後量越大。此結果與其他文獻使用不同研究方法結果吻合。

關鍵字

膠原蛋白 ； 三氟乙醇 ； 甘油 ； 旋光 ； 外差式光學偏光儀 ； 熱變性

並列摘要

Recently, many measurement methods have been used to study the structure change of protein after heating, such as differential scanning method, circularly polarized dichroism spectroscopy, X-ray diffraction crystallography method, etc. However, it is difficult for these methods to monitor the denaturation process of collagen solution in real time. In this study, a self-assembled optical heterodyne polarimeter that was capable of amplifying the optical rotation signal to 40-fold, and a precision thermoelectric cooler (TEC) were used to study the thermal denaturation phenomenon of collagen solution after heating.
Our results indicated that when collagen solution was heated from 25oC to 55°C, the thermal denaturation was at 40.2±0.2°C. When the collagen solution was cooled from 55°C to 25oC, about 66% of its structure was refolded back to triple helix again. However, both the concentration of the aqueous protein solution and the rate of heating with TEC system have some effects on the processes of denaturation, but they are not the main factors.
When collagen solution was mixed with Trifluoroethanol(TFE), we observed that the initial optical rotation value and the denaturation temperature were both decreased. This suggests that TFE will destroy the structure of collagen. Moreover, the destruction was saturated after the concentration of TFE exceeds 20% (v/v). At last, we added glycerol in the mixed solution of TFE and collagen. We observed that the denaturation temperature was increased when more glycerol was added. At high concentration of TFE, the protective effect is more significant than in low concentration. Not only these results can be applied to the processing of many proteins, but also are consistent with other studies using different methods.

參考文獻

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