利用外差式光學偏光儀研究十二烷基硫酸鈉 對小牛血清蛋白熱變性之影響

Using heterodyne optical polarimeter to study the effect on thermal denaturation of bovine serum albumin with sodium dodecyl sulfate

黃博郁(Bo-Yu Huang)

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(2012年)

https://doi.org/10.6843/NTHU.2012.00669

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，但皆無法即時的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20 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，設計了三部份的實驗來詳細探討BSA 水溶液在加熱後的熱變性現象。

實驗結果指出，BSA水溶液在加熱至約67.8正負0.3°C 時會產生明顯之熱變性現象，且此時蛋白質之結構是屬於可逆的，在降溫後其結構會因為蛋白質之復性作用而慢慢恢復。而隨著加熱溫度的提高，其結構會由可逆逐漸轉變為部分可逆，最後在大約超過75 °C 時結構會變為幾乎不可逆。而蛋白質水溶液之濃度、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。而在將BSA 水溶液和陰離子界面活性劑-十二烷基硫酸鈉以不同濃度比例相互混合後，在加熱時可由本研究之系統明顯觀測到其對蛋白質的結構具有一定程度之穩定作用，使其加熱到達原本的變性溫度67.8正負0.3°C 時並不會發生變性的現象，且其濃度莫耳比[SDS]/[BSA]約在10 左右時會出現最大之保護效果，大約將變性溫度延後了15°C，此結果可活用在許多蛋白質的加工上，也和部分文獻中使用不同方法所得之研究結果不謀而合。

關鍵字

外差式光學偏光儀 ； 小牛血清蛋白 ； 十二烷基硫酸鈉

並列摘要

In the past few years, many measurement methods have been used to study the structural change of protein after heating, such as differential scanning method,circularly polarized dichroism spectroscopy, X-ray diffraction crystallography method,etc. However, these methods are not easy to monitor the denaturation process of BSA aqueous solution in real time. In this study, a homemade optical heterodyne polarimeter that was capable of amplifying the optical rotation signal for 20-fold was built up by authors, and with the use of precision thermoelectric cooler (TEC), a
three-part experiments was designed to study the phenomenon of thermal denaturation of BSA aqueous solution after heating.

Our results indicated that when BSA aqueous solution is heated to about 67.8°C, the thermal denaturation was observed and the structure of BSA was reversible at this time by cooling down because of protein’s renaturation role. When the temperature was between 70-75°C, its structure was gradually changed from a reversible way to a partially reversible status, and finally the structure became almost
irreversible at about more than 75°C. However, although both the concentration of the aqueous protein solution and the rate of heating with TEC system have some effects on the temperature of denaturation, they are not the main factors.

When BSA solution was mixed with anionic surfactants – the sodium dodecyl sulfate (SDS), to create different concentration of solution, a certain extent of stable effect on the protein structure was significantly observed when heated by our TEC system. The mixed solution avoided the structure of protein unfolding at denaturation temperature of 67.76  0.34°C, and when its concentration molar ratio [SDS] / [BSA] was about 10, the protective effect reached its maximum, making the denaturation temperature delay about 15°C. These results can not only be applied to the processing of many proteins, but also were consistent with other studies using different methods.

並列關鍵字

heterodyne optical polarimeter ； bovine serum albumin ； sodium dodecyl sulfate

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