利用示差掃描熱量分析與雷射閃光熱擴散法 研究牛血清蛋白之熱變性

Studies of Thermal Denaturation of Bovine Serum Albumin using Differential Scanning Calorimetry and Laser Flash Method

洪慧芬(Huei-Fen Hong)

指導教授 ： 洪銘聰

國立中央大學/工學院/機械工程學系/碩士(2014年)

摘要

牛血清蛋白(bovine serum albumin,BSA)，是血清中的簡單蛋白質，常被用在分子生物學、免疫學、食品科學及蛋白質化學等生化研究上；相較於其他蛋白質，BSA能使細胞膜穩定，具有抑制脂質之過氧化、產生乳化及防凍等作用，另外有價格低及容易純化等優點，所以常當作實驗材料或基準蛋白質。BSA的結構是由蛋白質二級結構反覆摺疊而成的緊密立體球狀結構，當對原生態BSA加熱後，其動能提升，造成蛋白質分子鍵結強烈振動而破壞，最後凝聚形成無序組織的變性蛋白質。蛋白質熱變性的研究中，最常使用的方法是示差掃描熱量分析法(differential scanning calorimetry, DSC)，並藉由量測熱量與溫度關係探討蛋白質在變性狀態與原生態間的焓差、熵差及比熱差。然而，相關研究很少探討熱變性對熱傳輸性質的影響。文獻指出，因為生物組織的個體差異，以不同量測點、量測方法將使得熱傳導係數有很高的分散性，導致量測困難;是本研究覺得相關文獻較少的可能原因。
本研究中，使用DSC探討BSA水溶液在熱變性過程與熱量的關係，同時利用雷射閃光法(laser flash analysis, LFA)，研究熱變性對有效熱傳導係數及對有效熱擴散係數的時間相依性的影響。實驗量測5~20 wt%BSA水溶液之密度、比熱及有效熱擴散係數，計算出有效熱傳導係數隨溫度的變化。研究發現在水溶液中之BSA的含量越高，其熱變性過程所需的能量也越大，反應出的比熱變化也最大。在熱擴散分析中，LFA並無法正確地量測出熱變性過程的有效熱擴散係數，但可以反映出不同程度之熱變性對熱傳輸現象之影響。結果顯示，有效熱擴散係數隨BSA濃度增加而提高，在大於熱變性溫度後有明顯的提升，並與持溫時間呈線性成長；隨著BSA水溶液濃度的增加，熱變性過程對有效熱擴散係數的影響也更為劇烈。熱變性的程度對BSA溶液的傳輸特性具有一定的影響力。透過本研究探討蛋白質之熱變性將能了解生物分子熱傳輸現象在修復醫學及生醫檢測上且有潛在的應用與重要性。

關鍵字

牛血清蛋白 ； 熱變性 ； 熱傳輸

並列摘要

Bovine serum albumin, BSA, is often used in molecular biology, immunology, nutritional science or protein chemistry, due to its stability, inhibition of lipid peroxidation, emulsification and antifreeze effects. In addion, BSA’s low cost and easy to purify make it a good candidate for experiment and as a reference protein. BSA has a spherical structure that formed by the tightly folding of the secondary structures. In the elevated temperature, the native BSA gains kinetic and results in a strong vibration causing the damage of the molecule bonding. The 3D structure is then unfolds and turned into the denatured state. In order to study the thermal denaturation of protein, researchers commonly use differential scanning calorimetry method, DSC, to investigate the changes in enthalpy, entropy and specific heat by measuring the heat flow in the elevated temperature. The thermal denaturation effects on the heat transport properties of proteins, however, that is seldom investigated and may have potential impacts on biomedical applications.
In this study, we used DSC to explore the relationship between BSA solutions and the heat flow in the thermal denaturation process. Additionally, laser flash analysis, LFA, was use to study the effective thermal diffusivity of BSA solutions with various concentrations. The time dependence of the thermal diffusivity was also considered. Experiments had performed to measure the density, specific heat, and effective thermal diffusivity of BSA solutions with concentrations 5 ~ 20 wt%. The effective thermal conductivity was then calculated. We found that the energy required in the denaturation process increased with BSA concentration. Same trend can be found in the variation of specific heat. In the thermal diffusivity analysis, LFA couldn’t reveal the true thermal diffusivity when undego denaturation, but reflected the impact of different degrees of thermal denaturation process on heat transfer phenomena. The variation of thermal diffusivity caused by the thermal denaturation process was more dramatic with the increasing concentration. It also showed a linear relation between the thermal diffusivity and durtaion time. The change rate of thermal diffusivity increased with temperature and concentration, which means the increase in the degree of thermal denaturation process.

並列關鍵字

bovine serum albumin ； thermal denaturation ； heat transport

參考文獻

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