臺灣博碩士論文加值系統

螢光光譜、紫外線光譜、圓二色光譜及高效率液相層析圖譜已被用來探討在不同濃度鎂離子、不同酸度以及不同濃度三氟乙醇(TFE)環境下，α-水晶體蛋白之集結情況與其分子伴護功能。實驗的結果顯示出：在ANS的特性螢光分析光譜中，隨著鎂離子的增加，水晶體蛋白表面厭水性區域曝露也隨之增加；在色胺酸螢光分析光譜中，加入鎂離子的α-水晶體蛋白色胺酸的螢光強度都比未加鎂離子的α-水晶體蛋白的還要來的強；從α-水晶體蛋白加入鎂離子的分子伴護功能的測試中，發現鎂離子會影響其能力；α-水晶體蛋白在高效率液相層析圖譜(FPLC)中，出現540 kDa及20 kDa的吸收峰，並且發現540 kDa再經過一次分析，同樣會有540 kDa及20 kDa的吸收峰，而20 kDa卻只出現20 kDa的吸收峰；α-水晶體蛋白在不同酸度的層析圖譜中，我們發現酸度在4.0和5.0時的層析圖譜中只發現20 kDa的吸收峰，酸度在9.0時出現690 kDa的吸收峰及60 kDa的吸收峰；α-水晶體蛋白中加入三氟乙醇試劑，在三氟乙醇濃度大於10 %以後，則有渾濁的情況發生，從圓二色光譜圖發現隨著加入量的增加(0 %、2 %、4 %、8 %、10 %)其二級結構也隨著變化。其色胺酸螢光強度隨著三氟乙醇的增加而減少， 但是螢光強度都比正常的α-水晶體蛋白來的強。ANS的特性螢光強度隨著三氟乙醇的增加而減少。

Fluoroscence spectroscopy, circular dichroism spectroscopy (CD), and fast protein liquid chromatography (FPLC) have been used to study the aggregation of rat lens α-crystallin under the effects of Mg2+, trifluoroethanol and pH. The chaperone activity of α-crystallin toward ditiothreitol-induced insulin aggregation was measured under various concentration of Mg2+ and the activity was correlated with the structural change of α-crystallin. It was observed that ANS and tryptophan fluorescence emissions increased as the concentration of Mg2+ increased, indicating α-crystallin underwent a structural change with more hydrophobic region exposed. However, the change of chaperone activity did not correlate with the observed increase of fluorescence, suggesting that surface hydrophobicity and microenvironment of tryptophan are not necessarily relate to the chaperone function of α-crystallin under the influence of Mg2+. The aggregation study using FPLC showed a 20 kDa and a 540 kDa fractions, and the ratio of 20 kDa to 540 kDa was Mg2+ concentration dependent. Interestingly, re-chromatography of the 540 kDa fraction also gave a 20 kDa and a 540 kDa fraction, whereas the 20 kDa fraction remained no change after re-chromatographed. The aggregation also showed pH dependent. It was found that only 20 kDa fraction was observed at pH 4 and 5, while at pH 9.0 two fractions at 60 kDa and 690 kDa were observed. In the presence of trifluoroethanol, α-crystallin solution started to go opaque as the concentration of trifluoroethanol reached 10%. As trifluoroethanol concentration increased (less that 10 %), spectroscopic studies showedα-crystallin underwent a strnctural change, including loss of secondary structure, enhancement of tryptophan fluorescence and decrease of surface hydrophobicity.

中文摘要 i
英文摘要 iii
目錄 v
表目錄 viii
圖目錄 iX
第一章 緒論 1
一. 水晶體簡介 1
1. 水晶體的構造 1
2. 水晶體的形成 2
3. 水晶體的功能 5
4. 水晶體的透光性 6
5. 白內障 6
二. 水晶體之組成 8
1. β-水晶體蛋白 9
2. γ-水晶體蛋白 10
3. α-水晶體蛋白 12
3-1 α-水晶體蛋白的性質 12
3-2 α-水晶體蛋白的結構 13
3-3 不同環境對α-水晶體蛋白的影響 18
三.實驗目的 21
第二章 實驗 24
一、 儀器 24
二、 材料 26
三、 實驗方法 27
1. 水晶體蛋白的分離 27
2. ANS的螢光測定 27
3. 蛋白質色胺酸的螢光測定 29
4. 膠態粒子管柱層析分離測定 30
5. 水晶體蛋白分子伴護功能測試 30
6. α-水晶體蛋白在不同酸度的液相層析分析 31
7. 水晶體圓二色光譜宜的測定 31
8. 蛋白質分子量標準品的校正 32
第三章 結果與討論 33
一、 不同濃度鎂離子對α-水晶體蛋白結構的影響 41
二、 α-水晶體蛋白膠態粒子過濾層析分析 55
三、 α-水晶體蛋白在不同酸度存在下的液相層析圖譜 60
四、 α-水晶體蛋白在三氟乙醇試劑存在下的影響 63
第四章.結論 70
第五章 參考文獻 72

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