臺灣博碩士論文加值系統

由年紀四週的大白鼠水晶體純化出α水晶體蛋白低分子量和高分子量集結體(HMWA)，並於50-60℃下利用熱引發集結產生活體外之α水晶體蛋白HMWA作為活體內之對照。藉由許多的光譜測量來研究HMWA的結構和功能上的性質。根據Trp、non-Trp、ANS和MIANS螢光的增加以及far-UV和near-UV CD的增強，代表著天然的α水晶體蛋白與HMWA構形上的差異性。由這一些數據顯示天然的HMWA會比α水晶體蛋白更具疏水性，且有可能是因為α水晶體蛋白的部分展開所造成。由膠體過濾層析圖上顯示，將α水晶體蛋白於60℃下加熱1小時即可產生與活體內HMWA分子量相近之集結體。由Trp、ANS和MIANS螢光以及far-UV CD的結果顯示活體內與熱引發HMWA兩者具有非常相似的結構上性質，這或許也指出兩者可能具有相似的生成機制。利用DTT引發胰島素B鏈的集結之類似伴護活性測量顯示，α水晶體蛋白於50℃下預熱後具有比天然的α水晶體蛋白或HMWA更好的伴護功能。隨著溫度的升高會導致伴護活性的下降，且於60℃下所得到之HMWA具有低於活體內HMWA的伴護活性。ANS螢光和類似伴護活性的關連性顯示，表面疏水性並非決定α水晶體蛋白伴護功能的唯一決定因素。藉由10 mM和50 mM β-乙基硫醇修飾α水晶體蛋白上的半胱胺酸(Cysteine)殘基，反應24小時之後先於60℃下加熱1小時再進行膠體過濾層析和一維電泳。結果顯示在50 mM β-乙基硫醇的條件下依然具有熱引發HMWA的形成，但是卻沒有雙硫鍵的產生。代表著雙硫鍵的產生並非導致HMWA形成的(主要)因素，至少不是活體外熱引發HMWA形成的主要機制。我們的研究認為熱引發與活體內HMWA的形成是藉由部分地展開並且具有相似的機制，不過non-Trp螢光、near-UV CD及類似伴護活性的結果則指出兩著之間可能經由不一樣的展開過程。

α-Crystallin and high molecular weight aggregate (HMWA) were isolated from four weeks old Rat lenses. α-Crystallin was further heated at various temperatures (50-60℃) to induce thermal aggregation of HMWA, which was used to make a compare with in vivo HMWA. Spectroscopic measurement were performed to study the structure and functionality of both HMWA. Conformation differences of native HMWA were suggested based on the data of increased trytophan (Trp), non- tryptophan (non-Trp), 1-anilino- naphthalene-8-sulfonic acid (ANS), and 2-(4’-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) fluorescence intensity as well as the increased far-UV and near-UV circular dichroism (CD). These results indicated that native HMWA was more hydrophobic than α-crystallin, possibly resulting from the partial unfolding of native α-crystallin. Gel filtration chromatography showed that α-crystallin heat-induced HMWA prepared by preheating at 60℃ for an hour with the same molecular weight as that of in vivo HMWA. Trp, ANS, and MIANS fluorescence as well as far-UV CD measurements indicated that heat-induced HMWA and in vivo HMWA shared structural similarity, which further suggested the same aggregation mechanism. Chaperone-like activity was observed toward the aggregation of dithiothreitol (DTT)-induced insulin B-chain show that α-crystallin preheated at 50℃ has better activity than α-crystallin and native HMWA. With the increase of preheating temperature, the activity of α-crystallin decreased and it was observed that under 60℃, it was less active than native HMWA. The correlation between the ANS fluorescence and the chaperone-like activity suggests that surface hydrophobicity was not the sole determinant of the chaperone function of the α-crystallin. Cysteine modification of α-crystallin was carried out using 10 mM and 50 mM β-mercaptoethanol followed by heating at 60℃ for an hour, then was subjected to gel filtration and SDS-PAGE. Heat-induced HMWA remained formed from 50 mM modification of α-crystallin, whereas there was no disulfide bond was observed, indicated that disulfide bond formation was not the (main) factor leading to the formation of HMWA, at least in heat-induced HMWA formation. Our study suggests that heat-induced HMWA proceed similar mechanism as that of in vivo HMWA via partial unfolding, however, the unfolding process may differ as to show different non-Trp fluorescence, near-UV CD and chaperone-like activity as well.

中文摘要........................................................i
英文摘要.......................................................ii
目錄..........................................................iii
表目錄..........................................................v
圖目錄.........................................................vi
第一章 序論 1
一、水晶體的構造及功能..........................................1
二、水溶性和非水溶性水晶體蛋白..................................2
三、gamma (γ)水晶體蛋白.........................................4
四、beta (β)水晶體蛋白..........................................6
五、alpha (α)水晶體蛋白.........................................8
(一)、小熱休克性蛋白質家族(small heat shock protein family)..10
(二)、α水晶體蛋白的結構......................................12
(三)、α水晶體蛋白的功能......................................13
i.熱變性分析(heat denature assays).......................15
ii.雙硫鍵的還原分析(reduced disulfide bonds assays).......17
iii.紫外光輻射和氧化應力下蛋白質的變性分析(denature assay
of protein from UV irradiation and oxidative stress)...19
(四)、溫度對α水晶體蛋白伴護活性的影響........................22
六、alpha水晶體蛋白高分子量集結體(HMWA)........................23
七、研究動機...................................................25
第二章 實驗 31
一、材料.......................................................31
二、儀器設備...................................................33
三、實驗方法...................................................34
(一)、水晶體蛋白的純化.......................................34
(二)、一維與二維電泳分析.....................................36
(三)、螢光的測量.............................................36
(四)、圓二色光譜(Circular Dichroism spectra)的測量...........37
(五)、恰似伴護活性(chaperone-like activity)的測量............37
第三章 結果與討論 38
一、高分子量集結體(HMWA).......................................38
二、HMWA二維電泳...............................................40
三、圓二色光譜(Circular Dichroism spectra).....................42
四、螢光光譜...................................................43
五、類似伴護活性(chaperone-like activity)......................47
六、結論.......................................................48
參考文獻.......................................................68

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