臺灣博碩士論文加值系統

本研究利用Ascorbate-FeCl3-EDTA-H2O2為氧化系統，氧化a-水晶體蛋白。圓二色光譜儀和ANS 螢光放射光譜，被用來探討在氧化環境下a-水晶體蛋白的結構之改變及透析是否能夠幫助氧化後之a-水晶體蛋白回復原來構形；最後探討不同的氧化及透析的時間下，α-水晶體蛋白之二級結構與其分子伴護功能的關係。
由ANS螢光分析我們發現，氧化時間之增加對蛋白質的影響越大，氧化12小時後的α-水晶體蛋白螢光強度較正常的為高。透析會讓其三級結構慢慢改變，隨著透析時間的增加，透析10小時後的螢光強度比較接近正常的α-水晶體蛋白。由α-晶狀素防止胰島素B-chain聚集的實驗結果顯示，氧化後的α-水晶體蛋白會大大降低其分子伴護的能力，而透析可以增加其分子伴護功能。
由圓二色光譜分析我們知道，正常的α-水晶體蛋白以β-摺板為主要的構形，在波長192-196 nm有正的吸收峰，波長210-220 nm有負的吸收峰。而氧化會使其構形改變，吸收峰有變化。隨著透析時間的增加，二級結構會漸漸恢復，但並不會恢復到與原來完全一樣之構形。進一步由電腦程式SELCON 3的計算分析，得到不同氧化及透析時間下，α-水晶體蛋白二級結構的組成的比例。氧化5及10小時後a-螺旋結構比例增加20﹪，摺版結構降低10﹪，隨著透析時間增加會慢慢恢復其二級結構。在透析10小時後，α-螺旋結構恢復到18﹪與12﹪（正常的α-晶狀素蛋白為18﹪），β-摺版結構恢復到32﹪與33﹪（正常的α-晶狀素蛋為白35﹪）。其組成比例與原來正常的α-水晶體蛋白並不相同。
由以上結果我們推測α-晶狀素蛋白在氧化時因為β-摺版結構損失，及暴露於表面之疏水性區域的改變，而降低其分子伴護的功能。經由透析去除氧化因子，可以使其結構慢慢地回復到原來以β-摺版結構為主的構形，而恢復其分子伴護的功能。

In this study, circular (CD) and ANS fluorescence emission have been used to study the structural change of both the oxidized rat lens a-crystallin and the dialyzed oxidatively insulted a-crystallin by Ascorbate-FeCl3-EDTA-H2O2 oxidation system. The study of the relationship between secondary structure of a-crystallin and its chaperone activity under various times of oxidation then followed by dialysis was also studied. It was found that ANS fluorescence emission intensity increased as oxidation time increased. And the intensity was higher than normal a-crystallin when oxidation time was longer than 10 hours. Dialysis can result in the recovery of ANS fluorescence emission intensity. It was found that after dialysis of oxidized a-crystallin for 10 hours the intensity is almost the same as that of normal one. The study of chaperone activity of a-crystallin toward dithiothreitol-induced insulin-B aggregation showed the chaperone activity decreased for oxidized a-crystallin, however, dialysis resulted in the recovery of its activity. CD study shows that for normal a-crystallin there is a negative band centering at 217 nm, indicating that b-sheet form makes a dominant contribution to the total conformation. Oxidation caused the change of its secondary structure with the 217 nm band blue shifted and intensity decreased. With the increased of dialysis time, the secondary structure was gradually recovered, however, it did not recover completely to its original structure. With the analysis using SELCON 3 program, it is able to estimate the percentage of its secondary components. It was found that the percentage of a-helix increased 20﹪, whiles b-sheet decreased 10﹪for 5-hours and 10-hours oxidized a-crystallins. With the increase of dialysis time, it was observed that secondary components gradually recovered, with a-helix recovering back to 18﹪and 12﹪(18﹪for normal) and b-sheet recovering back to 32﹪and 33﹪(35﹪for normal) for 10-hour dialysis of oxidized a-crystallin. Our study has showed that the decreased of chaperone activity is due to the structural change caused by oxidation and dialysis is a way to regain its structure and chape

■中文摘要 Ⅰ
■英文摘要 Ⅲ
■目錄 Ⅴ
■表目錄 Ⅶ
■圖目錄 Ⅷ
第一章 緒論 1
一、水晶體簡介 1
1-1. 水晶體的位置 1
1-2. 水晶體的形成和結構 2
1-3. 水晶體的透明度 7
二、水晶體之蛋白質組成 8
2-1. g-水晶體蛋白 10
2-2. b-水晶體蛋白 13
2-3. a-水晶體蛋白 14
2-3.1 a水晶體蛋白的結構 14
2-3.2 a-水晶體蛋白的功能 19
2-4. 水晶體蛋白之高分子集結物 24
三、水晶體的生理代謝 25
3-1. 水晶體的生理代謝 25
3-2. 水晶體的氧化及白內障 28
四、研究動機 30
第二章 實驗 31
2-1 材料 31
2-2 儀器 32
2-3 實驗方法 33
2-3.1 水晶體蛋白的製備 33
2-3.2 蛋白質濃度的測定 34
2-3.3 氧化系統的配置 34
2-3.4 水晶體蛋白的氧化 35
2-3.5 圓二色光譜的測量 36
2-3.6 蛋白質二級結構的計算 36
2-3.7 ANS螢光放射光譜 37
2-3.8 分子伴護功能測量 37
第三章 結果與討論 38
3-1. 圓二色光譜圖 38
3-2. 由圓二色光譜圖計算蛋白質二級結構 40
3-3. ANS螢光放射光譜 42 3-4. a-水晶體蛋白防止胰島素B鏈聚集 44
第四章 結論 48
第五章 參考文獻 66
表目錄
表一 晶狀素蛋白性質比較 8
表二 a-水晶體蛋白與熱休克蛋白的比較 18
表三 氧化五小時後透析的a-水晶體蛋白之二級結構成分
計算分析的結果 55
表四 氧化十小時後透析的a-水晶體蛋白之二級結構成分
計算分析的結果 55
表五 氧化十二小時後透析的a-水晶體蛋白之二級結構成分
計算分析的結果 56
表六 氧化十四小時後透析的a-水晶體蛋白之二級結構成分
計算分析的結果 56
圖目錄
圖一 人類眼球略圖 1
圖二 水晶體的形成 2
圖三 晶狀體上皮細胞分化微觀圖 3
圖四 水晶體之Y接合線 3
圖五 水晶體的結構 4
圖六 水溶性蛋白質管柱層析分離圖 7
圖七 g-水晶體蛋白結構圖 9
圖八 g-水晶體蛋白4個區段間的三級構形 10
圖九 a-水晶體蛋白aA2及aB2的胺基酸序列 12
圖十 a-水晶體蛋白四級結構模型 14
圖十一 Carver 等人提出的a-水晶體蛋白四級結構模
型 15
圖十二 水晶體"幫浦-隙漏(pump-leak)"系統圖 16
圖十三 水晶體內葡萄糖的主要代謝途徑 22
圖十四 水晶體內榖麩胱甘的代謝 23
圖十五 蛋白質濃度測定之檢量線 25
圖十六 氧化5小時後的a-水晶體蛋白於透析過程中，
其遠紫外光區圓二色光譜圖 38
圖十七 氧化10小時後的a-水晶體蛋白於透析過程中，
其遠紫外光區圓二色光譜圖 38
圖十八 氧化12小時後的a-水晶體蛋白於透析過程中，
其遠紫外光區圓二色光譜圖 38
圖十九 氧化14小時後的a-水晶體蛋白於透析過程中，
其遠紫外光區圓二色光譜圖 38
圖二十 正常的a-水晶體蛋白對ANS螢光強度的影響
43
圖二十一 氧化5小時後透析的a-水晶體蛋白對ANS螢光強度的影響
圖二十二 氧化10小時後透析的a-水晶體蛋白對ANS螢光強度的影響
圖二十三 氧化12小時後透析的a-水晶體蛋白對ANS螢光強度的影響
圖二十四 氧化14小時後透析的a-水晶體蛋白對ANS螢光強度的影響
圖二十五 氧化5小時後透析的a-水晶體蛋白防止胰島素被DTT誘發集結沈澱的能力
圖二十六 氧化10小時後透析的a-水晶體蛋白防止胰島素被DTT誘發集結沈澱的能力
圖二十七 氧化12小時後透析的a-水晶體蛋白防止胰島素被DTT誘發集結沈澱的能力
圖二十八 氧化14小時後透析的a-水晶體蛋白防止胰島素被DTT誘發集結沈澱的能力

（1）N. S. Jaffe and J.Horwitz：Anatomy and embryology, in S. M. Podos and M. Yanoff(ed.), "Lens and Cataract", New York, Gower Medical Publishing, chapter 1, pp.1-7（1992）.
(2）R. M. Clayton：Developmental genetics of the lens, in H. Maisel (ed.)："The Ocular Lens", New York, Marcel Dekker, chapter 2, pp.61-92（1992）.
(3）廖邳銓 和 楊麟栩 ："一般眼科學"，南山堂出版社（1988）.
( 4）V. L. Taylor, K. J. Al-Ghoul, and M. J. Costello. Morphology of the normal human lens. Invest. Ophthalmol. Vis. Sci., 37, 1396-1410（1996）.
（5）M. Delaye and A. Tardieu. Short-range order of crystallin proteins account for eye lens transparency. Nature, 302, 415-417（ 1983）.
（6） J. J. Harding and K. J. Dillen. Structural protein of the mammalian lens. Exp. Eye Res., 22, 1-73（1976）.
（7） R. M. clayton：Structure of Lens Protein, in H. Maisel(ed.)："The Ocular Lens", New York, Marcel Dekker, chapter 4, pp.129-131（1992）.
（8）Y. Chirgadze, N. Nevskaya, E. Vernoslova, S. Nikonov, Y. Sergeev, E. Brazhnokov and A. Urzhumtsev. Crystal structure of calf lens gamma-crystallin IIIb resolution. Exp. Eye Res., 53, 295-304 （1991）.
（9）J. Bours.：Calf lens a-crystallin, a molecular chaperone, builds stable complex with b- and g-crystallins. Ophthalmic Res., 28（ suppl 1）, 23-31（1996）.
（10）G. Wistow, C. Slingsby and T. Blundell. Eye-lens protein : the three dimension structure of b-crystallin predicted from monomeric g-crystallin. FEBS Lett., 133, 9-16（1981）.
（11）M. K. Duncan, J. B. Mcdermott and J. P. Piatigorsky. Sequence and express of chicken bA2-and bB3-crystallins. Exp. Eye Res., 62, 111-119（1996）.
（12）H. J. M. Aarts, E. H. M. Jacsobs, G. van Willigen, N. H. Lubsen and J. G. G. Schoenmakers. Different evolution rats within lens-specific b-crystallin gene family. J. Mol. Evol., 28, 313-321（1989）.
（13）M. A. Martinus, E. A. Simone, J. J. Harding and W. W. de Jong. The influence of some post-translational Modifications on the chaperone-like activity of a-crystallin. Ophthalmic Res., 28（suppl 1）, 32-38（1996）.
（14）J. B. Smith, Y. Liu and D. L. Smith. Identification of Possible Regions of chaperone activity in lens a-crystallin. Exp. Eye Res., 63, 125-128（1996）.
（15）O. P. Lamba, D. Sinha, S. K. Shah and J. Renugopalakrishnan. Estimation of the secondary structure and conformation of bovine lens crystallins by infrared spectroscopy; quantitative analysis and resolution by Fourier self-deconvolution and curve fit. Biochim. Biophys. Acta, 1163, 113-123（1993）.
（16）R. J. Siezen and P. Argos. Structural similarity of lens crystallins. III. secondary structure estimation from circular dichroism and prediction from amino acid sequences. Biochim. Biophys. Acta, 748, 56-67（1983）.
（17） J. A. Carver, J. A. Aquilina and R. J. W. Truscott. An investigation into the stability of a-crystallin by NMR spectroscopy; evidence for a two-domain structure. Biochim. Biophys. Acta, 1164, 22-28（1993）.
（18）J. G. Bindels, R. J. Siezen and H. J. Hoenders. A model for the architecture of a-crystallin. Ophthalmic Res., 11, 441-452（ 1979）.
（19）A. Tardieu, D. Laport, P. Licinio, B. Krop and M. Delaye. Calf lens a-crystallin quaternary structure: a three-layer tetrahedral model. J. Mol. Biol., 192, 711-724（1986）.
（20）A. Carver, J. A. Aquilina, J. W. Truscott. A possible chaperone-like quaternary structure for a-crystallin. Exp. Eye Res., 59, 231-234（1994）.
（21）M. T. Walsh, A. C. Sen and B. Chakrabarti. Micellar subunit assembly in a three-layer model of oligomeric a-crystallin. J. Biol. Chem., 266, 20079-20084（1991）.
（22）G. J. Wistow. A possible quaternary structure for crystallins and small heat-shock proteins. Exp. Eye Res., 58, 729-732（1993 ）.
（23）E. R. Berman："Biochemistry of the Eye", New York, Plenum Press, chapter 5（1991）.
（24）G. Wistow. Domain structure and evolution in a-crystallins and aggregation. FEBS Lett., 181, 1-6（1985）.
（25） J. Clauwaert, J. F. Koretz, K. Thomson and R. C. Augusteyn. The effect of temperature on the renaturation of a-crystallin. Curr. Eye Res., 8, 397-403（1989）.
（26）N. C. Collier and M. J. Schlesinger. The dynamic state of heat-shock proteins in chicken embryo fibroblasts. J. Cell Biol., 103, 1495-1507（1986）.
（27）A. M. Leunissen and E. M. Christina. Evolution of the a-crystallin/small heat-shock protein family. Mol. Biol. Evol., 10, 103-126（1993）.
（28）J. Horwitz. a-crystallin can function as a molecular chaperone. Proc. Natl. Acad. Sci. USA., 89, 10449-10453（1992）.
（29）K. Wang and A. Spector. a-Crystallin can act as a chaperone under condition of oxidative stress. Invest. Ophthalmol. Vis. Sci. 36, 311-321（1995）.
（30）R. F. Borkman, G. Knight and B. Obi. The molecular chaperone a-crystallin inhibits UV-induced protein aggregation. Exp. Eye Res., 62, 141-148（1996）.
（31）A. C. John, A. N. Kerrie and J. A. Nicholls. Age-related change in bovine a-crystallin and high-molecular-weight Protein. Exp. Eye Res., 63, 639-647（1996）.
（32）Y. Zhucheng, C. Monica, L.S. David and B. S. Jean. Identification of the major components of the high molecular weight crystallins from old human lenses. Curr. Eye Res., 13, 415-421（1994）.
（33）O. P. Srivastava, K. Srivastava and C. Silney. Levels of crystallin fragments and identification of their origin in water soluble high molecular weight（HMW）proteins of human lenses. Curr. Eye Res., 15, 511-520（1996）.
（34）C. A. Paterson and N. A. Delamere：The lens, in W. M. Hart, Jr. （ed.）, "Adler's Physiology of the Eye", 9th ed., St. Louis, Mosby-Year Book, chapter 10, pp.359-369（1992）.
（35）E. R. Berman：Selected topics biochemistry relevant to the eye, in E. R. Berman（ed.）, "Biochemistry of the Eye.", New York, Plenum Press, chapter 2, pp38-40（1991）.
（36）N. A. Bruce, K. S. Mark and M. H. Tory. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA., 90, 7915-7922（1993）.
（37）J. W. Truscott and C. A. Robert. Oxidative changes in human lens proteins during senile nuclear cataract formation. Biochem. Biophys. Acta, 492, 43-52（1977）.
（38）L. Mikhail and B. J. Ortwerth . The generation of hydrogen peroxide by the UVA irradiation of human lens proteins. Photochem. Photobio, 62, 87-93（1995）.
（39）B. J. Ortwerth and P. R. Olesen. Glutathione inhibits the glycation and crosslinking of lens proteins by ascorbic acid. Exp. Eye Res., 47, 737-750（1994）.
（40）S. D. Varma, S. Ramachandran, P. S. Devamanoharan, S. M. Morris and A. H. Ali. Prevention of oxidative damage to rat lens by pryuvate in vitro: possible attenuation in vivo. Curr. Eye Res., 14, 643-649（1995）.
（41）A. Taylor, P. F. Jacques, T. Nowell, G. Perrone, J. Blumberg, G. Handelman, B. Jozwiak and D. Nadler. Vitamin C in human and guinea pig aqueous, lens and plasma in relation to intake. Curr. Eye Res., 16, 857-864（1997）.
（42）L. Packer. Protective role of vitamin E in biological system. Am J Clin Nutr , 53, 1050S-1055S（1991）.
( 43 ) Spector A, Garner WH. Hydrogen peroxide and human cataract. Exp Eye Res, 33, 673-681 ( 1981 )
( 44 ) Bhuyan DK. Peroxide concentration in normal and cataractous human lenses. Invest Ophthalmol Vis Sci, 33, 789 ( 1992 )
( 45 ) Ramachandran S, Morris SM, Devamanobaran P. Radio-isotope determination of hydrogen peroside in aqueous humor and urine. Exp Eye Res, 53, 503-506 ( 1991 )
( 46 ) Dische Z, Zil H. Studies on the oxidation of cysteine to cystine in lens proteins during cataract formation. Am J Ophthalmol, 34, 104-113 ( 1951 )
( 47 ) Bhuyan KC, Bhuyan DK. Evidence of increased lipid perosidation in cataracts IRC Med Sci., 9, 126-127 ( 1981 )
( 48 ) Worgul BV, David J.. Evidence of genotoxic damage in human cataractous lenses. Mutagen, 6, 495-499 ( 1991 )
( 49 ) Horwitz J.. Alpha crystallin can function as a molecular chaperone. Proc Natl Acad Sci USA, 89, 10449-10453 ( 1992 )
( 50 ) Sergei Yu. Venyamionv and Jen Tsi Yang. Determination of protein secondary structure. pp.70-103.
（51）賴建霖："大白鼠晶狀素蛋白在氧化環境下非活體之研究"成功
大學化學研究所碩士論文,（1997）
( 52 ) Sreerama ＆ Woody . A Self-consistent Method for the analysis of protein secondary structure from Circular Dichroism. Anal. Biochem., 209, 32-44 ( 1993 )
( 53 ) Sreerama et al.. Estimation of the number of a-helical and b-strand segments in proteins using CD spectroscopy. Protein Science, 8, 370-380 (1999 )
(54) Iwaki T. Kume-Iwaki A. Liem RK. Goldman JE. Alpha
B-crystallin is expressed in non-lenticular tissues and accumulates in
Alexander's disease brain. Cell. 57(1):71-8 ( 1989).
（55）K. Wang and A. Spector. The chaperone activity of bovine a-crystallin.
J. Biol. Chem., 269, 13061-13608（1994）.
（56）B. Raman and C. M. Roa. Chaperone-like activity and quaternary structure of a-crystallin.J. Biol. Chem., 266, 20079-20084（ 1991）.
（57）K. D. Biplsb, J. -N. L. Jack and C. Bir.eswar Heat-induced conformational change and increase chaperone activity of lens a-crystallin. Curr. Eye Res., 16, 303-309（1997）.
(58) Harding, J.J. and Carbbe, M.J.C.. The lens:Development,
proteins,metabolism and cataract. The Eye. Vol.1b.Pp.207-
492.New York, Academic Press.(1984)
(59) Ifeanyi, F. and Takemoto, L..Differential binding of a-
crystallin to bovine lens membrane. Exp. Eye Res.49,143-
7. (1989).
(60) Jack N.Liang and Xiao-yan Li. Interaction and aggregation
of lens crystallins. Exp. Eye Res.53,61-66. (1989)