不同化學變性劑、溫度和還原劑對溶菌

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姓名

周茂村(Mao-Tsun Chou) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

不同化學變性劑、溫度和還原劑對溶菌
(The studies of the lysozyme structure change with different chemical denaturants, temperatures and reduced reagent)

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摘要(中)

重組蛋白質在回收過程中，常會因為胜

摘要(英)

In the process of recombinant protein production, we often get inactive and insoluble inclusion bodies instead of target proteins due to high peptide concentration. In industrial application, denaturants such as urea or guanidine hydrochloride are used to solve these inclusion bodies to renature and purify/separate proteins and then denaturants are removed to get the protein we want.
Factors to stabilize protein structures include hydrophobic interactions, hydrogen bonding, electrostatic interaction and covalent disulfide bonding. Urea is mainly used to break hydrophobic interactions and guanidine hydrochloride is to destroy hydrophobic and electrostatic interactions. Sometimes, salts such as sodium chloride/lithium chloride are added in urea as electrostatic interaction destroyer to enhance the denaturant power. Another commonly used reduced reagent (dithiothreitol, DTT) is to reduce disulfide bonding within protein. All these different mechanisms bring various influences to protein structures.
In our study, we examine secondary and tertiary structure changes of lysozyme in various conditions (different concentrations of urea / guanidine hydrochloride / salts (sodium chloride), experimental temperature, with/without reduced reagent) with the aid of circular dichroism and fluorescence spectrophotometer to investigate the relationship between above factors and spectrum structure.
When reduced reagent exists, guanidine hydrochloride causes greater changes in protein structure than urea and sodium chloride. Without reduced reagent, only high concentration of guanidine chloride causes significant protein structure changes. The results of fluorescence experiments indicates that tryptophan inside protein didn’’t expose easily when disulfide bond remained unbroken.
Besides, from temperature manipulation experiments, we see the denature reaction with denaturants and reducing agents added is an exothermic reaction. At the mean time, the entropy of system decreases and melting temperature (Tm) is lower than that in buffer solution.
From the above experiments, we know that if the disulfide bond is not reduced, we have to use denaturant with the capacity to break hydrophobic and electrostatic interactions in high concentration to induce significant changes in lysozyme structure. When disulfide bond is reduced, denaturants causes changes of lysozyme structure easier that indicate lysozyme is a hard protein itself.
It’s possible to investigate the relationship between activity and structure of enzymes with the aid of ultraviolet spectrometer. In further studies, comparing reaction enthalpy in various conditions measured by isothermal titration calorimeter with thermodynamic data from spectrometer, we can learn the pathway of lysozyme denature process to see it’s two state or multi-state phase transformation reaction.

關鍵字(中)

★ 溶菌
★ 螢光光譜儀
★ 圓二色光譜儀
★ 二硫代蘇糖醇
★ 胍鹽酸
★ 尿素
★ 還原劑
★ 結構
★ 變性劑
★ 溫度

關鍵字(英)

★ temperature
★ denaturant
★ structure
★ lysozyme
★ fluorescence spectrophotometer
★ circular dichroism
★ reduced reagent
★ urea
★ guanidine hydrochloride
★ dithiothreitol

論文目次

目錄
中文摘要......................................................................Ⅰ
Abstract......................................................................Ⅲ
目錄..........................................................................Ⅴ
圖目錄........................................................................Ⅷ
表目錄......................................................................ⅩⅠ
第一章緒論..................................................................1
第二章實驗原理與文獻回顧....................................................3
2.1 蛋白質介紹............................................................3
2.1.1 蛋白質的組成、功能及穩定結構之作用力..............................3
2.1.2 蛋白質結構........................................................9
2.1.3 穩定態的構形與特性...............................................14
2.1.4 蛋白質摺疊模型...................................................15
2.2 蛋白質之變性.........................................................17
2.2.1 破壞蛋白質穩定力量的方式.........................................17
2.2.2 變性劑與還原劑對蛋白質分子之作用.................................18
2.3 常見的分析方法.......................................................20
2.4 圓二色光譜儀.........................................................21
2.4.1 圓二色光譜儀發展歷史.............................................21
2.4.2 光的吸收與光學活性的關係.........................................24
2.4.3 圓二色光譜儀測量原理.............................................28
2.4.4 蛋白質或多胜

參考文獻

1. Armstrong, F. B. Biochemistry. Oxford, 1989.
2. Bailey, P. D. An introduction to peptide chemistry. John Wiley & Sons, Ltd., 1990.
3. Berges, H., Kassah, E., Conte, D., Adjadj, E., Houee-Levin, C. "Ab-Initio calculations on arginine-disulfide complexes modeling the one-electron reduction of lysozyme. Comparison to an experimental reinvestigation." J. Phys. Chem., 101(42), 7809-7817, 1997.
4. Beychok, S., Warner, R.C. "Denaturation and electrophoretic behavior of lysozyme." J. Am. Chem., 81, 1892, 1959.
5. Bhattacharjya, S., Balaram, P. "Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg-white lysozyme." Protein Sci., 6, 1065-1073, 1997.
6. Blake, C. C. F., Koenig, D. F., Mair, G. A., North, A. C. T., Phillips, D. C., Sarma, V. R. "Structure of hen egg-white lysozyme." Nature, 22, 757-761, 1965.
7. Bovey, F.A. Polymer conformation and configuration. Academic Press Inc., 1969.
8. Boyer, R. F. Biochemistry. Brooks/Cole Publishing Company, 1999.
9. Campbell, M. K. Biochemistry. Saunders College Publishing, 1995.
10. Canfield, R. E. "Chapter 1: Introduction", In Lysozyme. Osserman, E. F., Canfield, R. E., Beychok, S. (Eds). Academic press. 1974.
11. Carrect, R. H., Grisham, C. M. Biochemistry. Saunders College Publishing. 1999.
12. Copeland, R. A. Methods for protein analysis. Chapman & Hall, 1994.
13. Charney, E. The molecular basis of optical activity: Optical rotatory dispersion and circular dichroism. John Wiley & Sons, Inc., 1979.
14. Cunningham, F. E., Lineweaver, H. "Stabilization of egg-white proteins to pasteurizing temperatures above 60℃." Food. Technol., 19, 136, 1965.
15. Crabbé, P. In ORD and CD in chenistry and biochemistry. Academic Press Inc., 1972.
16. Darby, N. J., Creighton, T. E. Protein structure. IRL Press at Oxford University Press, 1993.
17. Day, R. A., Underwood, A. L. Quantitative analysis. Prentice Hall, 1991.
18. Dickinson, E. Matsumura, Y. "Proteins at liquid interfaces: role of molten globule state." Colloids Surfaces B., 3(1-2), 1-17, 1994.
19. Dill, K.A. "Theory for the folding and stability of globular-proteins." Biochemistry, 24(6), 1501-1509, 1985.
20. Fasman, G. D. Circular dichroism and the conformation analysis of biomolecules. Plenum Press. 1996.
21. Fersht, A. Structure and mechanism in protein science. W. H. Freeman and Co. 508-539, 547-553, 1999.
22. Freire, E. "Thermal denaturation methods in the study of protein folding." Meth. Enzymol., 259, 144-168, 1995.
23. Gorin, G., Wang, S. -F., Wilson, B. M. "Assay of lysozyme by its lytic action on Micrococcus lysodeikticus cell." Anal. Biochem., 39, 113, 1971.
24. Gorin, L., Felic, F. "Influence du manganede sur la stabilite du lysozyme. I. Influence du manganese ser le vitesse d’’ inactivation irreversible du lysozyme par la chaleur." Biochem. Biophys. Acta., 10, 128,1953.
25. Greenfield, N.J. "Review: Methods to estimate the conformation of proteins and polypeptides from circular dichroism data." Anal. Biochem., 235, 1-10, 1996.
26. Hamaguchi, K. "Conformation and enzymatic activity of lysozyme." Tampakushitsu. Kakusan Kosa., 13, 98, 1968.
27. Hash, J. H. "Chapter 9: Lysozyme chalarposis", In Lysozyme. Osserman, E. F., Canfield, R. E., Beychok, S. (Eds). Academic press. 1974.
28. Hermans, J. "Methods for the study of reversible denaturation of proteins and interpretation of data." Methods Biochem. Anal., 13, 81-111, 1965.
29. Imoto. T., Yagishita, K. "A Simple activity measurement of lysozyme." Agr. Biol. Chem., 35, 1154, 1971.
30. Jirgensons, B., Philadelphia, A. K., Evanston, G. F. S., Berlin, H. G. W. Optical rotatory dispersion of proteins and other macromolecules. Springer-Verlag New York Inc., 1969.
31. John, D. M., Weeks, K. M. "For the record: van’’t Hoff enthalpies without baselines." Protein Sci., 9, 1416-1419, 2000.
32. Karplus, M., Weaver, D. L., Nature, 260, 404-406, 1976.
33. Kauzmann, W. "Some factors in the interoretation of protein denaturation." Adv. Protein Chem., 14, 1-63, 1959.
34. Kuwajima, K. "The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure." Protein. Struct. Funct. Genet., 6(2), 87-103, 1989.
35. Laurents, D. V., Baldwin, R. L. "Characterization of the unfolding pathway of hen egg white lysozyme." Biochemistry, 36, 1496-1504, 1997.
36. Lightner, D. A., Gurst, J. E. Organic conformational analysis and stereochemistry from circular dichroism spectroscopy. John Wiley & Sons, Inc., 2000.
37. Liu, Y., Sturtevant, J. M. "The observed change in heat capacity accompanying the thermal unfolding of proteins depends on the composition of the solution and on the method employed to change the temperature of unfolding." Biochemistry, 35, 3059-3062, 1996.
38. Lumry, R., Bioltonen, R., Brandts, J. F. "Validity of the "two-state" hypothesis for conformational transitions of proteins." Biopolymers, 4, 917-944, 1966.
39. Makki, F., Durance, T. D. "Thermal inactivation of lysozyme as influenced by pH, sucrose and sodium chloride and inactivation and preservative effect in beer." Food Res. Int., 29(7), 635-645, 1996.
40. Mathews, C. K., Holde, K. E. V., Ahern, K.G. Biochemistry. Addison Wesley Longman, Inc. 2000.
41. Matsubara, M., Nohara, D., Sakai, T. "Difference between guanidinium chloride and urea as denaturants of globular protein: The possibility of application to improve refolding process." Chem. Pharm. Bull., 40, 550-552, 1992.
42. Mckee, T. "Biochemistry." Wm.C. Brown Publisher, 1996.
43. Muesing, R. A., Nishida, T. "Disruption of low- and high-density human plasma lipoproteins and phospholipid dispersions by 1-anilinonaphthalene-8-sulfonate." Biochemistry, 10(15), 2952-2962, 1971.
44. Nakanishi, K., Berova, N., Woody, R. W. Circular dichroism principles and applications. VCH Publishers, Inc., 1994.
45. Neurath, H., Greenstein, J. P., Putnam, F. W., Erickson, J.O. "The chemistry of protein denaturation." Chem. Rev., 32, 157-265, 1943.
46. Nölting, B. Protein folding kinetic. Springer-Verlag Berlin Heidelberg, 1999.
47. Ohgushi, M., Wada, A. " ’’Molten-globule state’’: A compact form of globular-proteins with mobile side-chains." FEBS Lett., (164(1), 21-24, 1983.
48. Osserman, E. F., Canfield, R. E., Beychok, S. "Sections 3: biological and clinical studies", in Lysozyme. Osserman, E. F., Canfield, R. E., Beychok, S. (Eds). Academic press. 1974.
49. Phillips, D. C. "Chapter 2: The properties of lysozyme", in Lysozyme. Osserman, E. F., Canfield, R. E., Beychok, S. (Eds). Academic press. 1974.
50. Proctor, V. A. Cunningham, F. E. "The chemistry of lysozyme and its use as a food preservative and a pharmaceutical." CRC Crit. Rev. Food Sci. Nutr., 26(4) 359-395, 1988.
51. Ptitsyn, O. B. Protein folding. Creighton, T. E. (Ed.), Freeman, 243, 1992.
52. Ptitsyn, O. B., Pain, R. H., Semisotnov, G. V., Zerovnik, E., Razgulyaev, O. I. "Evidence for a molten globule state as a general intermediate in protein folding." FEBS Lett., 262(1), 20-24, 1990.
53. Raford, S. E., Dobson, C. M., Evans, P. A. "The folding of hen lysozyme involves partially structure intermediates and multiple pathways." Nature, 358, 302-307, 1992.
54. Rodger, A., Nordén, B. "Circular dichroism and linear dichroism." Oxford, 1997.
55. Rothwarf, D. M., Scheraga, H. A. "Role of non-native aromatic and hydrophobic interaction in the folding of hen egg white lysozyme." Biochemistry, 35, 13797-13807, 1996.
56. Sadana, A. "Review: protein refolding and inactivation during bioseparation: Bioseparation implication." Biotechnol. Bioeng., 48(5), 481-489, 1995.
57. Sandow, A. "The anti-bacterial activity of egg-white." Proc. Soc. Exp. Biol. Med., 24, 172, 1926.
58. Sasahara, K., Demura, M., Nitta, K. "Partially unfolded equilibrium state of hen lysozyme studied by circular dichroism spectroscopy." Biochemistry, 39, 647506482, 2000.
59. Saxena, V. P., Wetlaufer, D. B., "Formation of three-dimensional structure in proteins. I. Rapid nonenzymic reactivation of reduced lysozyme." Biochemistry, 9, 5015-5023, 1970.
60. Shakhnovich, E. I., Finkelstein, A. V. "Theory of cooperative transitions in protein molecules. 1. Why denaturation of globular protein is a 1st-order phase-transition." Bioplyomers, 28(10), 1667-1680, 1989.
61. Sherman, R. D., Charles, M. P. "Factors affecting the activity of egg white lysozyme." Arch. Biochem. Biophys., 40, 364, 1951.
62. Smith, A. L. Shirazi, H. M., Mulligan, S. R. "Water sorption isotherms and enthalpies of water sorption by lysozyme using the quartz crystal microbalance/heat conduction calorimeter." Biochem. Biophys. Acta., 1594, 150-159, 2002.
63. Summers, C. A., Flowers Ⅱ, R. A. "Protein renaturation by the liquid organic salt ethylammonium nitrate." Protein Sci., 9, 2001-2008, 2000.
64. Teale, F. W., Weber, G. "Ultraviolet fluorescence of the aromatic amino acids." Biochem. J., 65, 476-482, 1957.
65. Tedford, L. -A., Smith, D., Schaschke, C. T. "High pressure processing effects on the molecular structure of ovalbumin, lysozyme and b-lactoglobin." Food Res. Int., 32, 101-106, 1999.
66. Townes, J. K. "Moisture content in proteins: its effects and measurement." J. Chromatogr. A., 705, 115-127, 1995.
67. van den Berg, B. Chung, E. W., Robinson, C. V., Dobson, C. M. "Characterization of the dominant oxidative folding intermediate of hen lysozyme." J. Mol. Biol., 290, 781-796, 1999.
68. Velluz, L., Legrand, M., Grosjean, M., Optical circular dichroism. Academic Press, Inc., 1965.
69. Wescott, C. R., Klibanov, A. M. "The solvent dependence of enzyme specificity." Biochem Biophys. Acta., 1206(1), 1-9, 1994.
70. West, S. M., Guise, A. D., Chaudhuri, J. B. "A comparison of the denaturants urea and guanidine hydrochloride on protein refolding." Food Bioprod. Process., 75, 50-56, 1997.
71. Wetlaufer, D. B. Adv. Protein Chem., 17, 303-390, 1962.
72. Yang, J. T., Wu, C. -S. C., Martinez, H.M. "Calculation of protein conformation from circular dichroism." Meth. Enzymol., 130, 208-269, 1986.
73. Yashitake, S., Shinichiro, A. "Use of egg white lysozyme in the food industry." New. Food. Inc., 19, 17, 1977.
74. 江建民 "應用螢光及旋光技術於生命薄膜的研究." 科儀新知, 18(6), 44-57, 1997.
75. 林敬二、林宗義儀器分析 (上冊). 美亞書版股份有限公司, 1992.
76. 李雅雯 "Ribonuclease A於逆微胞系統中的復性及其界面現象之研究." 中央大學化學工程研究所碩士. 2001.
77. 郭春芳 "以circular dichroism探討溫度、pH值及鹽濃度對多醣構形之影響." 台灣大學食品研究所碩士. 2000.
78. 陳美方 "酸鹼度和相對黏度對於神經毒蛋白的開放與回疊路徑的探討." 台灣大學化學研究所碩士. 2000
80. 陳致真 "以旋光法量葡萄糖溶液濃度之統計設計與研製." 台灣大學電機工程研究所碩士. 1997.
81. 孫逸民、陳玉舜、趙敏勳、謝明學、劉興鑑儀器分析. 全盛圖書有限公司. 1997.
82. 賀孝雍儀器分析. 曉園出版社有限公司. 1991.
83. 黃世權 "澱粉分解酶摺疊程序之研究." 中原大學化學工程研究所碩士. 2001.
84. 程郁郁 "含假異胞嘧啶及其衍生物之去氧寡核酸鏈在水溶液中形成核酸三螺旋之研究." 中國文化大學應用化學研究所碩士. 1997.
85. 董有蘭化學與生命. 國立編譯館. 1999.
86. 趙世彬、鄒煦杕、蔡東璣、陳河吉、周大中、李文齡生物化學. 藝軒圖書出版社. 1998.
87. 劉茂宏、張勝善 "雞蛋白溶菌

指導教授

陳文逸(Wen-yih Chen)

審核日期

2002-7-4

推文