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研究生: 馬怡婷
Yi-Ting Ma
論文名稱: 鹽類效應對溶菌酶溶液性質影響之研究
Studies of salt effect on properties of lysozyme solutions
指導教授: 洪伯達
Po-Da Hong
口試委員: 蔡協致
Hsieh-Chih Tsai
程君弘
Chun-Hung Cheng
學位類別: 碩士
Master
系所名稱: 工程學院 - 材料科學與工程系
Department of Materials Science and Engineering
論文出版年: 2019
畢業學年度: 107
語文別: 中文
論文頁數: 53
中文關鍵詞: 溶菌酶雲點溫度蛋白質溶液相圖液-液相分離膠體系統相界線
外文關鍵詞: Hofmeister series, DLVO theory, depletion interaction, metastable, anion-specific, chaotropes, kosmotropes, protein-poor liquid phase, protein-rich liquid phase
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蛋白質分子間的相互作用力為使蛋白質溶液中呈現多樣化聚集現象的主因,而相互作用力的大小會受到溶液環境條件的影響。因此,本研究我們固定鹽類溶液為3wt% NaCl,緩衝溶液為0.1M NaAc,pH 4.5的條件下建立一球形蛋白質溶液相圖。利用加裝linkam控溫裝置和CCD的光學顯微鏡,紀錄觀察結晶溶解情形,得到溶解度數據。利用雲點測量儀偵測透光強度隨溫度的變化,測得雲點數據。然後,將兩個實驗數據分別帶入理論式子擬合,而得到液-固相界線和液-液相界線。蛋白質溶液便可依據濃度和溫度的不同,劃分成三個區域:透明均一區、液-固共存區、液-液分離區。

添加不同種鹽類實驗中,我們固定蛋白質濃度為85 mg/ml,緩衝溶液為0.1M NaAc,pH 4.5,選用四種鹽類 (NaCl, NaBr, NaI, NaClO4) 測定雲點溫度隨鹽類濃度的變化。由實驗結果得知,雲點溫度呈現的行為相當複雜。在低濃度鹽類區,陰離子對雲點溫度的影響性會遵循inverse Hofmeister series;在較高濃度鹽類區,順序會反轉成direct Hofmeister series。我們試著以chaotropic陰離子特定吸附現象和由離子改變表面張力大小的行為去解釋雲點溫度呈現的複雜行為。


The interaction between protein molecules is the main reason for the diversified aggregation phenomenon in protein solution, and the interaction force is affected by the environmental conditions of the solution. Therefore, in this study, we established a phase diagram for spherical protein solution, which fixed a salt solution of 3wt% NaCl and a buffer solution of 0.1M NaAc at pH 4.5. Optical microscope equipped with linkam temperature control device and the CCD was used to record and observe the dissolution of the crystal to obtain the solubility data. The cloud point data was measured by detecting the change of the light transmission intensity with temperature by using a cloud point measuring device. Then, the two experimental data were respectively fitting into the theoretical equation to obtain liquid-solid phase boundary and the liquid-liquid phase boundary. According to different concentration and temperature, the protein solution can be divided into three regions: homogeneous region, liquid-solid coexistence region and liquid-liquid separation region.

In the experiment of adding different kind of salts, we fixed the protein concentration of 85 mg/ml and a buffer solution of 0.1M NaAc at pH 4.5. Four kind of salts (NaCl, NaBr, NaI, NaClO4) were used to determine the change of cloud point temperature with salt concentration. It’s known from the result of the experiment that the behavior of cloud point temperature is quite complicated. In low salt concentration region, the effect of anions on cloud point temperature follows an inverse Hofmeister series. At higher salt concentration region, the order reverts to a direct Hofmeister series. We tried to explain the complex behavior of cloud point temperature by the specific adsorption phenomenon of chaotropic anions and the behavior of changing the surface tension by ions.

論文提要內容 I Abstract II 誌謝 III 目錄 IV 圖表目錄 VI 論文符號表 VIII 第一章 前言 1 1.1蛋白質結構 1 1.2膠體系統 4 1.2.1帶電膠體溶液的穩定性 5 1.2.2 Depletion interaction 7 1.2.3膠體系統相圖 8 1.3 蛋白質的聚集行為 10 1.4 Hofmeister series 10 1.5 離子分散力 12 1.6 相轉變 12 1.7研究目的 15 第二章 實驗方法 16 2.1蛋白質溶液與鹽類溶液的製備 16 2.2蛋白質濃度標定 18 2.3實驗方法 19 2.3.1光學顯微鏡 19 2.3.2雲點測量儀 (Cloud Point Measuring Device) 20 2.3.3 相界線的測定 (Determination of Phase Boundaries) 20 第三章 結果與討論 22 3.1蛋白質溶液相圖 22 3.2 蛋白質聚集形態 (Morphology) 26 3.3不同鹽類種類和濃度對雲點溫度的影響 30 3.4不同種鹽類液-液相分離的聚集形態 33 第四章 結論 37 參考文獻 38

1. Branden, C.; Tooze, J., “Introduction to protein structure, 2nd”, Garland publishing, New York (1998).
2. Grigsby, J.J.; Blanch, H.W.; Prausnitz, J.M., “Cloud point temperatures for lysozyme in electrolyte solutions: effect of salt type, salt concentration and pH”, Biophys. Chem., 91, 231-243 (2001).
3. Gunton, J. D.; Shiryayev, A.; Pagan, D. L., “Protein condensation: Kinetic Pathways to Crystallization and Disease”, Cambridge University Press, New York (2007).
4. Broide, M. L.; Tominc, M. T.; Saxowsky, M. D., “Using phase transitions to investigate the effect of salts on protein interactions”, Phys. Rev. E, 53, 6325 (1996).
5. Muschol, M.; Rosenberger, F., “Liquid-liquid phase separation in supersaturated lysozyme solutions and associated precipitate formation/crystallization”, J. Chem. Phys., 107, 1953 (1997).
6. Berland, C. R.; Thurston, G. M.; Kondo, M.; Broide, M. L.; Pande, J.; Ogun, O.; Benedek, G. B., “Solid-liquid phase boundaries of lens protein solutions”, Proc Natl Acad Sci USA, 89, 1214-1218 (1992).
7. Tanaka, S.; Yamamoto, M.; Ito, K.; Hayakawa, R.; Ataka, M., “Relation between the phase separation and the crystallization in protein solutions”, Phys, Rev. E, 56 (1997).
8. Zhang Y.; Cremer, P. S., “The inverse and direct Hofmeister series for lysozyme”, Proc Natl Acad Sci USA, 106, 15249-15253 (2009).
9. Lekkerkerker, H. N. W.; Tuinier, R., “Colloids and the depletion interaction”, Springer Science + Business Media (2011).
10. 李以圭, 陸九芳 “電解質溶液理論”,清華大學出版社 (2005)
11. Wolde, P. R.; Frenkel, D., “Enhancement of Protein Crystal Nucleation by Critical Density Fluctuations”, Science, 277, 1975-1978 (1997).
12. Anderson, V. J.; Lekkerkerker, H. N. W., “Insights into phase transition kinetics from colloid science”, Nature, 416, 811-815 (2002).
13. Kunz, W., “Specific ion effects”, World Scientific (2010).
14. Zhang Y.; Cremer, P. S., “Interactions between macromolecules and ions: the Hofmeister series”, Curr Opin Chem Biol, 10, 658-663 (2006).
15. Collins, K. D., “Charge Density-Dependent Strength of Hydration and Biological Structure”, Biophys. J., 72, 65-76 (1997).
16. Ninham, B. W.; Yaminsky, V., “Ion-binding and ion specificity: The Hofmeister effect and Onsager and Lifshitz theories”, Langmuir, 13, 7, 2097-2108 (1997).
17. Boström, M.; Williams, D. R. M.; Ninham, B. W., “Specific ion effects: why DLVO theory fails for biology and colloid systems”, Phys. Rev. Lett., 87, 168103 (2001).
18. Boström, M.; Tavares, F. W.; Finet, S.; Skouri-Panet, F.; Tardieu, A.; Ninham, B. W., “Why forces between proteins follow different Hofmeister series for pH above and below pI”, Biophys. Chem., 117, 217-224 (2005).
19. Finet, S.; Skouri-Panet, F.; Casselyn, M.; Bonneté, F.; Tardieu, A., “The hofmeister effect as seen by SAXS in protein solutions”, Curr Opin Colloid Interface Sci, 9, 112-116 (2004).
20. Boström, M.; Williams, D. R. M.; Ninham, B. W., “Special ion effects: why the properties of lysozyme in salt solutions follow a Hofmeister series”, Biophys. J., 85, 686-694 (2003).
21. Boström, M.; Williams, D. R. M.; Ninham, B. W., “Ion specificity of micelles explained by ionic dispersion forces”, Langmuir, 18, 16, 6010-6014 (2002)
22. Boström, M.; Lonetti, B.; Fratini, E.; Baglioni, P.; Ninham, B. W., “Why pH titration in protein solutions follows a Hofmeister series”, J. Phys. Chem. B, 110, 14, 7563-7566 (2006).
23. Salis, A.; Pinna M. C.; Bilaničová, D.; Monduzzi, M.; Nostro, P. L.; Ninham, B. W., “Specific anion effects on glass electrode pH measurements of buffer solutions: bulk and surface phenomena”, J. Phys. Chem. B, 110, 6, 2949-2956 (2006).
24. Voinescu, A. E.; Bauduin, P.; Pinna, M. C.; Touraud, D.; Ninham, B.W.; Kunz, W., “Similarity of salt influences on the pH of buffers, polyelectrolytes, and proteins”, J. Phys. Chem. B, 110, 17, 8870-8876 (2006).
25. Boström, M.; Williams, D. R. M.; Ninham, B. W., “Surface tension of electrolytes: Specific ion effects explained by dispersion forces”, Langmuir, 17, 15, 4475-4478 (2001).
26. Boström, M.; Kunz W.; Ninham, B. W., “Hofmeister effects in surface tension of aqueous electrolyte solution”, Langmuir, 21, 6, 2619-2623 (2005).
27. Israelachvili, J. N., “Intermolecular and surface forces”, Academic Press, pp. 213-259 (1991).
28. Zhang, Y. J.; Furyk, S; Bergbreiter, D. E,; Cremer, P. S., “Specific ion effects on the water solubility of macromolecules: PNIPAM and the Hofmeister series”, J Am Chem Soc, 127, 14505-14510 (2005).
29. Zhang Y.; Furyk, S.; Sagle, L. B.; Cho, Y.; Bergbreiter, D. E.; Cremer, P. S.; “Effects of Hofmeister anions on the LCST of PNIPAM as a function of molecule weight”, J Phys Chem C, 111, 8916-8924 (2007).
30. Cho, Y.; Zhang, Y.; Christensen, T.; Saqle, L. B.; Chilkoti, A.; Cremer, P. S., “Effects of Hofmeister anions on the phase transition temperature of elastin-like polypeptides”, J. Phys. Chem. B, 112, 44, 13765-13771 (2008).
31. Chen, X.; Yang, T.; Kataoka, S.; Cremer, P. S., “Specific ion effects on interfacial water structure near macromolecules”, J. Am. Chem. Soc., 129, 40, 12272-12279 (2007).
32. Pegram, L. M.; Thomas Record Jr., M., “Thermodynamic origin of Hofmeister ion effects”, J. Phys. Chem. B, 112, 31, 9428-9436 (2008).
33. Jarvis, N. L.; Scheiman, M. A., “Surface potentials of aqueous electrolyte solutions”, J. Phys. Chem., 72, 1, 74-78 (1968).
34. Pegram, L. M.; Thomas Record Jr., M., “Partitioning of atmospherically relevant ions between bulk water and the water/vapor interface”, Proc Natl Acad Sci USA, 103, 14278-14281 (2006).
35. Pegram, L. M.; Thomas Record, M., “Hofmeister salt effects on surface tension arise from partitioning of anions and cations between bulk water and the air-water interface”, J. Phys. Chem. B, 111, 19, 5411-5417 (2007)
36. Wu, T.; Jiang, Q.; Wu, D.; Hu, Y.; Chen, S.; Ding, T.; Ye, X.; Liu, D.; Chen, J., “What is new in lysozyme research and its application in food industry? A review”, Food Chem., 274, 698-709 (2018).
37. Pusey, M. L.; Gernert, K., “A method for rapid liquid-solid phase solubility measurements using the protein lysozyme”, J. Cryst. Growth, 88, 419-424 (1988).
38. Rosenberger, F.; Meehan, E. J., “Control of nucleation and growth in protein crystal growth”, J. Cryst. Growth, 90, 74-78 (1988).
39. Howard, S. B.; Twigg, P. J.; Baird, J. K.; Meehan, E. J., “The solubility of hen egg-white lysozyme”, 90, 94-104 (1988).
40. Segrè, P. N.; Prasad, V.; Schofield, A. B.; Weitz, D. A., “Glasslike kinetic arrest at the colloidal-gelation transition”, Phys. Rev. Lett., 86, 6042 (2001).
41. Ries-Kautt, M. M.; Ducruix, A. F., “Relative effectiveness of various ions on the solubility and crystal growth of lysozyme”, J. Biol. Chem., 264,745-748 (1989).
42. 王昱凱, 碩士論文 “蛋白質溶液聚集形為之研究”, 台灣科技大學高分子工程研究所 (2007).
43. Salvetti, G.; Tombari, E.; Mikheeva, L.; Johari, G. P., “The endothermic effects during denaturation of lysozyme by temperature modulated calorimetry and an intermediate reaction equilibrium”, J. Phys. Chem. B, 106, 23, 6081-6087 (2002).
44. Yan, H.; Frielinghau, H.; Nykanen, A.; Ruokolainen, J.; Saiani, A.; Miller, A. F., “Thermoreversible lysozyme hydrogels: properties and an insight into the gelation pathway”, Soft Matter, 4, 1313 (2008).
45. 陳詩佳, 碩士論文“蛋白質溶液聚集形為與形態學之研究”, 台灣科技大學高分子工程研究所 (2008).
46. Taratuta, V. G.; Holschbach, A.; Thurston, G. M.; Blankschtein, D.; Benedek, G. B., “Liquid-liquid phase separation of aqueous lysozyme solutions: effects of pH and salt identity”, J. Phys. Chem., 94, 5, 2140-2144 (1990).
47. Cacioppo, E.; Pusey, M. L., “The solubility of the tetragonal form of hen egg white lysozyme from pH 4.0 to 5.4”, J. Cryst. Growth, 114, 286-292 (1991).
48. Fiobelo, F. L.; Galkin, O.; Vekilov, P. G., “Spinodal for the solution-to-crystal phase transformation”, J. Chem. Phys., 123, 014904 (2005).
49. Sedgwick, H.; Kroy, K.; Salonen, A.; Robertson, M. B.; Egelhaaf, S. U.; Poon, W. C. K., “Non-equilibrium behavior of sticky colloidal particle: beads, clusters and gels”, Eur. Phys. J. E., 16, 77 (2005).
50. Tanaka, H.; Nishikawa, Y., “Viscoelastic phase separation of protein solutions”, Phys. Rev. Lett., 95, 078103 (2005).
51. Velev, O. D.; Pan, Y. H.; Kaler, E. W.; Lenhoff, A. M., “Molecular effects of anionic surfactants on lysozyme precipitation and crystallization”, Cryst. Growth Des., 5, 351 (2005).
52. Haasen, P., Materials Science and Technology, Vol. 5., “Phase Transformation in Materials”, VCH (1991).

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