臺灣博碩士論文加值系統

本實驗目的在探討逆相液相層析法中影響peptides及蛋白質分離的一些重要參數包括：有機溶劑、離子對試劑及壓力等。其中並針對壓力影響作進一步的熱力學探討以瞭解壓力所造成之作用力改變。
在有機溶劑方面，分別比較以甲醇和氰甲烷作為有機沖提溶劑時，對核糖核酸A ( ribonuclease A )、溶菌 ( lysozyme )、α-乳白蛋白 (α-lactalbumin )在碳十八（C18）靜相上滯留機制的影響。這三種蛋白質的胺基酸數目及分子量很相近，主要的差異在於其疏水性大小。結果發現甲醇的分離效率都比氰甲烷來得差，而溶劑影響的差異在越疏水的蛋白質上越不明顯，可能是因為疏水性蛋白質與有機溶劑媒合（solvation）的作用力較大，當有機溶劑濃度高於可將蛋白質脫附的臨界濃度後，蛋白質表面能夠被媒合（solvation）得很完全而減少與靜相的作用以利其沖提。在離子對試劑方面，本實驗分別利用蛋白質以及由苯胺基丙酸（phenylalanine）及離胺酸（lysine）所組成之一系列不同長度的相似peptides（homologous），來探討磷酸、甲酸、三氟乙酸、七氟化丁酸作為離子對試劑時的影響。結果顯示分析物的滯留時間隨離子對試劑之疏水性增加而增加，而且越親水的peptides，其滯留行為受到離子對試劑疏水性的影響越明顯。較親水的peptides因本身與靜相的疏水性作用力不夠大，必須配合疏水性較強的離子對試劑，才能與靜相有充分的作用而加以分離。此外針對兩種不同來源的溶菌（lysozyme），它們在129個胺基酸中只相差了7個，比較不同動相組成對其分離效率的影響，實驗數據顯示以氰甲烷與三氟乙酸所組成的動相，在等位沖提下具有最好的分離效率。
在壓力影響方面，已知壓力由23bar升高至318bar時，蛋白質的滯留時間增加了2-3倍，為了更進一步瞭解壓力所造成的改變，利用不具立體結構的peptides來與具有複雜立體結構的蛋白質作比較。由實驗結果推論，壓力造成蛋白質立體結構、離子解離及靜相結構的影響是可忽略的。壓力主要是造成疏水性作用力的改變，使平衡往溶質體積小的靜相移動，導致蛋白質的滯留時間增加。而且利用容量因子之自然對數（lnk''）與壓力作圖所得之斜率，計算出分析物在動相與靜相中的莫耳體積差，發現當越疏水的peptides如苯胺基丙酸（phenylalanine）與靜相之疏水性作用力越大時，其莫耳體積變化也會越明顯，所求得之莫耳體積差約為－10 cm3/mol，而親水性的peptides如離胺酸（lysine）與靜相作用力較弱，其莫耳體積差就明顯變小，約為－1.5 cm3/mol，然後根據20胺基酸之間的疏水性大小，可間接估算其餘胺基酸之莫耳體積差。除此之外，比較溶菌（lysozyme）及苯胺基丙酸（phenylalanine）的莫耳體積差，可發現溶菌（lysozyme）大約是透過表面上十多個疏水性相當於苯胺基丙酸（phenylalanine）的胺基酸來與靜相進行吸附。

In this study, the parameters affecting protein separation on reversed-phase liquid chromatography, such as organic solvent、ion-pairing agent and the pressure were investigated. For the effect of pressure, the thermodynamic principals were applied to infer the cause of observed results.
For organic solvent effect, three model proteins, ribonuclease A、lysozyme and α-lactalbumin which have the similar molecular weight and number of amino acids but different hydrophobicity, were judiciously chosen for this investigation. It was found that the separation efficiency using methanol as organic modifier is always worse than using acetonitrile. Moreover the efficiency difference between methanol and acetonitrile were smaller for the more hydrophobic proteins. It could be that the hydrophobic proteins were easily solvated by organic solvents such that less interaction with stationary phase once desorbed. For ion-pairing agent effect, two homologous series, poly-L-phenylalanine and poly-L-lysine, in addition to the three model proteins were used to investigate the effect of four ion-pairing agents : phosphoric acid、formic acid、trifluoroacetic acid and heptafluorobutyric acid. It was found that the retention time was increased with the hydrophobicity of ion-pairing agents. Moreover, the retention time of the hydrophilic peptides was more sensitive to the change of ion-pairing agents. The more hydrophilic peptides resulted in a better separation efficiency when paired with a more hydrophobic ion-pairing agent. Furthermore, the separation of lysozymes from chicken and turkey which are only different in 7 amino acids out of total 129 amino acids, had a better resolution and efficiency ehen under an isocratic elution using the mobile phase composed of acetonitrile and trifluoroacetic acid.
For pressure effect, it was demonstrated that the retention time of the lysozyme was increased by as much as two to three times as the absolute pressure on the viewing window was increased from 23 to 318 bar. Since many factors such as conformational changes of proteins, ionization, and hydrophobic interactions may all contribute to the protein retention and are subjective to change with the pressure, it is desirable to find out the major cause of the pressure effect. In this study, a homologous series of poly-aminoacid which have no secondary structures was investigated. It was found that the more hydrophobic peptides resulted in a more pronounced pressure-induced retention and consequently, a greater volume change ( △V＝Vsta－Vmob ) which was calculated from the first derivative of the lnk'' versus pressure polts. For example, the volume change of hydrophobic poly-L-phanylalanine was determined to be around minus 10 cm3/mol per phenylalanine, and the volume change of hydrophilic poly-L-lysine is about minus 1.5 cm3/mol per lysine. It is believed that perturbations in solute ionization、conformation change and stationary phase structural change have a minor impact under the investigated conditions, and the pressure-induced shift of the equilibria regarding hydrophobic ad-desorption is the major cause of the observed increase of protein retention. Assuming a linear relationship between the volume change and the hydrophobicity of amino acids, the volume change of other amino acids could be estimated. Moreover from comparing the volume changes of lysozyme and phenylalanine, it was predicted that about tens phenylalanine-equivalent residues on the lysozyme surface were involved in the hydrophobic association with the chromatographic ligands.

中文摘要………………………………………………………….Ⅰ
英文摘要………………………………………………………….Ⅲ
表目錄…………………………………………………………….Ⅷ
圖目錄…………………………………………………………….Ⅸ
第一章 緒論
1-1 研究背景……………………………………………………………1
1-2 本章參考文獻………………………………………………………5
第二章 毛細管液相層析線上偵測系統之建立
2-1 研究背景……………………………………………………………8
2-2 層析及偵測系統裝置………………………………………………8
2-2-1 毛細管柱之製備……………………………………………10
2-2-2 毛細管柱效能測試…………………………………………11
2-2-3 層析及偵測系統……………………………………………13
2-3 毛細管液相層析高壓系統之建立………………………………..15
2-4 Moment Calculation………………………………………………..17
2-5 本章參考文獻……………………………………………………..19
第三章 動相組成對peptides及蛋白質分離之影響探討
3-1 研究背景…………………………………………………………..22
3-2 實驗部分…………………………………………………………..24
3-2-1 試劑…………………………………………………………24
3-2-2 儀器裝置……………………………………………………25
3-3 有機溶劑對蛋白質滯留行為的影響……………………………..26
3-4 離子對試劑對蛋白質及peptides分離的影響…………………...31
3-4-1 核糖核酸A、溶菌、α-乳白蛋白之分離比較………31
3-4-2 疏水性peptides（poly-L-phenylalanine）之分離比較………33
3-4-3 親水性peptides（poly-L-lysine）之分離比較……………….36
3-5 不同來源之溶菌的分離比較…………………………………..39
3-6 結論………………………………………………………………..44
3-7 本章參考文獻……………………………………………………..45
第四章 壓力對peptides及蛋白質滯留行為之探討
4-1 研究背景…………………………………………………………..59
4-2 實驗部分…………………………………………………………..60
4-2-1 試劑…………………………………………………………60
4-2-2 儀器裝置……………………………………………………61
4-3 理論……………………………………………………………….62
4-4 壓力對peptides在等位沖提下滯留行為的影響………………..64
4-4-1 壓力對容量因子之影響探討………………………………64
4-4-2 壓力對莫耳體積變化之影響探討…………………………67
4-5 壓力對溶菌(Lysozyme)滯留行為的影響………………….…..72
4-6 結論………………………………………………………………..77
4-7 本章參考文獻……………………………………………………..79
第五章 未來展望……………………………………………....89
附錄
一、 利用兩點線上偵測系統探討動相組成對溶菌（lysozyme）
沖提的效應……………………………………………………….91
二、 壓力對溶菌（lysozyme）在碳十八管柱中滯留行為之探討
…………………………………………………………………….95

1. Fausnaugh, J. L.; Kennedy, L. A.; Regnier, F. E., J. chromatogr., 1984, 317, 141-155.
2. Regnier, F. E.; Gooding, K. M., Anal. Biochem., 1980, 103, 1-25.
3. Hancock, W. S.; Sparrow, J. T. in Cs. Harvath (Editor), High-Performance Liquid Chromatography, Vol. 3, Academic Press, New York, 1983, p. 49.
4. Hearn, M. T. W. in Cs. Harvath (Editor), High- Performance Liquid Chromatography, Vol. 3, Academic Press, New York, 1983, p. 87.
5. Dolan, J. W.; Snyder, L. R. , LC.GC., 1987, 5, 970-978.
6. Hodges, R. S.; R. Parker, J. M.; Mant, C. T.; Sharma, R. R., J. Chromatogr., 1988, 458, 147-167.
7. Lundell, N.; J. Chromatogr., 1993, 639, 97-115.
8. Lundell, N.; Markides, K., J. Chromatogr., 1993, 639, 117-124.
9. Guo, D.; Mant, C. T.; Hodges, R. S., Pept. Res., 1990, 3, 8-15.
10. Vissers, J. P. C, J. Chromatogr.A, 1999, 856, 117-143.
11. Boardman, N. K.; Partridge, S. M., J. Biochem., 1955, 59, 543-554.
12. Jennissen, H. P., Biochemistry., 1976, 15, 5683-5692.
13. Jennissen, H. P., J. Chromatogr., 1978, 159, 71-83.
14. Regnier, F. E. LC, Lig. Chromatogr. HPLC Mag., 1983, 1, 350-356.
15. Kerlavage, A. R.; Weitzmann,C. J.; Hasan,T.; Cooperman, B. S.,
J. Chromatogr., 1983, 266, 225-237.
16. Welling, G. W.; Nijmeier, J. R.; van der Zee, R; Groen, G.; Wilterdink, J.
B.; Welling-Wester, S., J. Chromatogr., 1984, 297, 101-109.
17. Regnier, F. E., Science 1983, 222, 245-252.
18. Lau, S. Y. M.; Taneja, A. K.; Hodges, R. S., J. Chromatogr., 1984, 317,
129-140.
19. Hearn, M. T. W.; Grego, B, J. Chromatogr., 1981, 203, 349-363.
20. Snyder, L. R.; Stadalius, M. A.; Quarry, M. A., Anal. Chem., 1983, 55,
1412A-1430A.
21. Snyder, L. R., J. Chromatogr., 1978, 16, 223.
22. Nyiredy, S. In Chromatography; Kaiser, O.; Incom: Duesseldort,
Germany, 1997, 231-239.
23. Barford, R. A.; Sliwinski, B. J.; Breyer, A. C.; Robbert, H. L.,
J.Chromatogr. 1982, 235, 281-288.
24. Hearn, M. T. W.; Grego, B., J. Chromatogr., 1983, 255, 66-77.
25. Horvath, Cs.; Melander, W.; Molnar, I.; Molnar, P., Anal. Chem., 1977,
49, 2295.
26. Horvath, Cs.; Melander, W.; Molnar, I., J. Chromatogr., 1976, 125, 129.
27. Guo, D.; Mant, C.T.; Hodges, R.S., J. Chromatogr., 1987, 386, 205-222.
28. Pearson, J.D.; McCroskey,M.C., J. Chromatogr. A, 1996, 746, 277-281.
29. Deelder, R.S. ; Linssen, H.A.J. ; Konijnendijk, A.P.; van den Berg,
J.H.M., J. Chromatogr., 1979, 185, 241.
30. Deelder, R.S.; van den Berg, J.H.M., J. Chromatogr., 1981, 218, 327.
31. Knox, J.H.; Hartwick, R.C., J. Chromatogr., 1981, 204, 3.
32. Tomlimson, E ; Riley, C.M.; Jefferies, T.M., J. Chromatogr., 1979, 173,
89.
33. Hung, C.T.; Taylor, R.B., J. Chromatogr., 1980, 202, 333.
34. Hung, C.T.; Taylor, R.B., J. Chromatogr., 1981, 209, 175.
35. Luries, I.S.; Demchuk, S.M., J. Liq. Chromatogr., 1981, 4, 337.
36. Melander, W.R. Horvath, Cs., in Hearn, M.T.W.(Editor), Ion-pair
Chromatography, Marcel Dekker, New York, 1985, Ch. 2.
37. Bartha, A.; Vigh, Gy., J. Chromatogr., 1987, 395, 503.
38. Bennett, H.P.J., J. Chromatogr., 1983, 266, 501-510.
39. Martin, M.; Blu, G.; Guiochon, G., J. Chromatogr. Sci., 1973, 11, 641-
648.
40. Martire, D. E., J. Liq. Chromatogr., 1987, 10, 1569-1588.
41. Martire, D. E.; Bioehm, R. E., Anal. Chem., 1989, 61, 471-482.
42. Bilingmeyer, B. A.; Hooker, R. P.; lochmuller, C. H.; Rogers, L. B.,
Sep. Sci. 1972, 7, 131-139.
43. Prukop, G.; Rogers, L. B., Sep. Sci., 1978, 13 , 59-70.
44. Tanaka, N.; Yoshimura,T.; Araki, M., J. Chromatogr. 1987, 406, 47-256.
45. Bylina, A.; Ulanowicz, M., Chem. Anal., 1998, 43, 955-959.
46. Mant,C.T; Hodge,R.S.(Editor), High-Pergormance Liquid
Chromatography of Peptides and Protein Separation, Analysis, and
Conformation, CRC Press, London, 1991, p18.
47. Mant,C.T; Hodge,R.S.(Editor), High-Pergormance Liquid
Chromatography of Peptides and Protein Separation, Analysis, and
Conformation, CRC Press, London, 1991, p294.