臺灣博碩士論文加值系統

摘要

在這個研究中，我們設計了三組不同鏈長的胜肽鏈，第一組為由五個胺基酸組成的GW-X-WG系列，其目的是為了以逆相層析(RPC)與疏水性作用層析(HIC)測量每一個胺基酸在胜肽鏈中(-X-)的疏水性。第二組為九個胺基酸組成的GWDWDWGWG系列，其目的是為了確定所計算的胺基酸疏水性是否有加成性。第三組為十一個胺基酸組成的GELELKLKLEG系列，其目的在探討長胜肽鏈在疏水管柱中的滯留行為與其空間構型之間的關係。

本實驗所求得的胺基酸疏水性與其他學者所建立的胺基酸疏水性間皆具有不錯的相關性。從各胺基酸與疏水基材作用時之熱力學參數發現Tryptophan與C-18基材間存在著很強的直接作用力；相反的，亂度的變化對含有脂肪支鏈的胺基酸(Ala、Vle、Ile、Leu)與C-18基材間的作用具主要影響。

由長胜肽鏈(九個胺基酸胜肽與十一個胺基酸胜肽)在疏水管柱中的滯留行為與圓極偏光光譜，我們發現胺基酸排序不同並不會對九個胺基酸胜肽之滯留行為與溶液中構型產生改變，但對於十一個胺基酸胜肽之滯留行為與溶液中構型則產生明顯差異。另外，根據實驗所得之熱力學參數我們推測溶液中構型的差異將使得吸附前後亂度的變化不一致，進而影響這組胜肽鏈的滯留行為。

Abstract

In this study, we have designed three series of peptides of different length. The 5-residue peptides of the sequence GW-X-WG, where X is substituted by 10 amino acids(G, A, V, I, L, W, D, E, K, H). Measuring the retention time of the 5-residue peptides in reversed-phase chromatography (RPC) and hydrophobic interaction chromatography (HIC), we can determine the hydrophobicity of amino acids in peptide(-X-).The 9-residue peptides of the sequence GWDWDWGWG and 11-residue peptides of the sequence GELELKLKLEG are designed to examine the hydrophobicity of long-chain peptide can be predicated by summating hydrophobicity of each amino acid residue. Additionally, circular dichroism(CD) are used to investigate the effect of conformation to the retention behavior of long-chain peptides.

The hydrophobicity of amino acids in this study correlates moderately well with the hydrophobicity of other scholars. Plots of the logarithmic retention factor against the reciprocal temperature (10~50℃), we can estimate the thermodynamic parameters of the interaction between amino acids and hydrophobic ligands. The results reveal that the interaction between tryptophan and hydrophobic C-18 ligands is particularly strong. We also find that the hydrophobic adsorptions of aliphatic amino acids are entropy driven.

When the long-chain peptides don’t possess secondary structure, we have shown that the use of amino acid residues hydrophobicity to predict long-chain peptide hydrophobicity can be reliable. For the 9-residue peptides, the same composition but differing sequences were shown to have the same conformation and the same retention behavior. By contrast, the 11-residue peptides, which have the same composition but differing sequences were shown to have the different conformation and the different retention behavior. According to the thermodynamic parameters, 11-residue peptides which have the different retention behavior are due to the different entropy change, we suppose that the difference in conformation will cause the different entropy change of adsorptions.

總目錄

中文摘要--------------------------------------------------------------- Ⅰ
英文摘要--------------------------------------------------------------- Ⅱ
總目錄------------------------------------------------------------------ Ⅳ
圖目錄------------------------------------------------------------------- Ⅶ
表目錄------------------------------------------------------------------- Ⅸ

第一章 緒論--------------------------------------------------------------1
第二章 基本原理與文獻回顧------------------------------------------------ 3
2.1 疏水作用力之簡介及其在生物體中的角色------------------------ 3
2.2 生化分子疏水性之量測方法------------------------------------ 7
2.2.1 胺基酸在水相及乙醇中的溶解度--------------------- 7
2.2.2 兩液相分配方法------------------------------------------ 8
2.2.3 以暴露於水溶性蛋白表面的機率定義親疏水值---- 12
2.2.4 以層析系統量測胺基酸疏水性------------------------- 14
2.2.4.1 以RPC測量胺基酸的親疏水性--------------------- 15
2.2.4.2 以HIC測量胺基酸的親疏水性--------------------- 17
2.3 構型對滯留行為的影響------------------------------------------ 21
2.4以層析方法量測疏水性吸附之熱力學參數------------------- 26
第三章 實驗藥品、設備、方法------------------------------------- 29
3.1 實驗藥品---------------------------------------------------------- 29
3.1.1 胜肽鏈------------------------------------------------------- 29
3.1.2 層析方面-------------------------------------------------- 30
3.1.3 圓極偏光光度計方面------------------------------------ 30
3.2 實驗設備---------------------------------------------------------- 30
3.2.1 一般設備-------------------------------------------------- 30
3.2.2 高效能液相層析------------------------------------------ 31
3.2.3 圓極偏光光度計------------------------------------------ 31
3.3 實驗方法------------------------------------------------------------ 32
3.3.1 高效能液相層析實驗------------------------------------ 32
3.3.2 圓極偏光光度計------------------------------------------ 34
第四章 結果討論---------------------------------------------------- 36
4.1滯留因子與移動相中有機相比例或鹽濃度間的關係-------- 36
4.1.1以逆相層析系統量測各胺基酸在胜肽鏈之疏水貢獻----- 37
4.1.1.1 截距(lnk0)與胜肽疏水性之關係------------------------- 40
4.1.1.2 斜率(S)與胜肽疏水性之關係---------------------------- 42
4.1.2以疏水性層析系統量測各胺基酸在胜肽鏈之疏水貢獻-- 44
4.1.2.1截距(lnk0)與胜肽疏水性之關係-------------------------- 47
4.1.2.2斜率(m)與胜肽疏水性之關係---------------------------- 48
4.2 計算胺基酸在鏈中間的疏水性------------------------------------ 51
4.2.1以五個胺基酸胜肽定義胺基酸的lnk0及S----------------- 51
4.2.2以九個胺基酸胜肽定義胺基酸的lnk0及S----------------- 52
4.2.3與其他學者建立之胺基酸疏水性比較---------------------- 55
4.3 由胺基酸疏水性預測長胜肽鏈之疏水性--------------------------- 58
4.4 構型對長胜肽鏈滯留行為的影響------------------------------------ 62
4.5 疏水性吸附時之熱力學參數------------------------------------------ 66
4.5.1 胺基酸吸附時之寒0與巽0------------------------------ 67
4.5.2 九個胺基酸胜肽之熱力學參數--------------------------- 72
4.5.3 十一個胺基酸胜肽之熱力學參數------------------------ 75
第五章 結論------------------------------------------------------------ 82

參考文獻--------------------------------------------------------------- 85


圖目錄

圖2.1 二十種常見胺基酸及其結構---------------------------------- 5
圖2.2 穩定蛋白質結構的作用力------------------------------------ 6
圖2.3 兩液相分配系統示意圖---------------------------------------- 9
圖2.4 Nozaki and Tanford的hydrophobicity scale與Yunger and Cramer的LOG(P)之關係圖。--------------------------------------------- 10
圖2.5 兩成分混合樣品經過管柱層析分離的示意圖------------ 14
圖2.6 胺基酸在SynChropak propyl中Logk’與ammonium sulfate莫耳濃度間的關係，DNS =dansyl ------------------------- 18
圖2.7 KLLK與KALK形成-helix結構時胺基酸的相對位置---- 22
圖2.8 Krause所設計的胜肽鏈形成-helix結構時胺基酸的相對位置-------------------------------------------------------------------------------- 23
圖2.9胜肽鏈與其D-amino acids置換系列之圓極偏光光譜------- 24
圖2.10 胜肽鏈在RPC中滯留時間與由CD所定義之helicity之關係--------------------------------------------------------------------------------- 24
圖2.11 Chen設計的helical與random coil胜肽鏈之圓極偏光圖譜--------------------------------------------------------------------------------- 25
圖2.12 胺基酸在Sepherogel HIC中的van’t Hoff plot ------------- 26
圖2.13蛋白質的吸附機制--------------------------------------------- 30
圖4.1 30℃時GW-X-WG系列在C-18管柱中滯留因子的對數(lnk’)與有機溶劑比例()間的關係圖--------------------------------------- 39
圖4.2 在23%ACN與0%ACN下胜肽鏈疏水性之關係圖------------ 41
圖4.3 在23%ACN時胜肽鏈lnk’與斜率S之關係圖------------------- 43
圖4.4 在逆相層析23%ACN時胜肽鏈lnk0/S與lnk’之關係圖------ 44
圖4.5 30℃時GW-X-WG系列在C-4管柱中滯留因子的對數(lnk’)與鹽濃度(MS)間的關係圖-------------------------------------------------
45
圖4.6 胜肽鏈在逆相層析中lnk0與lnk’ (2.1M AS)之關係圖-------- 48
圖4.7 胜肽鏈在疏水性層析中m與lnk’(2.1M AS)之關係圖-------- 49
圖4.8在2.1M AS時胜肽鏈lnk0/m與lnk’之關係圖--------------------- 50
圖4.9 30℃時九個胺基酸胜肽系列在C-18管柱中滯留因子的對數(lnk’)與有機溶劑比例()間的關係圖-----------------------------------
53
圖4.10 九個胺基酸胜肽在23~27%ACN下實際疏水性與預測水性之比較----------------------------------------------------------------------
59
圖4.11十一個胺基酸胜肽在23~27%ACN下實際疏水性與預測水性之比較--------------------------------------------------------------- 60

圖4.12九個和十一個胺基酸胜肽在23~27%ACN下疏水性之比較--------------------------------------------------------------------------- 62
圖4.13 九、十一個胺基酸胜肽在層析實驗條件時的圓極偏光圖譜 64
圖4.14九個和十一個胺基酸胜肽在30℃，23%ACN時之圓極偏光圖譜---------------------------------------------------------------------------
65
圖4.15 五個胺基酸胜肽 (GW-X-WG)在23% ACN之van’t Hoff plot-------------------- --------------------------------------------------
67
圖4.16 五個胺基酸胜肽 (GW-X-WG)在23~27% ACN之寒0--- 69
圖4.17 各胺基酸在23% ACN之[寒0 (-X-)-寒0 (-G-)] ----------------- 70
圖4.18 五個胺基酸胜肽 (GW-X-WG)在23~27% ACN之T巽0- 71
圖4.19 各胺基酸在30℃、23%ACN時之T[巽0 (-X-)-巽0 (-G-)]--------- 72
圖4.20 九個胺基酸胜肽在23% ACN之van’t Hoff plot ----------- 73
圖4.21 DWGWD與DWDWG在30℃、23~27%時的寒0與T巽0------------------------------------------------------------------------------
74
圖4.22 DWGWH與DWHWG在30℃、23~27%時的寒0與T巽0------------------------------------------------------------------------------
74
圖4.23 十一個胺基酸胜肽在23% ACN之van’t Hoff plot -------- 75
圖4.24 以Logarithmic equation迴歸GELE與GELK的van’t Hoff plot -------------------------------------------------------------------------------
77
圖4.25 以Quadratic equation迴歸GELE與GELK的van’t Hoff plot------------------------------------------------------------------------------- 78
圖4.26 十一個胺基酸胜肽在30℃、23~27% ACN之寒0與T巽0------------------------------------------------------------------------------ 80

表目錄

表2.1 Hydrophobicity scale-------------------------------------------- 8
表2.2 胺基酸側鏈疏水性常數(谼)------------------------------ 11
表2.3 Janin建立的各胺基酸之疏水性--------------------------------- 13
表2.4 Guo量測胜肽鏈滯留時間的操作條件-------------------------- 16
表2.5 Guo建立的各胺基酸在pH2與pH7之retention coefficients-- 17
表2.6 Gehas以HIC斜率定義之疏水性------------------------------ 19
表2.7 胺基酸在HIC中的滯留行為--------------------------------- 20
表2.8 胜肽鏈名稱與胺基酸序列--------------------------------------- 21
表2.9 以D-amino acids置換KLALKLALKALKAALKLA-amide- 23
表2.10 胺基酸進行疏水性吸附時的熱力學參數-------------------- 27
表2.11 胜肽鏈與RPC-C4管柱間作用的熱力學參數---------------- 28
表4.1 短鏈胜肽在逆相層析中之lnk0與S-------------------------------- 40
表4.2 短鏈胜肽在逆相層析中之lnk0與m------------------------------- 47
表4.3胺基酸在胜肽鏈中相對於G的lnk0(-X-)與S(-X-)-------------------- 52
表4.4 九個胺基酸胜肽其胺基酸組成及其胺基酸排列順序--------- 52
表4.5 九個胺基酸胜肽其lnk0與S----------------------------------------- 54
表4.6 兩個胜肽鏈所求得的lnk0(-G-)與S(-G-)及其平均值--------------- 55
表4.7 各胺基酸在在鏈中間時的lnk0、S與lnk0/ S---------------------- 55
表4.8 其他學者量測胺基酸疏水性所選用的系統--------------------- 56
表4.9 lnk0(-X-)與其他學者所建立的胺基酸疏水性---------------------- 56
表4.10九個胺基酸胜肽其胺基酸組成及其胺基酸排列順序--------- 58
表4.11十一個胺基酸胜肽其胺基酸組成及其胺基酸排列順序------ 60
表4.12 五個胺基酸胜肽在23% ACN之寒0與巽0------------------ 68
表4.13 GELE與GELK在23% ACN時以Logarithmic equation(L)與Quadratic equation(Q) 迴歸出的熱力學參數---------- 78

參考文獻

Biswas, K. M.; Devido, D. R.; Dorsey, J. G. ‘‘Evaluation of Methods for Measuring Amino Acid Hydrophobicities and Interactions.’’ Journal of Chromatography A., 1000, 637-655, 2003.

Ben-Naim, A. Hydrophobic Interactions, Plenum Press: New York, 1980; pp 1-15.

Chabanet, C.; Yvon, M. ‘‘Prediction of Peptide Retention Time in Reversed-Phase High-Performance Liquid Chromatography.’’ Journal of Chromatography., 599, 211-225, 1992.

Chen, Y.; Mant, C. T.; Hodges, R. S. ‘‘Temperature Selectivity Effects in Reversed-Phase Liquid Chromatography due to Conformation Differences between Helical and Non-helical Peptides.’’ Journal of Chromatography A., 1010, 45-61, 2003.

Chothia, C. ‘‘The Nature of the Accessible and Buried Surfaces in Proteins.’’ Journal of Molecular Biology., 105, 1-12, 1976.

Fauchere, J. L.; Pliska, V. ‘‘Hydrophobic parameters of amino acid side chains from the partitioning of N-acetyl-amino acid amides.’’ European Journal of Medicinal Chemistry., 18, 369, 1983.

Gehas, J.; Wetlaufer, D. B. ‘‘Isocratic Hydrophobic Interaction Chromatography of Dansyl Amino Acids：Correlation of Hydrophobicity and Retention Parameters.’’ Journal of Chromatography., 511, 123-130, 1990.

Guo, D.; Mant, C.; Taneja, A. K.; Parker, J. M. R.; Hodges, R. S. ‘‘Prediction of Peptide Retention Times in Reversed-Phase HPLC：I. Determination of Retention Coefficients of Amino Acid Residues of Model Synthetic Peptides.’’ Journal of Chromatography., 359, 499-517, 1986.

Haidacher, D.; Vailaya, A.; Horvath, C. ‘‘Temperature Effects in Hydrophobic Interaction Chromatography.’’ Proceedings of the National Academy of Sciences of the United States of America., 93, 2290-2295, 1996.

Hearn, M. T. W. HPLC of Peptides and Proteins: Separation, Analysis and Conformation; Hodges, R. S., Mant, C. T. Eds., CRC Press: Florida, 1991, pp 105-111.

Hearn, M. T. W.; Zhao, G. ‘‘Investigations into the Thermodynamics of Polypeptide Interaction with Nonpolar Ligands.’’ Analytical Chemistry., 71, 4874-4885, 1999.

Hodges, R. S., Mant, C. T. HPLC of Peptides and Proteins: Separation, Analysis and Conformation, CRC Press: Florida, 1991.

Horvath, C.; Melander, W.; Molnar, I. ‘‘Solvophobic Interactions in Liquid Chromatography with Nonpolar Stationary Phases.’’ Journal of Chromatography., 125, 129-156, 1976.

Janin, J. ‘‘Surface and Inside Volumes in Globular Proteins.’’ Nature., 277, 491-492, 1979.

Kauzmann, W. ‘‘Some Factors in the Interpretation of Protein Denaturation.’’ Advances in Protein Chemistry., 14, 1-63, 1959.

Krause, E.; Beyermann, M.; Dathe, M.; Rothemund, S.; Bienert, M. ‘‘Location of an Amphipathic -helix in Peptides Using Reversed-Phase HPLC Retention Behavior of D-Amino Acid Analogs.’’ Analytical Chemistry., 67, 252-258, 1995.

Lee, B.; Richards, F. M. ‘‘The Interaction of Protein Structures: Estimation of Static Accessibility.’’ Journal of Molecular Biology., 55, 379-400, 1971.

Mckee, T.; Mckee, J. R. Biochemistry, 2nd ed., McGraw-Hill Publishers: New York, 1996, pp 135.

Meek, J. L. ‘‘Prediction of Peptide Retention Times in High-Pressure Liquid Chromatography on the Basis of Amino Acid Composition.’’ Proceedings of the National Academy of Sciences of the United States of America., 77, 1632-1636, 1980.

Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry, 3rd ed., Worth Publishers: New York, 2000; pp 119.

Nozaki, Y.; Tanford, C. ‘‘The Solubility of Amino Acids and Two Glycine Peptides in Aqueous Ethanol and Dioxane Solutions.’’ The Journal of Biological Chemistry., 246, 2211-2217, 1971.

Queiroz, J. A.; Tomaz, C. T.; Cabral, J. M. S. ‘‘Hydrophobic Interaction Chromatography of Proteins.’’ Journal of Biotechnology., 87, 143-159, 2001.

Rothemund, S.; Krause, E.; Beyermann, M.; Dathe, M.; Engelhardt, H.; Bienert, M. ‘‘Recognition of {helical Peptide Structures Using HPLC retention data for D-Amino Acid Analogues: influence of Peptide Amphipathicity and of Stationary Phase Hydrophobicity.’’ Journal of Chromatography A., 689, 219-226, 1995.

Southall, N. T.; Dill, K. A.; Haymet, A. D. J. ‘‘A View of Hydrophobic Effect.’’ Journal of Physical Chemistry B., 106, 521-533, 2002.

Tanford, C. ‘‘Contribution of Hydrophobic Interactions to the Stability of Globular Conformation of Proteins.’’ Journal of the American Chemical Society., 84, 4240-4247, 1962.

Tanford, C. The Hydrophobic Effect：Formation of Micelles and Biological Membranes., 2nd ed., Wiley-Interscience, John Wiley & Sons: New York, 1980, pp 21-28.

Wieprecht, P.; Rothemund, S.; Bienert M.; Krause, E. ‘‘Role of Helix Formation for the Retention of Peptides in Reversed-Phase HPLC , Journal of Chromatography A., 912, 1-12, 2001.

Yamasaki, Y.; Uchida, K.; Tomizawa, H.; Ishikawa, O.; Inouye, K. ‘‘Separation of Amino Acid and Peptides by High Performance Hydrophobic Interaction Chromatography.’’ J. Liq. Chrom. & Rel. Technol., 22, 1965-1974, 1999.

Yunger, L. M.; Cramer, R. D. 3rd. ‘‘Measurement of Correlation of Partition Coefficients of Polar Amino Acids.’’ Molecular Pharmacology, 20, 602-608, 1981.

陳柏勳（民國八十七年），”疏水鏈長短與密度對蛋白質在疏水作用層析的影響”，碩士論文，私立中原大學化學工程研究所。

林伯勳 (民國九十年)，” 胜肽鏈疏水性質之熱力學探討”，碩士論文，私立中原大學化學工程研究所。

李碧芬 (民國九十一年)，” 胜肽鏈中脂肪支鏈氨基酸的疏水表現之探討”，碩士論文，私立中原大學化學工程研究所。