利用奈米孔洞導電離子交換樹酯在電雙層臨界點上濃縮peptide

Trapping peptide at critical-point double-layer of nanoporous conducting granules

蔡佳君(Chia-chun Tsai)

指導教授 ： 王少君

國立中正大學/理學院/化學暨生物化學系/碩士(2012年)

摘要

本實驗室先前的研究中，曾利用一個陽離子交換樹酯顆粒，將其置於一適當的電場下(100 V/cm)來濃縮酸性的螢光染料分子。此染料分子的濃縮倍率達105倍到106倍。藉由此研究我們觀察到染料的解離度對濃縮倍率造成的影響，我們發現當緩衝溶液pH值達到染料分子之pKa + 2或更高時，染料分子已近乎完全解離，其濃縮倍率達到最大。而後進一步以此濃縮器來濃縮蛋白質，觀察不同pH值之下螢光蛋白質濃縮效率，發現於含有較高電荷態的pH值下亦具有較高的濃縮倍率（可達104倍）。
本研究接續探討合成螢光多肽(FITC-Angiotensin I)在不同pH值時的濃縮情形。本研究探討pH值範圍為pH 6.03 ~ pH 10.95，在pH 6.03時平均濃縮倍率只有4倍，隨著pH值增加濃縮倍率漸增，在pH 7.7時濃縮倍率趨近最大值，可達16218倍。由實驗結果可知濃縮倍率主要受mobility所影響，當分子因溶液pH值改變使得帶電荷量越多，mobility越大，濃縮效果也越好。當pH值大於8.5以後，濃縮倍率有略微下降的現象。推測原因可能是在較鹼性的環境下OH-增加，因為OH-的mobility較高，使得OH-優先與臨界點的正電荷中和，使螢光負離子移動至臨界點的分子數量較少，導致濃縮倍率下降。以胺基酸側鏈帶電荷量去估算FITC-Angiotensin I 的等電點理論值約為5.85，且一併估算出在本研究所探討各pH溶液中，FITC-Angiotensin I的帶電量。我們發現在pH 6與pH 7.5之間，濃縮倍率隨著帶電量的絕對值，呈現指數形式的漸增關係。

關鍵字

電雙層臨界點 ； 濃縮 ； 多肽

並列摘要

Our previous work using an ion exchange resin granule to concentrate acidic fluorescence dyes when this resin granule is placed in a moderate electric field (100 V/cm). The concentration factor of dyes using this device was found enormous 105 to 106. In the past we also studied the effects of dye dissociation in how concentration factor was influenced by the dissociation degrees of dye and concluded that the concentration factor reaches the maximum when the buffer solution pH exceeded pKa + 2 to make the dye molecules fully dissociated. In addition, using this solute trapping device to concentrate proteins, having observed the concentrate effects of fluorophore-tagged protein under different pH solutions, we found in pH conditions, when protein posses higher charge states, a higher concentration factor (up to 104 times) was achieved.
In this study, we continue the above works to observe the concentration effects of the dye-tagged peptide, fluorescein isothiocyanate (FITC) tagged angiotensin I, in various pH values. The pH value range is pH 6.03 ~ pH 10.95. The average concentration factor in the pH 6.03 is found only four folds. However, when the solution pH increases, the concentration factor increases. The concentration factor approaches the maximum at pH 7.7, up to 16,200 times. The increasing concentration factor mainly attributes to the increasing mobility, when the trapped solute FITC-tagged angiotension I is with higher charge states in more basic solution. The concentration factor slightly declined when solution pH is greater than 8.5. Since that the concentration of high mobility anions, hydroxide increases in strongly alkaline conditions, we speculate that OH- ions compete more favorably than FITC-tagged peptide while both migrating into the trapping area to neutralize accumulated cations by asymmetric fluxes. As a result, the concentration factors of dye-tagged angiotension I in strongly basic solution are somewhat lower than the factor in moderate basic conditions. Finally, having assigned the charge states of FITC-tagged angiotension I in the above pH conditions, we found the concentration factor increases exponentially with respect to the absolute values of solute charge.

並列關鍵字

peptide ； critical-point double-layer ； concentration

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