本實驗的主要目的，就是希望在逆流層析中找到新的動靜相系統 來分離蛋白質，利用含有水核的反微胞，可使蛋白質在兩相分佈，依 逆流層析中有機-水相的特殊分佈，靜相是帶電的反微胞有機相，動 相是pH 值和鹽類濃度的梯度變化，類似於 Ion exchange 的原理，藉 由改變蛋白質和靜相之間的電荷作用力而分離蛋白質混合物。
雖然我們實驗中的蛋白質總回收率尚無法達到100 %，但仍然可 以得到較高濃度和蛋白質相對濃度90 %以上的蛋白質，且反微胞靜 相製備相當容易而且價格低廉，只需少量的反微胞就可達到蛋白質製 備分離效果。
在本實驗中只調整了動相的組成，未來還可改變反微胞組成，添 加其他的萃取劑等，希望以此為基礎，嘗試從發酵液、細胞培養液等 生物樣品中分離蛋白質，提供一項新的蛋白質純化方式。
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表2-1 ( A )、界面活性劑依電荷分類 (Anionic surfactant)
脂肪酸鹽 ─COONa 脂肪酸鹽 ─COONa 磺酸鹽 ─SO3Na
磷脂鹽 ─OPO3Na
陽離子型界面活性劑 (Cationic surfactant)
N+ (Amphoteric surfactant)
R3N+─CH2COO
-非離子型界面活性劑 (Nonionic surfactant)
CH2-O-CH2-O-CH2-OH
表2-2 ( A )、親水、疏水基團的HLB參數
( B )、不同 HLB 值之界面活性劑應用範圍.
( A )
Hydrophilic Group number
Hydrophobic Group number
─SO4Na 38.7 ─CH─ 0.475
─COOK 21.1 ─CH2─ 0.475
─COONa 19.1 ─CH3 0.475
─SO3Na 11.0 =CH─ 0.475
─OH (sorbitan ring)
0.5
( B )
HLB range Applications 1~6 W/O 乳化劑
6~9 潤濕劑 (wetting agent) 8~18 O/W 乳化劑
13~15 洗滌劑 (Detergent) 15~18 加溶劑 (Solubilizer)
表2-3 ( A )、常用的疏水性的陰(陽)離子型界面活性劑 SDBS：sodium dodecylbenzene sulfonate
Name MW Type cmc
(ppm) hexadecyltrimethylammonium bromide 364.6 cationic 350
cetyl pyridinium chloride 358 cationic 525 sodium dodecylbenzene sulfonate 348.5 anionic 500 sodium 2-diethylhexyl sulfosuccinate 444 anionic 1110
Name Formula MW type λmax
Dye surfactant solvent KC
(mM-1)
表4-1、實驗二各收集管的蛋白質濃度
Tube Number 肌紅蛋白 (ppm) 細胞色素 C (ppm) 溶菌酶 (ppm)
1 0 0 0 2 0 0 0 3 186.0 0 0 4 55.8 0 0.2 5 6.3 6.3 0.2 6 1.3 9.0 0.2
7 1.3 25.4 1.8
8 1.6 40.6 1.6
9 3.4 51.0 1.5
10 3.4 38.8 2.6 11 0.4 26.1 2.3 12 4.9 22.3 1.7 13 28.3 3.6 26.0 14 10.1 15.9 12.3
15 0 3.9 25.2
16 0 1.2 19.5
17 0 0.4 10
表4-2、實驗二各收集管中的蛋白質蛋白質相對濃度 Collector Concentration
(ppm)
表4-4、實驗三各收集管的蛋白質濃度
Tube Nunber 肌紅蛋白 (ppm) 細胞色素C (ppm) 溶菌酶 (ppm)
1 0 0 0 2 25.8 0 0 3 194.2 7.1 4.9
4 50.1 19.5 5.3
5 6.7 20.7 0.2 6 3.0 23.5 0.2 7 4.6 48.5 3.4 8 2.3 59.8 0.2 9 3.0 44.0 0.2 10 2.3 16.4 1.2 11 2.3 14.5 0.2 12 3.6 11.4 1.1 13 9.1 17.9 3.0
14 7.8 12.4 11.8
15 6.9 8.8 34.3 16 3.8 3.9 91.3 17 4.1 2.2 50.9 18 0 0 38.7 19 0 0 19.2 20 0 0 7.1
表4-5、實驗三各收集管中的蛋白質相對濃度 Collector Concentration
(ppm)
圖2-1、界面活性劑分子
圖2-2、界面活性劑濃度和溶液的物理性質
親水端 親油端
圖2-3、微胞結構
( a )球形結構 ( b )雙層結構 ( c )柱狀結構 ( d )層狀結構
( a )微胞 ( b )反微胞
圖2-4、微胞與反微胞環境示意圖
(a) 微胞：界面活性劑親水端向外 (oil in water) (b) 反微胞：界面活性劑疏水端向外 (water in oil)
圖2-5、界面活性劑、水、有機溶液三相圖
圖2-6、液相-液相層析方法 (A) 傳統步驟式液相萃取。
(B) 逆流萃取 (CCE)，兩相都是動相，分別往反方向移動。
(C) 液相-液相層析 (LLC)，靜相是以固體支持物滯留在管柱中。
(D) 逆流層析 (CCC)，靜相滯留不需固體支持物 (Support-free)，
而是以重力場、離心力場將靜相滯留在管柱中。
(A) (B)
圖2-7、流體靜力平衡(HSES)示意圖 (A) 靜相為下層相，動相為上層相 (B) 靜相為上層相，動相為下層相
(A)
(B)
圖2-8(A)、抽取河水的螺旋裝置示意圖 (B)、螺旋管柱裝置模型
圖2-9、螺旋管柱中的輕重兩相移動情形
圖2-10、反扭轉機構轉動系統
分別是行星軸同步式、非行星軸同步、行星軸非同步式等三種，再變 化成七個模型
行星軸非同步 Nonsynchronous 行星軸同步
Synchronous
非行星軸同步 Nonplanetary
Bring Container to Central Axis
Ⅰ ω ω´ Ⅵ
ω 2ω+ ω´
Ⅱ ω ω
Ⅲ ω
ω
Bring Container to Central Axis
ω 2ω ω ω´
Ⅳ ω Ⅶ
2ω+ ω´
Ⅴ
圖2-11、高速逆流層析的旋轉機構
(A) 螺管支持物不僅以角速度 ω 繞著中心軸旋轉，同時自身也以相 同的角速度往相同方向旋轉
(B) 螺管支持物的同步行星運動可防止流體導入管與導出管扭轉
圖2-12、Ⅳ型同步行星式逆流層析的混合在管柱內的分佈情形
A B
圖3-1、逆流層析裝置圖
圖3-2、HPLC 實驗裝置圖
圖4-1、蛋白質在含有反微胞的逆流層析管柱中示意圖 (a)正向萃取-蛋白質分佈偏向進入反微胞
(b)反向萃取-蛋白質分佈偏向回到水中 (a)
(b)
mv
694.8
+MS, 1.2-1.2min (#69-#71)
0.0
400 600 800 1000 1200 1400 1600 m/z
616.2 1+
+MS, 0.0-0.1min (#3-#6)
0
400 600 800 1000 1200 1400 1600 m/z
616.6
+MS, 0.6-1.4min (#34-#76)
0.0
400 600 800 1000 1200 1400 1600 m/z
圖4-4、以 ESI-MASS 分析圖 4-3 中的樣品
圖4-5、肌紅蛋白中的heme分子 (MW=616)
0 20 40 60 80 100 120
0 20 40 60 80 100
percentage (%)
Time (min)
Mobile phae A1 pH 7 Mobile phae B1 pH 12
圖4-6、實驗一的逆流層析動相組成
動相A1：含有50mM Tris-HCl、0.1M KCl、pH 7 緩衝溶液 動相B1：含有50mM K2HPO4、 0.1M KCl、pH 12 緩衝溶液
mv
mv
minutes
0 20 40 60 80
0 500 1000
圖4-9、實驗二的逆流層析訊號
動相組成：A2 & B2 動相流速 1 mL/min UV 偵測器：280 nm
0 2 4 6 8 10 12 14 16 18
7 8 9 10 11 12
pH
Tubes
圖4-10、實驗二各收集管的 pH 值
(b)
cytochrome c
lysozyme myoglobin
圖4-11、HPLC 分析實驗二收集管中的蛋白質含量 (a)蛋白質的濃度(ppm)
(b)蛋白質的蛋白質相對濃度(%)
0 20 40 60 80 100 120
0 2 4 6 8 10 12 14 16
pH gradient curve
0 5 10 15
1 3 5 7 9 11 13 15 17
(a) cytochrome c
lysozyme
( b) cytochrome c lysozyme myoglobin
圖4-15、HPLC 分析實驗三收集管中的蛋白質含量 (a)蛋白質的濃度(ppm)
(b)蛋白質的蛋白質相對濃度(%)
6 0 0 0 8 0 0 0 1 0 0 0 0 1 2 0 0 0 1 4 0 0 0 1 6 0 0 0 1 8 0 0 0 2 0 0 0 0 cytochrome c
(b)
cytochrome c
(c)
lysozyme