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2 色層分析法 Chromatography
2.1 色層分析原理 Basic principles
極性不同的分子在兩相中有不同的分佈比例
2.2 膠体過濾法 Gel filtration
依樣本分子量之差異來做分離純化
2.3 離子交換法 Ion exchange
利用樣本分子的表面帶電性質差異來進行分離
2.4 親和層析法 Affinity chromatography
利用分子間的親和性大小不同來進行分離
2.5 HPLC 及 FPLC 改善介質的材質及吸附容量可增加速度及解析力
1
Round filter paper
Long rectangle paper
Thin layer
(TLC)
Column
Samplecapacity increased
Largercapacity
Development
Adapted from Scope RK (1987) Protein Purification – Principles and Practice p.9
Paper partition chromatography (PPC)
Martin & Synge (1952)
■層析法演進 Historical review2
sample
sample
■色層分析法的基本機制 Essential mechanism
●極性相似的兩分子間,其親和力較強。
Polar → Polar
Non-polar → Non-polar
Like
Dissolves
Like
3
永久的偶極性
+d
-d
O
lplp
LL
Two-phase separation system
流動相
Mobilephase
固定相
Stationaryphase
樣本分子依其分子極性選擇親和兩相之一
ADSORPTION PARTITION
Liquid - Solid Liquid - Liquid
Two-phaseA B C
每次分離樣本都要做一次選擇
One Plate of
Separation
Theoreticalplate
number
SPolar
L
Non-Polar
PolarNon-polar
Sample Sample
2.1 色層分析法原理 Principle of chromatography4
圖 2.1
One plate
■層析法的板數概念 One separation, one plate
B
BB
B B
B B B
B
BBA
A
A
AA
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B B
B B
B B
B
B
B
80
20
10
90
64
16
1
9
16
4
9
81
51
13
0
1
3
1
8
73Polar
Non-polar
100A
+
100B
5
■常用層析法 Common chromatographic methods6
Partition Adsorption
小分子
Paper partition
chromatography (PPC)
Thin-layer chromatography
(TLC) a)
Gas chromatography (GC) b)
Reverse phase
Ion exchange
TLC a)
GC b)
大分子
Gel filtration
Reverse phase
Ion exchange
Affinity chromatography
Hydrophobic interaction
Hydroxyapatite
a, b) 這兩種層析法均可進行 partition 或 adsorption,取決於所用展開液的成分與比例表 2.1
2.2
2.32.4
2.4.42.3.2
2.4.4
■薄層層析法操作 Thin-layer chromatography
Thin-layer plate
Sample spotting Add developer Developing
Activated
Not-activated
7
(Adsorption)
(Partition)
2.2.1 原理概述 Basic principles
是一種 partition 層析法
2.2.2 膠体介質 Gel materials
是一種長鏈的大分子糖類聚合物
2.2.3 膠体管柱 Gel and column
管柱性質、影響因素及管柱系統
2.2.4 管柱操作 Column operation
裝填並操作一支膠体過濾管柱
2.2.5 問題及解決 Problem and solution
常見的錯誤要預先避免
2.2 膠体過濾法 Gel filtration8
Smaller molecules are retarded
Largermolecules
Small molecules
■膠体過濾法是一種 Partition層析法
Sample
Stationary
phase
(liquid)
Mobilephase(liquid)
Stokes radiusMolecular sizeand shape
Elute out faster
Gel filtration is a partition type chromatography (liquid-liquid)
9
圖 2.2
■
各種色析膠体
Pharmacia
Bio-Rad
Sephadex
Sepharose
Sephacryl
Sephacel
BioGel P
BioGel A
glucose (dextrose)
agarose
glucose + acrylamide
cellulose
acrylamide
agarose
FPLC Superose, Superdex
Mono Q, Mono S
Gel material is a polymer of carbohydrate or acrylamide
10
FastPerformanceLiquidChromatography
■
膠体的構成
Ph
arm
acia
(19
91
) G
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tion
–P
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.35
Sephadex
glucose unit
Space for filtration
Sephadex is the polymer of glucose with chemical cross-linking
11
■膠体的使用範圍 Sephadex G
●MW ranged from ten to several hundred thousands
Pharmacia (1991) Gil Filtration – Principles and Methods p.37
G-200 could be smashed flat easily
From powder to gel form
1 g → 20 mL gel1 g → 7.5 mL
(its backbone support is too weak)
Small proteins
12
G-50
G-25
G-10
■
膠体的構成
Ph
arm
acia
(19
91
) G
il F
iltra
tion
–P
rin
cip
les a
nd
Me
tho
ds p
.22
Sephacryl
Acrylamide
Cross-linking
Larger space,
Stronger support for backbone
13
■洋菜膠体的成膠反應 Agar gel formation
Pharmacia (1991) Gil Filtration – Principles and Methods p.38, 39
Even stronger backbone
Much larger space
14
洋菜、寒天、瓊脂
■膠体的使用範圍 Sepharose
●MW ranged from ten thousands to several millions
Pharmacia (1991) Gil Filtration – Principles and Methods p.40
2% gel6% gel
Large proteins
Sepharose is rigid
Sephadex
Sephacry
Cross-linking
15
■膠体過濾法的膠球 Gel filtration beads16
■管柱內膠体的組成區隔 Spaces in a column
Adapted from Pharmacia (1991) Gil Filtration – Principles and Methods p.11
Vt Vo Vt - Vo
管柱總体積
膠体外体積
膠体內体積
Mobile phase (L) Stationary phase (L)
17
底面積
(total volume) (void volume)
h
■膠体過濾的溶離圖譜 A typical chromatogram
XY
ZEnzyme
activity
Vit. B12
Vo之前不應有物質溶離出來
Vt之後不應有物質溶離出來
目標酵素 (E) 最好不要落在主要蛋白質峰 (Y) 的範圍內
Elution Volume (mL)
A
VoVe
Vt0
E
18
圖 2.4
Blue Dextran
■
介質的粗細影響解析力
Poor separation
Peak flattened
Best separation
0
50
100
0.5 1.0 1.5
Superfine
Fine
Corse
Ve/Vt
0
50
100
0
50
100
Rela
tive a
mount
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Bead size is critical to the resolution of gel chromatography
19
Dispersion
■溶離液擴散進出膠球 Diffuse in and out bead
溶離液或樣本分子,由膠体粒子外圍均勻向內或向外擴散。Sample or buffer is diffusing in and then out of the gel particle
Diffusion
20
圖 2.3A
擴散 瀰散
■溶離液對膠球的擴散或瀰散 Two types of gel
若膠体粒子較大,則由外圍擴散
到內部的距離長,通過粒子所需
要的時間拉長,降低分離效果。
Dispersion (瀰散) 方式的膠体
因通透性佳,溶離液可直接流
入膠体,不再靠擴散作用。
Small particle size reduces the diffusion
time and enhances its resolution
Dispersion type gels let the sample
molecules flow directly through the gel
body, and have better resolution
21
圖 2.3B
■粒子粗細影響溶離液流動 Bead size vs flow rate
膠体的顆粒越小其解析力越大但流速變慢
Adapted from Scope RK (1987) Protein Purification – Principles and Practice p.192
Smaller particles reduce the space between the beads, and prevent the
turbulence as the buffer flows, but the flow rate might be retarded
22
■
不同介質的運用選擇
Sephacryl S-200
Elution volume
Sephacryl S-300
高分子量
分離較好
色帶較慢
溶離出來
低分子量
分離較好
色帶較快
溶離出來
Absorb
ance
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0
50
100
0
50
100
Choose the gel which brings your target protein out of the column earlier
23(10~1,500 kDa)
(5~250 kDa)
■樣本体積的影響 Sample volume at 1% of Vt
● Sample volume at 1~2% of total gel volume
Adapted from Pharmacia (1991) Gil Filtration – Principles and Methods p.46
Superdex 200
Transferrin (81 kD)
& IgG (160 kD)
0 1 2 3 4 5
0.5
1.0
1.5
Sample volume (% of Vt)
Resolution
a
cb
R =a
(b+c)/2
24
a越大分離越開b, c 越小越好
R = 1表示剛好分開
20 25
25
■
膠体管柱的最佳化調整
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(19
91
) G
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Me
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.59
Elution time (h)
Concentr
ation
85
2
0 1 2 3
0 10 20 30Increaseflow rate
Reducecolumn height
A B C
A B C
AB C
25
85
Flow rate (mL/cm2/h)
Bed height (cm)標準分離條件都可分得很好
若加快流速時間省十倍解析力下降
流速已加快再縮短管柱時間再縮短效果則更差但 C 沒問題
管柱長度
溶離速度
■
色析管柱的形狀
10 cm2
30 cm
10 cm
30 cm2
300 cm3
矮胖型Short and fat
細長型Long and slim
●矮胖型管柱不能容忍分離不佳Fat column cannot tolerate poor separation
但其流速及容量均較大But it has better flow rate and higher capacity
Adapted from Scope RK (1987) Protein Purification – Principles and Practice p.195
26
色帶有點歪斜但不影響結果
色帶有點歪斜就會影響分離
■
液相層析管柱系統 A
緩衝液Buffer
梯度製造器Gradient mixer
幫浦Pump
記錄器Recorder
監視器Monitor
管柱Column
膠体Gel
膠体裝填
分劃收集器Fraction collector
(1)
(2)
(3) (4)
(5)
adapter
reservoir
轉接器Connector
The whole family of liquid chromatography apparatus
27
圖 2.5
Buffer starts here
■膠柱裝填方法 Packing column step by step
緩衝液平衡
Equilibratedin buffer
溫度平衡Temperatureequilibrated
靜置膠体Gel stands o/n
清洗膠体Wash gel well
預估体積Estimate
gel volume 裝填膠体Pouring gelsmoothly
暫停流洗Stop elution
X
繼續流洗Keep eluting
Put on reservoir
已堆積Sedimentation
沈積中In progressing
上清Supernatant
加壓流洗Elute under
High pressure
Put on adaptor
緊密堆積
檢查管柱是否暢通
1
2 Gel
GelCheck flow rate of empty column
Pack gel tightly
28
圖 2.6
A
B
C
D
E
■色析膠体的裝填 Packing column
一口氣倒入膠体,勿陷入氣泡。Pour gel slurry smoothly (non-stop), avoid trapping any bubble P
ha
rma
cia
(19
91
) G
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iltra
tion
–P
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les a
nd
Me
tho
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29
■ General principle for column chromatography
Let target protein eluted out the column earlier
Finer bead has better resolution, but slower flow
Use larger column size yet consider practical need
Fast flow reduces resolution, slow brings diffusion
Apply 1% of the total gel volume for sample
Slim column for gel filtration, fat column for others
Gel selection
Bead size
Column size
Flow rate
Sample volume
Column shape
Pack tightly Pack the gel tightly for better resolution
30
2.3 離子交換法 Ion exchange
2.3.1 原理概述 Basic principles
離子與固相擔体帶電基團間的爭奪戰 (Ion wars)
2.2.2 交換介質 Exchange materials
是帶有電荷基團的多醣長鏈聚合物 (膠球)
2.3.3 緩衝液與層析系統 Buffer system
緩衝液對離子交換法之影響極大 (Why?)
2.3.4 管柱操作方法 Column operation
如何操作一支離子交換管柱
2.3.5 色層焦集法 Chromatofocusing
依蛋白質等電點之差異來進行分離
31
■陰離子交換法 Anion Exchange
Solidsupport
+ + ++
++
+
++
++ + +
++
+
Sample
Mobilephase
Stationary phase
Cation groups
Counterion
Anion
+Ion exchange is an adsorption chromatography
32
圖 2.7
吸附
■質子可以吸著或脫離一基團
Proton: the smallest and most abundant particle in the living cell controlling the pH and the charge of a molecule
H+
lone pair electrons
HH
H+
NHH
NAmino
H+
Ampholyte: a molecule contains both positively and negatively charged groups
Carboxylic CO
O
H
CO
O
Proton can enter or leave a functional group relatively freely
33
離子及它的產地
■選擇離子交換介質
+Net Charge of a Protein
Buffer pH
pI
-
3
4
5
6
7
8
9
10
0
Anion exchange
Cation exchange
Use buffer having only one pH unit difference to the
pI of target protein
34
+
(1) 環境 pH 影響帶電性質
(2) 蛋白質帶電性乃可變異
(3) 帶電性質決定分子行為
■陰離子交換膠体的 pH 變化
Support
OH-
OH-
pI = 6
pH = 5
pH = 7-
+
Donnan Effect
OH-
OH-
++
+
+
+
The pH change of the microenvironment surrounding the ion exchange gel particle
pH = 8
Counterion
35
圖 2.9
陰離子交換膠體附近的 pH 其實比較高
使用的緩衝液 pH 比 pI 高 1 pH
相反用陽離子交換的情況也一樣 (pH 更低)
其實是更高
■離子取代優先順序 Displacing order of ions
++ +
++
++
++ +
++
++
+ +++
>
>
電荷高者(higher charge)
離子大者(larger ion)
濃度大者(higher concentration)change pH, NaCl gradient
>
Key properties determining the order of ion replacement
36
圖 2.8
■陰離子交換法分離範圍
100
50
25
75 64kD
pI
The approximate range of proteins isolated by chromatography
37
離子交換法只能分離出大約 1 pH 範圍的蛋白質
再用第二種方法縮小範圍
目標蛋白質
二次元電泳展開樣本大部份蛋白質之分布
■離子交換介質 Common ion exchange materials38
表 2.2
陰陽強弱Resin /
Polystyrene
Glycan /
Cellulose (= X)
Mono
bead
Anio
n E
xchan
ger
StrongDowex-1
-+NR3
TEAE-X
(QAE-X) -+NR3
QDowex-2
WeakDowex-3
-+NHR2 DEAE-X -O(CH2)2+NHR2
IR-45
Cation
Exch
an
ge
r
Strong Dowex-50 -SO3- Phospho-X -PO4
SWeak IRC-150 -COO- CM-X -CH2COO
X = Sephadex, Sepharose, Sephacel or cellulose
選擇:陰離子或陽離子交換?吸附力強弱?何種材質? (樹脂、多糖、Monobead)
■膠体的組成與外型 Support material and bead
CelluloseSephacelMonobead
Pharmacia (1980) Separation News: 5
● Homogeneous bead shape is critical to its resolution and flow rate
Ph
arm
acia
(19
91
) Io
n E
xch
an
ge
Chro
ma
togra
phy –
Prin
cip
les a
nd
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.24
39
■不同膠体的吸著容量有很大差異
DEAE-Sephadex A-25
DEAE-Sephadex A-50
DEAE-Sepharose CL-6B
DEAE-Sephacel
Lactalbumin Albumin Ferritin
191
10
45
38
31
102
115
86
2
1
4.3
8.6
Buffer:
0.01 M Tris-HCl, pH 8.0
(mg / mL gel)
Adapted from Pharmacia (1991) Ion Exchange Chromatography – Principles and Methods p.64
Each gel has different adsorption capacity toward different target proteins
40
三者吸附量之差異是可以解釋的,如何切入思考?
分子量
pI
■離子交換法預備試驗 Determine the conditions
5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50
10 20 30 40 50 60 70 80 90 100
pH
NaCl
pH = 7
0.15 MNaCl
mg/mLprotein
7.0
0.15
50
Enzyme + Buffer + Gel
Sup
Spin-down
Incubation Spin Sup + Spindown
Adapted from Pharmacia (1991) Ion Exchange Chromatography – Principles and Methods p.70 Capacity
41
■膠柱裝填方法 Packing column step by step
緩衝液平衡
Equilibratedin buffer
溫度平衡Temperatureequilibrated
靜置膠体Gel stands o/n
清洗膠体Wash gel well
預估体積Estimate
gel volume 裝填膠体Pouring gelsmoothly
暫停流洗Stop elution
X
繼續流洗Keep eluting
Put on reservoir
已堆積Sediment
沈積中In progressing
上清Supernatant
加壓流洗Elute under
High pressure
Put on adaptor
緊密堆積
檢查管柱是否暢通
1
2 Gel
GelCheck flow rate of empty column
Gel should be equilibrated completely before packing
A
B
C
D
E
42
■液相層析管柱系統 A
緩衝液Buffer
梯度製造器Gradient mixer
幫浦Pump
記錄器Recorder
監視器Monitor
管柱Column
膠体Gel
膠体裝填
分劃收集器Fraction collector
(1)
(2)
(3) (4)
(5)
adapter
reservoir
轉接器Connector
The whole family of liquid chromatography apparatus
43
■鹽梯度的兩種方式 Two ways for making gradient
連續梯度 階段梯度
高限溶液
低限溶液
兩種方式均可,各有特點。Both methods have their specific applications
Eliminate dead
volume
Pharmacia
連續梯度Continuous
連續梯度階段梯度Step-wise
Dead volume膠柱面Gel surface
Upper-limitLower-limit
44
圖 2.10
離子鍵在鹽溶液中不易形成提高鹽濃度分別流洗蛋白質
Dead volume 會破壞拉好的鹽梯度
■連續與階段梯度比較
0.1
0.2
0.3
0.4
0.1
0.2
0.3
0.4
0 50 100 150 200
0 50 100 150 200
Elution volume
連續梯度 Continuous gradient
階段梯度 Step-wise gradient
Pro
tein
concentr
ation
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Continuous vs step-wise
45
■鹽濃度效果比較
0 20 40 60 80 100
0 20 40 60 80 100
0.1
0.2
0.3
0.4
0.1
0.2
0.3
Elution volume
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NaCl
Pro
tein
concentr
ation
Effect of salt concentration
46
■溶離体積會影響解析度
0.5 M NaCl
300 mL
0.5 M NaCl
200 mL
0.5 M NaCl
100 mL
Elution volume
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Rela
tive
absorp
tion
100
50
0
0 100 200 300
100
50
0
100
50
0
Resolution improved
but protein diluted
Effect of elution volume
47
■管柱長短的影響
0.03 0.04 0.06 0.1 0.2
0.5
1.0
0
0.03 0.04 0.06 0.1 0.2
0.5
1.0
0
Acetate concentration (M)
5 cm column
10 cm column
A
B
A
B
Effect of column length
Pro
tein
concentr
ation
48
A 好像被拖延了
■離子交換的梯度溶離有濃縮效果
Salt gradient elution begins
Sample
applied
Ad
ap
ted fro
m S
co
pe
RK
(19
87
) P
rote
in P
urification –
Prin
cip
les a
nd
Pra
ctice p
.11
2
Salt gradient of ion exchange can concentrate the proteins in sample elution
Salt elutes and brings the proteins moving forward,
but proteins are adsorbed again in the front and retarded
49
樣本最前線會反覆吸住膠體
■離子交換法操作要點 Summary on operation
Equilibration Used gel should be regenerated and equilibrated well
Elution Eluted with NaCl in continuous or step-wise gradient
Dead volume Eliminate any dead volume in the column
Buffer pH Keep target proteins in weak charged state
Non-specific Wash out contaminants with low NaCl concentration
Gradient Use proper concentration and volume in elution
Elute through Elute target protein through column and adsorb others
Sample Equilibrated in buffer, don’t overload column capacity
50
■離子交換法實例 Two-step cellulase purification
0.5
100 20 30
0.5
100 20
Retention time (min)
Mono Q HR 5/5
1 mL/min
20 mM Tris, pH 7.6
Crude cellulase 2.5 mg
0.5 M NaCl
0.2 M NaCl
Mono S HR 5/5
1 mL/min
20 mM acetate,
pH 3.6
Q
S--
-
+++
Adapted from Pharmacia (1991) Ion Exchange Chromatography – Principles and Methods p.127
FPLC (fast performance liquid chromatography)
51
■離子交換法實例
0.1
0.2
0.3
0
0.4
100 200 300
0.1
0.2
0.3
0
NaCl
Elution volume
Pro
tein
conte
nt
/ A
ctivity
DEAE Sepharose CL-6B
SS
SDS-PAGE
A typical example
52
■色層焦集法如何拉出 pH 梯度
6 9
9
8
7
6
Volume
實際結果 無法拉出 pH 梯度
Buffering effect 9
8
7
6
Volume
假如含有許多緩衝分子
● Polybuffer含有連續 pKa的緩衝分
子 (ampholyte) 可拉出連續 pH 梯度
6
9pH
9
8
7
6
?9
8
7
6
How chromatofocusing creates its pH gradient by Polybuffer
If there are manybuffering molecules
in the buffer…
● Polybuffer contains ampholyte which is a mixture of chemicals having continuous pKa
53
9
5
2.3.5 色層焦集法 Chromatofocusing
其介質也是一種離子交換介質
但所使用的 Polybuffer 可拉出穩定的 pH 梯度
Using ion exchange bead, but eluted with Polybuffer to create a pH gradient
54
Taller thanShorter than
++
++
■色析焦集法的焦集機制
8
7
6
5 +
+
+
+
+-
0+
Po
lyb
uff
er
焦集在等電點Protein focused at its pI
超越等電點 (帶負電) 被吸附Protein moves beyond its pI will be
negatively charged and retarded
在低 pH 處帶正電被排斥Protein at lower pH is positively
charged and repelled by the beads
Focusing mechanism for a sample protein in chromatofocusing
55
陰離子交換介質
+Net Charge of a Protein
Buffer pH
Isoelectric point,pI
-
3
4
5
6
7
8
9
10
0 -圖 7.1
+
■環境影響分子的帶電性質
56
●抹香鯨肌紅蛋白 (pI = 8.2)追過馬肌紅蛋白 (pI = 7.4)
PharmaciaThe early protein might be caught up by a later protein in chromatofocusing
57
2.4 親和層析法 Affinity chromatography
2.4.1 原理概述 Basic principles
要有一對具有高親和性的分子
2.4.2 親和吸著劑 Affinity adsorbent
有很多親和性吸著劑可利用
2.4.3 金屬螯合層析法 Metal chelating
利用分子與金屬的吸引力
2.4.4 疏水性層析法 Hydrophobic interaction
利用分子間的疏水性引力
2.4.5 液相分配 Liquid partition
分子在兩液相間的分配比例不同
58
■親和層析法的四項要素 Four essential factors
(2)
SpecificBindingSubstance (B)
溶離
Elution
(4)
配体Ligand (A)
耦合反應 (3)
Coupling Reaction
雜質contaminants
B
B
B
B
(1)
Solid Matrix
B
59
圖 2.12
A
Solid matrix = Sepharose
(1) 固相擔體(2) 專一配對(3) 耦合反應(4) 溶離方法
B
■親和層析法的作用機理 How it works
固相擔体Solid support
(1) (2) (3)
A
B
Sample
Washing Elution
X B
親和基團配体 ligand
XB
A A
60
圖 2.11
Activity
■典型的親和層析操作 Typical elution pattern
Elution volume
Protein
pH 2.05
*
61
*超出管柱容量
■專一性結合力量的構成因素 Specific interactions
I. Conformational Match:
Van der waals interaction
II. Interaction Forces:
(1) Hydrogen bond
(2) Hydrophobic interaction
(3) Electrostatic interaction
(4) Van der Waals interaction
+Kd
兩分子間因構形互補所造成的吸引力是由凡得瓦爾力所貢獻
+ -
=O…H-N-
Two types of forces contribute to the specific affinity between proteins
62
■構形互補所造成的吸引力 B
A
Van der Waal interactions
between non-polar surfaces
vdw vdw
凡得瓦爾鍵數目夠多即足以造就親和力
63
-d +d
-d +d -d +d
-d+d -d+d
vdw
非極性電子分布均勻
有時電子分布偏向一邊
造成短暫耦極 引發鄰近原子耦極
產生短暫凡得瓦爾力
也有可能轉另一方向
可能回到原來的狀態
■生化分子的反應基團 Some functional groups
-NH2胺基-COOH酸基
-HC=O醛基
-O-醚
氰基 -C N
-OH醇基 -SH硫醇基
-CH3烷基
●●●●●
●●●
Inert
●●
●
●
The functional groups in a living cell are mild in reactivity
64
■可與各種配体基團反應的介質
* EDC = N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide HCl
Pharmacia
Affinity materials are designed to react with functional groups on ligands
65
表 2.3
配体基團 親和性介質 反應基團 反應方式
-NH2
CNBr-activated Sepharose 4B -C≡N 直接反應
CH-Sepharose 4B 或其活化型-COOH 加 EDC*
N-OH-succinimide 直接反應
Epoxy-activated Sepharose 6B oxirane 直接反應
-COOH AH-Sepharose 4B -NH2加 EDC*
-OH Epoxy-activated Sepharose 6B oxirane 直接反應
-SH
Epoxy-activated Sepharose 6B oxirane 直接反應
Thiopropyl-Sepharose 6B -S-S-R DTT 活化
Activated Thio-Sepharose 4B -G-S-S-R 直接反應
■親和性介質的反應基團 Functional groups
- O-C-C-C-O-C-C-C-C-O-C-C - COOH
Sepharose
- OH
- O- C N
NH2 -Protein
- NH -C-C-C-C-C-C- NH2
- NH -C-C-C-C-C-C- COOH
- O - C - NH - Protein
NH
Epoxy
N-Succinimide
Carboxylic
Amino
CNBr activated
- NH -C-C-C-C-C-C- C - O-N
O
O
O
spacer arm effect
- O - C NCNBr
66
■各種親和性介質及其專一性基團Pharmacia
Affinity adsorbents with special affinity ligands
67
表 2.4
配 体 (A) 親和性分子 (B) 說 明
抗体 對應之抗原 免疫吸著劑,大多自行合成
基質或抑制劑 對應之酵素 酵素的專一性結合
Protein A 部分 IgG 單株抗体純化
Con A 醣蛋白 對 α-D-葡萄糖、甘露糖基有專一性
Heparin 凝血蛋白等 Heparin Sepharose CL-6B
Chelator (NTA)-Ni2+ His-tag 表現蛋白質 (His)6 與兩價陽離子間的親和力
Oligo (dT) mRNA Oligo (dT)-cellulose
Cibacron-Blue NAD(P)+ 結合酵素 Blue Sepharose CL-6B
AMP 或ADP等 同上 5'AMP-, 2', 5'ADP-Sepharose 4B
單糖及其衍生物 Lectin 用來純化 lectin
■金屬螯合層析法 Using metal-chelating affinity
NiSolid
support
His
His
His
Protein
Metal Chelate Affinity Chromatography
imidazole
elution-O-C-C-C-O-C-C-C-N
OH
C-COOH
C-COOHOH
His6 Expressed proteinTransition metals 過渡元素
Tag
d orbiter
68
圖 2.13
■親和層析法實例 Materials used in an example
Chitin(solid support)
CHOM(ligand, bait)雞蛋粘多糖蛋白
Chicken ovamucoidTrypsin inhibitor
Trypsin(target)O
O
CH2OH
OH
NH2
O
O
CH2OH
OH
NH
O=CH3
glutaraldehyde
69
Stryer L (1995) Biochemistry 4/e
SDS-PAGE
■以親和層析法純化 Trypsin purification
CHOM
Trypsin
A B C&D
Disc-PAGE
A B C = D
以膠体電泳檢定親和層析
操作過程各步驟的樣本
Check by PAGE →
Chitin
70
相同的樣本卻有不同的色帶?
■ Hydroxyapatite 吸著劑
(Ca5(PO4)3OH)2 磷酸鈣陶土
可選擇 NaCl或磷酸等不同溶離條件Elution by NaCl or phosphate gradient leads to different results
71
具有非常多樣的吸附特性有負電荷磷酸也有鈣離子
氫氧基磷灰石人骨的主要成分
University of Liverpool, ChemTube3D (left) Bio-Rad CHT (right)
■蛋白質表面的極性或非極性分布
Stryer L (1995) Biochemistry 4/e Fig. 21-36
Superxoide
dismutase
(SOD)
Activie site
Hydrophobic area
72
蛋白質表面的非極性區域多寡不同可利用來作為分離純化的手段 (HIC)
Polarsolvent
■疏水性及反相層析法 Hydrophobic interaction
HIC (liquid-solid) Reversed phase chromatography (liquid-liquid)
Using non-polar groups as a stationary phase Using ion-exchanger
Polarsolvent
Non-polararea
Hydrophobic group(solid)Polar area
Non-polararea
(liquid)
Non-polarsolvent
++
+++++++ ++
Polar phase
C18 = -C18H37
C6 = -C6H13
Phenyl = -C6H6
73
圖 2.14
有各種不同應用方式最常被使用在 HPLC
溶離液由極性轉成非極性 (或相反)
■Hydrophobic interaction chromatography(HIC)
Elution volume (mL)
0
10
20
30
40
50
5
10
15
20
25
100 200 300 400
Eth
yle
ne
gly
co
l (%
)
Ammonium sulfate (% sat.)
Pro
tein
concentr
ation BA
74
BA = beta amylase
Adapted from Pharmacia - Hydrophobic Interaction Chromatography
由極性到非極性的梯度
蛋白質表面非極性區域很小
非極性區較大
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