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

il F

iltra

tion

–P

rin

cip

les a

nd

Me

tho

ds p

.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

Ad

ap

ted fro

m P

ha

rma

cia

: G

il F

iltra

tion

–P

rin

cip

les a

nd

Me

tho

ds

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

Ad

ap

ted fro

m P

ha

rma

cia

: G

il F

iltra

tion

–P

rin

cip

les a

nd

Me

tho

ds

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

Ad

ap

ted fro

m P

ha

rma

cia

(19

91

) G

il F

iltra

tion

–P

rin

cip

les a

nd

Me

tho

ds p

.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

il F

iltra

tion

–P

rin

cip

les a

nd

Me

tho

ds

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

Me

tho

ds p

.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

Ad

ap

ted fro

m P

ha

rma

cia

: Io

n E

xch

an

ge

Chro

ma

togra

ph

y –

Prin

cip

les a

nd

Me

tho

ds

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

Ad

ap

ted fro

m P

ha

rma

cia

: Io

n E

xch

an

ge

Chro

ma

togra

ph

y –

Prin

cip

les a

nd

Me

tho

ds

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

Ad

ap

ted fro

m P

ha

rma

cia

: Io

n E

xch

an

ge

Chro

ma

togra

ph

y –

Prin

cip

les a

nd

Me

tho

ds

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