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

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■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