•A mammalian cell has 50,000-100,000 proteins
(including PTMs)
•Selective techniques isolate one (affinity)
• Non-selective techniques isolate large number/all
•Proteomics require isolation of proteins at high resolution (selective)
•Must be amenable to high throughput (HT)
1

Proteins can be isolated on the grounds of the following properties:
•Mass/density (hydrodynamic)
•Size (gel filtration)
•pI (pI precipitation of IEF)
•Charge (Ion-exchange)
•Solubility (acetone of ammoniumsulfate ppt)
•Structure (affinity)
myosin insulin ( ββββ -chain) myoglobin
2

Gal4 homodimer
Human insulin ( ββββ -chain)
Ribbon Electrostatic surface
3

-
+
ν =
Eq f
• ν Is velocity of the molecule
•E is electeric field strength (volts/cm
•q is net charge on molecule
•f is frictional coefficient
Glycine, Gly, G no charge
Hydrophobicity = 0.67
MW 57Da pK a pK a
COOH = 2.35
NH
2 pI=5.97
= 9.6
Alanine, Ala, A no charge
Hydrophobicity = 1.0
MW 71Da pK a pK a
COOH = 2.34
NH
2 pI = 6.01
= 9.69
Valine, Val, V no charge
Hydrophobicity = 2.3
MW 99Da pK a pK a
COOH = 2.32
NH
2 pI = 5.97
= 9.62
Leucine, Leu, L no charge
Hydrophobicity = 2.2
MW 113Da pK a pK a
COOH = 2.36
NH
2 pI = 5.98
= 9.60
Isoleucine, Ile, I no charge
Hydrophobicity = 3.1
MW 113Da pK a pK a
COOH = 2.36
NH
2 pI = 6.02
= 9.68
Proline, Pro, P no charge
Hydrophobicity = -0.29
MW 97Da pK a pK a
COOH = 1.99
NH
2 pI = 6.48
= 10.96
4

Phenylalanine, Phe, F no charge
Hydrophobicity = 2.5
Absorbs UV
MW 147Da pK pK a a
COOH = 1.83
NH pI=5.48
2
= 9.13
Tyrosine, Tyr, Y weak charge
Hydrophobicity = 0.08
Absorbs UV
MW 163Da pK pK a a
COOH = 2.20
NH pI=5.66
2
= 9.11
Tryptophan, Trp, W no charge
Hydrophobicity = 1.5
Absorbs UV
MW 186Da pK pK a a
COOH = 2.38
NH pI=5.89
2
= 9.39
Serine, Ser, S no charge
Hydrophobicity = -1.1
MW 87Da pK pK a a
COOH = 2.21
NH
2 pI = 5.68
= 9.15
Threonine, Thr, T no charge
Hydrophobicity = -0.75
MW 101Da pK pK a a
COOH = 2.11
NH
2 pI = 5.87
= 9.62
Asparagine, Asn, N no charge
Hydrophobicity = -2.7
MW 114Da pK a pK a
COOH = 2.02
NH
2 pI = 5.41
= 8.08
Glutamine, Gln, Q no charge
Hydrophobicity = -2.9
MW 128Da pK a pK a
COOH = 2.17
NH
2 pI = 5.65
= 9.13
5

Cysteine, Cys, C weak charge
Hydrophobicity = 0.17
MW 103Da pK a pK a
COOH = 1.96
NH
2 pI = 5.07
= 8.18
Methionine, Met, M no charge
Hydrophobicity = 1.1
MW 131Da pK a pK a
COOH = 2.28
NH
2 pI = 5.74
= 9.21
Acidic
Aspartic acid, Asp, D negative charge
Hydrophobicity = -3.0
MW 115Da pK pK a
COOH = 2.19
a
NH
2 pI = 2.77
= 9.60
Glutamic acid, Glu, E negative charge
Hydrophobicity = -2.6
MW 129Da pK pK a
COOH = 2.19
a
NH
2 pI = 3.22
= 9.67
Basic
Histidine, His, H
Weak positive charge
Hydrophobicity = -1.7
MW 137Da pK a pK a
COOH = 1.82
NH
2 pI = 7.59
= 9.17
Lysine, Lys, K positive charge
Hydrophobicity = -4.6
MW 128Da pK a pK a
COOH = 2.18
NH
2 pI = 9.47
= 8.95
Arginine, Arg, R positive charge
Hydrophobicity = -7.5
MW 156Da pK a pK a
COOH = 2.17
NH
2
= 9.04
pI = 10.76
6

A.A.
F
G
H
I
K
A
C
D
E
P
Q
R
S
T
V
L
M
N
W
Y
Amino acid pK a values
Carboxylic acid
2.3
1.8
2.0
2.4
2.3
2.0
2.0
2.2
1.8
2.1
2.6
2.3
2.4
2.2
1.8
2.4
1.8
2.4
2.2
Amine
9.9
10.8
10.0
9.6
9.2
8.8
10.6
9.1
9.0
9.2
9.1
9.8
9.2
9.7
9.2
10.4
9.6
9.4
9.1
Side Chain
-
8.6
4.5
-
-
-
9.8
-
12.5
-
-
-
-
-
-
-
6.8
-
11.1
(1) HA H + + A -
= [H + ][A -
[H+] = K a
[HA]/[A ]
- log[H + ] = - logK a
- log[HA]/[A ] pH = pK a
+ log[A ]/[HA] pH = pK a
+ log(R) pH - pK a
= log(R)
10 (pH - pKa) = R
(3)
(4)
(5)
(6)
(7)
(8)
7

(1) [A ]/[HA] = R
-
A
T
= [A ] + [HA]
Substitute eq. 2 in eq. 3
A
T
= [HA]R + [HA]
= [HA](1 + R)
[HA] = A
T
(1 + R)
Substitute rearranged eq. 2 in eq. 6
[A ] = A
T
R
(1 + R)
(3)
(4)
(5)
(6)
(7)
How to calculate the charge of an amino acid at a given pH value
•Lets choose K
•and lets choose pH 7.2
•K has three dissociable groups that can contribute to the charge:
•an α -NH
2
, an α -COOH and an ε -NH
2 group
•The pKa values of the three groups are 9.2, 2.2 and 11.1
10 (pH-pKa) = R
Lets start with the amine:
∴ 10 (7.2-9.2) = 10 -2
Since the in the HA species
[HA] = A
T
α –NH
(1/(1+R))
Assume A
T
= 1
∴ [HA] = 1/(1+ 10 -2 ) = 1/1.01 = 0.99
+
Thus the α –NH
2 is almost fully protonated at pH 7.2, and will contribute a charge of
+0.99 to the amino acid
8

How to calculate the charge of an amino acid at a given pH value continued…
•Lets look at the α –COOH next
•The pKa value of the –COOH is 2.2
10 (pH-pKa) = R
∴ 10 (7.2-2.2) = 10 5
The dissociated –COO- is the charged species
We are therefore interested in the A- species and not the HA
[A ] = A
T
(R/(1+R))
Assume A
∴
= 1
∴ [A ] = 10 5 /(1+ 10 5 ) = 100,000/100,001 = 0.999
Thus the α COOH is almost fully deprotonated at pH 7.2, and will contribute a charge of -0.999 to the amino acid
How to calculate the charge of an amino acid at a given pH value continued…
•Lets look at the ε –NH
2 next
•The pKa value of the ε –NH
2 is 11.1
10 (pH-pKa) = R
∴ 10 (7.2-11.1) = 10 -3.9
The protonated
[HA ] = A
T
ε –NH
3
+ is the charged species
We are therefore interested in the HA species and not the A -
Assume A
∴
(1/(1+R))
T
= 1
[HA] = 1/(1+ 10
Thus the ε –NH
-3.9
) = 1/1.0001259 = 0.999
2 is almost fully protonated at pH 7.2, and will contribute a charge of
+0.999 to the amino acid
The net charge of lysine at pH7.2 is therefore the sum of the three charges:
α -NH
2
+ α -COOH + ε –NH
2
+ 0.99 - 0.999 + 0.999 = + 0.99
9

1. Calculate R for each dissociable group
2. Calculate [A ] or [HA]
3. Calculate fractional charge pH
R
1.7
COOH
[A-] charge
R
8.3
SH
[A-] charge
R
10.8
0 0.019952623 0.019562304 -0.019562304
5.01187E-09 5.01187E-09 -5.01187E-09 1.58489E-11
NH2
[HA] charge
Total charge
1 0.199526231 0.166337531 -0.166337531
5.01187E-08 5.01187E-08 -5.01187E-08 1.58489E-10
1
1
2 1.995262315 0.666139425 -0.666139425
5.01187E-07 5.01187E-07 -5.01187E-07 1.58489E-09 0.999999998 0.999999998
1 0.980437691
1 0.833662419
0.333860073
3 19.95262315 0.952273279 -0.952273279
5.01187E-06 5.01185E-06 -5.01185E-06 1.58489E-08 0.999999984 0.999999984
4 199.5262315 0.995013121 -0.995013121
5.01187E-05 5.01162E-05 -5.01162E-05 1.58489E-07 0.999999842 0.999999842
0.047721693
0.004936604
5 1995.262315 0.999499064 -0.999499064
0.000501187
0.000500936
-0.000500936
1.58489E-06 0.999998415 0.999998415
-1.58489E-06
6 19952.62315 0.999949884 -0.999949884
0.005011872
0.004986879
-0.004986879
1.58489E-05 0.999984151 0.999984151 -0.004952611
7 199526.2315 0.999994988 -0.999994988
0.050118723
0.047726721
-0.047726721
0.000158489 0.999841536 0.999841536 -0.047880173
8
9 19952623.15
10 199526231.5 0.999999995 -0.999999995
50.11872336
0.980437696
-0.980437696
0.158489319 0.863193111 0.863193111
13
14
1995262.315 0.999999499 -0.999999499
12 19952623150
1.99526E+11
1.99526E+12
0.99999995
1
1
-0.99999995
-1
0.501187234
5.011872336
5011.872336
0.333860575
0.833662469
0.999800514
-0.333860575
-0.833662469
-0.999800514
0.001584893 0.998417615 0.998417615
11 1995262315 0.999999999 -0.999999999
501.1872336
0.998008711
-0.998008711
1.584893192
0.38686318
0.38686318
15.84893192 0.059350943 0.059350943
-0.33544246
0.015848932 0.984398338 0.984398338 -0.849264081
-1.11724458
-1.61114553
-1.94044957
-1 50118.72336
0.999980048
-0.999980048
158.4893192 0.006270012 0.006270012 -1.993710035
-1 501187.2336
0.999998005
-0.999998005
1584.893192 0.000630559 0.000630559 -1.999367445
!
"
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 p H
10

The peptide DKG pKa = 9.8
H O
H
3
N +
CH
2
COO pKa = 3.9
H
H O
CH
2
CH
2
CH
2
CH
2
NH
3
+
H
C
α
O
C
H CH
3
O pKa = 2.4
pKa = 10.5
pH
N terminus pKa = 9.8
Aspartic acid R-group
Pka=3.9
Lysine R-group pKa=10.5
C terminus pKa=2.9
Total charge
0
R
1 1
R
0.000125877 -0.000125877
1 0.999999998 0.999999998 0.001257343 -0.001257343
R
1 1
1
R
0.001257343 -0.001257343 0.998616781
1 0.012432735 -0.012432735 0.986309922
2 0.999999984 0.999999984 0.012432735 -0.012432735 0.999999997 0.999999997
0.11181577
-0.11181577
0.875751492
3 0.999999842 0.999999842
0.11181577
-0.11181577
0.999999968 0.999999968 0.557311634 -0.557311634 0.330872565
4 0.999998415 0.999998415 0.557311634 -0.557311634 0.999999684 0.999999684 0.926412444 -0.926412444 -0.483724394
5 0.999984151 0.999984151 0.926412444 -0.926412444 0.999996838 0.999996838 0.992119316 -0.992119316 -0.918534922
6 0.999841536 0.999841536 0.992119316 -0.992119316 0.999968378 0.999968378 0.999206302 -0.999206302
-0.99135724
7 0.998417615 0.998417615 0.999206302 -0.999206302 0.999683872 0.999683872 0.999920573 -0.999920573 -0.999443004
8 0.984398338 0.984398338 0.999920573 -0.999920573 0.996847691 0.996847691 0.999992057 -0.999992057 -1.003064939
9 0.863193111 0.863193111 0.999992057 -0.999992057
0.96934657
0.96934657
0.999999206 -0.999999206 -1.030644692
10 0.38686318
0.38686318
0.999999206 -0.999999206 0.759746927 0.759746927 0.999999921 -0.999999921
-1.2402522
11 0.059350943 0.059350943 0.999999921 -0.999999921 0.240253073 0.240253073 0.999999992 -0.999999992 -1.759746839
12 0.006270012 0.006270012 0.999999992 -0.999999992
0.03065343
0.03065343
0.999999999 -0.999999999 -1.969346561
13 0.000630559 0.000630559 0.999999999 -0.999999999 0.003152309 0.003152309
14 6.30918E-05 6.30918E-05 1 -1 0.000316128 0.000316128
1
1
-1
-1
-1.99684769
-1.999683872
You can also use the program “What charge?” to calculate charge versus pH curves of any amino acid, peptide or protein, based on the entered sequence. You can download the program from http://www.uovs.ac.za/faculties/documents/04/112/Software/11476-what_charge.zip
11

1.0
0.0
-0.5
-1.0
-1.5
-2.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 p H
-
- -
- -
-
-
-
SDS
- - -
-
-
-
protein
•Sodium dodecylsulfate (SDS)
•Amphipatic molecule
•0.1% SDS gives one SDS molecule per 2 amino acid residues
•Thus charge per unit length of all proteins are the same
• Proteins of the same size will have the same charge
•Proteins separated mainly by size
•Very basic proteins such as histones behave anomalously
12

•The composition of polyacrylamide gels is expressed as:
•%T grams acrylamide plus gram bis-acrylamide per 100ml solution
•%C grams bisacrylamide per 100ml solution
•Typical gels %T range from 3% to 20%
•%C from 3 to 6%
•The higher the acrylamide percentage, the smaller the gel pores
•The higher the bis-acrylamide percentage, the samller the gel pores
•Larger molecules cannot migrate through small gel pores
•Percentage acrylamide has range within which molecules can be separated
•3% gels for protein of 1000kDa
•20% gels for proteins of 10kDa
•Polyacrylamide gel electrophoresis = PAGE
13

glycine protein
Cl -
Stacking gel
3%T pH 6.8
Running gel
8-16%T pH8.8
•Sample buffer is
•glycine
•Cl
•and protein
•When glycine enters the gel, at pH6.8 it is a zwitterion
•To maintain current, the Cl and proteins migrate
•The pores are very large, so the proteins can move fast
•The glycine follows
•This tends to concentrate the proteins in thin bands before they strike the running gel, where separation occurs
•The glycine becomes charged in the running gel, and carries current with Cl -
•Proteins separated in pH gradient formed by ampholytes
•Where protein is at pH below pI, it is (+)-charged, and move towards anode
•Where protein is at pH above pI, it is (-)-charged and moved towards cathode
•At pH = pI, protein has no net charge, and is immobile
•Proteins migrate to a pH region where they have no net charge
•Proteins separated in terms of pI
•Ampholytes poured into gel before setting
•Buy pre-fabricated Immobilised pH gradient (IPG) strips
•Wide range (pH3-10) or narrow range (pH8-9) strips
14

Two-dimensional gel electrophoresis (2DGE)
Run IEF in the first dimension and SDS-PAGE in the second dimension
1 st dimension
Resolved by pI
SDS-PAGE
Example of a 2D Gel
240 µ g of E. coli 2DGE, pH range 4 – 7, mass range 10 – 120kDa.
15

Resolution of 2DGE
More proteins in proteome than can be resolved by standard 2DGE system
Use 30cmX30cm gels (resolve 10 spots)
Run multiple IEF strips at overlapping or adjacent pH ranges pH 3-12 pH 5-6
Sensitivity of 2DGE
•Proteins levels differ by 10 5 -10 9 in a cell
•Superabundant proteins are present at 1,000,000 copies per cell
•Abundant proteins are present at 100,000 copies per cell
•Rare at proteins are present at 1000 copies per cell
•Thus, very rare proteins may not be visible on 2DGE gels
•Very abundant proteins may obscure less abundant proteins
•Solve sensitivity by increasing resolution
•Narrow range IPGs
•Prefractionation or affinity depletion
16

Spot-picker robot for 2DGE gels
An LC/MS/MS system
17

δ +
+
+
+
+
δ -
+
+
+
+
+
+
-
+
-
•Most proteins are highly soluble under physiological conditions (0.15-0.2M salt and neutral pH)
•Solubility affected by:
•Polar interactions with solvent molecules
•Ionic interactions with the salt ions present
•Repulsive electrostatic repulsions between
•Like charges
•Small, heterogenous aggregates
+
+
+
+
-
+
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
-
+
+
+
+
+
-
-
+
+
+
18

+
+
+ pH < pI
-
+
+
+
+
-
-
•Repulsion
+
+
pH pI
+
+
+
-
-
+
+
-
-
+ -
+
-
-
-
•Little repulsion
•Hydrophobic interaction predominates
•Also electrostatic attraction between opposite charges
•A protein is least soluble close to its pI
-
+
pH > pI
-
-
-
-
+
-
-
-
-
•Repulsion
-
-
+
Attracting electrostatic charges
-
+
+
+ -
+
-
+
-
+
+
+
-
-
-
+
+
-
-
+ salt
+ -
+
-
+
-
+
+
-
+
-
-
-
+
+
+ +
+
-
-
+
+
-
-
+ -
+
+
-
-
-
Ions screen electrostatic charge
Attractive forces weaken
+
19

+
+ -
+
-
+
-
-
+ salt + -
+
-
+
+
-
+
+
+
+
-
+
-
-
-
-
+
-
-
Water molecules ordered next to exposed hydrophobic patches
Ions compete for water molecules
Hydrophobic patches exposed
Aggregation results
Ring of ordered water molecules
Hydrophobic droplet
•Volume of sphere = 4 π r 3 /3
•Surface of sphere = 4 π r 2
•1ml sphere has surface area of 4.8cm
2
•2 spheres of 1ml will have combined area of 9.6mm
•If the two droplets fuse, the combined volume is 2ml
2 •The total surface area of the 2ml droplet is 7.7cm
∴ if the two droplets fuse, the surface area will be less the number of water molecules that must be highly ordered is less the number of disordered water molecules increase increase in entropy
20

More disordered water molecules
• •
• ∆ G = ∆ H - T ∆ S
∆ S is the change in entropy (order)
•Change from order to disorder gives a positive ∆ S, and ∴ a ∆ G
•Processes where ∆ G < 0 (i.e., negative) occur spontaneously
•At higher temperature, the disorder in the free water molecules increase
∴ transfer of a water molecule from the ordered environment next to the hydrophobic droplet to the disordered state, will have a larger ∆ S component
•Thus, it becomes very favourable to fuse the droplets at higher temperatures
Na +
Cl -
Cl -
Cl -
Na +
Cl -
Na +
Na +
Cl -
More disordered water molecules
•At higher salt concentrations, weak interactions between the dis ordered water molecules are further disrupted, i.e., the molecules become even less ordered
•transfer of a water molecule from the ordered environment next to the hydrophobic droplet to the disordered state, will have a larger ∆ S component at higher salt
•it becomes very favourable to fuse the droplets at higher salt concentrations
21

Salting out described by: log
10
(solubility) = A – m (salt concentration)
A = constant that depends on pH and temperature m = independent of pH and temperature
•Salt must not interact with protein (chaotropic)
•Protein aggregation increases with increase in temperature
•Anions with decreasing tendency to salt out lysozyme:
•phosphate > sulphate > acetate > Cl > Br > nitrate > perchlorate
•Cations with decreasing tendency to salt out lysozyme:
• ammonium > K + > Na +
• This series is known as the Hofmeister series
% Saturation
0-40
% Enzyme precipitated
4
% Protein precipitated
25
Purification factor
First trial
60-80 32
80 supernatant 2
32
21
Conclusion: Enzyme precipitated more in 40-60 than 60-80; try 45-70
Second trial 0-45 6 32
45-70 90
70 supernatant 4
38
30
Conclusion: Good recovery, but poorer purification; try 48-65
Third trial 0-48 10 35
48-65 75
65 supernatant 15
25
40
1.0
2.4
3.0
22

•Organic solvents decrease dielectric constant of solvent
•Displaces water molecules from and solvates hydrophobic patches
+
+ -
+
-
+
+
+
-
-
+
-
-
-
soluble
+
-
-
+ organic solvent
-
-
•Solvent displaced water molecules from hydrophobic patches
•Increase in hydrophobic interaction
•Also evidence of electrostatic attraction
•Aggregation
•Ethanol
•Acetone
•Methanol
• i -propanol
• n -propanol
•Dioxan
• 2 -methoxyethanol
•Precipitation must be performed below 10°C, otherwise proteins denature
•Some organic polymers such as polyethylene glycol
(PEG) acts like organic solvents, but are effective at much lower concentrations
23

Increasing temperature
Internal hydrophobic interactions stabilizes protein
•Elevated temperature causes increased molecular vibration
•This allows organic solvent molecules to interact with internal hydrophobic regions
•Internal hydrophobic interactions become disrupted
Protein unfolds and denatures
24

Stationary phase
Sample of 3 different sized molecules
(Stokes radius)
Excluded
Partially included
Totally included
Mobile phase
Elution profile
Time
V o
V e
V t t
V > V t t
???
V t
= V o
+ V i
For a column, V o
= 2,000,000)
V and is difficult to measure
V i i can be determined by eluting blue dextran (M is internal volume plus particle volume (inaccessible to all), can be calculated from V t
-V o
Excluded molecules elute at V o r
Molecules that have access to full V
What if elution volume V e
> V t i elute at V t
? Inert stationary phase?
25

The partition coefficient (K d
) for a molecule is given by
Total volume accessible to molecule
V e
- V o
V t
- V o
Total internal volume
•If K
•If K
•If K
•K d d d
= 1, molecule has full access
> 1, there is adsorption to the column matrix is not the “true” partition coefficient, since V includes gel particle volume i
V e1
V e2
W
1
W
2
W
1
+ W
2
∴ The difference in elution volume divided by the average peak width, W
If R = 1, we have baseline resolution
26

•A chromatographic column can be thought of as a number of independent partition “cells”
•Each such partition cell is known as a “theorical plate”
•Column resolution depends on the number of theorical plates (N) on the column
5.55 V e
2
W 2
•Where W is peak width, and V e is the elution volume
•The height equivalent of a theoretical plate (HETP) is given by the column length divided by the number of theoretical plates (N)
• HETP =
=
L
N
LW 2
5.55 V e
2
•Where L is the column height, W is peak width, and V elution volume is the
27

•The theoretical plate height is related to the elution speed of the mobile phase
•H is a function of
•A, diffusion of the molecule: the longer the molecule is on the column, the more it will diffuse
•B, non-equilibrium, the longer partitioning between the stationary and mobile phase takes, the higher the plate height
•C, column effects, including packing quality, particle
υ
υ + C
van Deemter relation
5
4
A (diffusion)
B (non-equilibrium)
C (column quality)
A+B+C terms
3
2 Optimum flow rate
1
0
0 5 10
Elution rate ( ν )
15 20
•A = 4, B = 0.2, C = 1
•As flow rate increases
•Effect of diffusion decreases
•Effect of non-equilibrium increases
•Optimum flow rate (arrow) minimizes these two terms
28

Trade name
Sephadex
Supplier
Pharmacia
Sephacryl
Polyacrylamide
(Bio-Gel P)
Sepharose
Bio-Gel A
Ultragel
Pharmacia
Bio-Rad
Pharmacia
Bio-Rad
Composition
Cross-linked dextran
Comment
Cannot be manufactured with exclusion limit > 600,000Da
(particle not rigid, and collapses)
N,N’methylenebisacrylamide cross-linked dextran
N,N’methylenebisacrylamide cross-linked acrylamide
Agarose
Higher exclusion limits than
Sephadex
Similar to Sephadex
Very high exclusion limits
Combined polyacrylamide-agarose gels
Higher flow-rates than agarose
V i poor good
Log
10
(M r
) poor
29

Ribbon Electrostatic surface
30

Acidic
Aspartic acid, Asp, D negative charge
Hydrophobicity = -3.0
MW 115Da pK pK a
COOH = 2.19
a
NH
2 pI = 2.77
= 9.60
Glutamic acid, Glu, E negative charge
Hydrophobicity = -2.6
MW 129Da pK pK a
COOH = 2.19
a
NH
2 pI = 3.22
= 9.67
Basic
Histidine, His, H
Weak positive charge
Hydrophobicity = -1.7
MW 137Da pK a pK a
COOH = 1.82
NH
2 pI = 7.59
= 9.17
Lysine, Lys, K positive charge
Hydrophobicity = -4.6
MW 128Da pK a pK a
COOH = 2.18
NH
2 pI = 9.47
= 8.95
Arginine, Arg, R positive charge
Hydrophobicity = -7.5
MW 156Da pK a pK a
COOH = 2.17
NH
2
= 9.04
pI = 10.76
a
31

•IEX separates proteins on the basis of their net surface charge
•An ion-exchange (IEX) resin is a resin than is negatively or positively charged at a given pH
•Proteins with the opposite charge will bind to this resin
•Cation exchanger binds positively charged proteins
•Anion exchanger binds negative charged proteins
•Electrostatic attraction between charged protein z
1 charged group z
2
, is given by
F = z
1 z
2
Dr 2 and IEX resin
•The larger the (opposite) charges and the closer they are, the stronger the attraction
• Ion exchange columns are typically eluted by increasing the ionic strength (salt concentration) of the buffer
Resin matrices are composed of different materials
•Cellulose
•Agarose
•Dextran
•Synthetic polymers
•Polyacrylamide
IEX matrices will also act as gel filtration media
32

+1
Q
Anion exchangers
0
S
CM
Cation exchangers
-1
8 9 10 pH
•DEAE (diethylaminoethyl) and CM (carboxymethyl) are weak exchangers
•Sulfonyl (S) and quaternary ammonium ions (Q) are strong exchangers
Anion exchangers Strength
Quaternary ammonium strong
Diethylaminoethyl
Diethylaminopropyl weak weak
Functional group
3
-O-CH
2
-CH
2
-NH + -(CH
2
-CH
3
)
2
O-CH
2
-CHOH-CH
2
-NH + -(CH
2
-CH
3
)
2
Cation exchangers Functional group
Sulfopropyl (S)
Methyl sulfonate (SP)
Carboxymethyl (CM) strong strong weak
O-CH
2
-CHOH-CH
2
-O-CH
2
-CH
2
-CH
2
-SO
3
-
O-CH
2
-CHOH-CH
2
-O-CH
2
-CHOH-CH
2
-SO
3
-
-O-CH
2
-COO -
33

v
Cation exchangers
-
+
0 v
Anion exchangers pH v v v v v v
34

1. Activation of the matrix
2. Attachment of the ligand to the activated group
Many activated matrices are commercially available
• CNBr-activated agarose Sepharose 4B
• 6-aminohexanoic acid (CH)-agarose
• 1,6-diaminohexane (AH) agarose
• Carbonyldiimidazole (CDI)-agarose
• Epoxy-agarose
gel
OH
CNBr gel
O
O
C NH
NH
2
R
OH gel
O C
NH
2
+
NH R
35

C-myc
HA
VSV-G
HSV
V5
FLAG
Tag Sequence
EQKLISEEDL
YPYDVPDYA
YTDIEMNRLGK
QPELAPEDPED
GKPIPNPLLGLDST
DYKDDDDKG
VSV-G: vesicular stomatitis virus G protein
HSV: Herpes simplex virus
•Based on the affinity of metal ion for basic amino acid residues
•Ions mostly used include Ni 2+ , Co 2+ , Cu 2+ , Zn 2+ and Fe 2+
•Metal ion is immobilized by partial coordination binding by IDA, NTA and TED and TED
• Affinity is caused by metal ion not being fully coordinated
•Solvent and buffer molecules coordinate weakly
•Basic amino acid residues such as histidine, tryptophan and cysteine coordinate much more strongly
•One can fuse a stretch of 6 contiguous histidine residues (6× his tag) to the C-terminal or N-terminal of a protein, and isolate the recombinant fusion protein on a Ni 2+ -agarose column
• Protein is eluted with
•imidazole (structural analog of histidine)
•elution buffer pH can be dropped to 6, where histidine is protonated and cannot bind coordinately to Ni 2+
36

OH
CH N
CH
2
COO -
CH
2
COO -
Imido diacetic acid (IDA)
OH
CH
2
CH CH
2
CH
CH
2
COO -
N
CH
2
COO -
CH
2
COO -
Nitrilo triacetic acid (NTA)
OH
CH
2
CH CH
2
N
CH
2
CH
2
N
CH
2
COO -
CH
2
COO -
CH
2
COO -
Tris(carboxymethyl) ethylene diamine (TED)
Histidine
C NH
CH2
HC NH
Imidazole
37

•Although one-step IMAC yield pure protein, it is seldom homogenous
•TAP tag is based on having two tandem affinity tags in protein
•Most frequent is calmodulin binding protein (CBP) followed by protein A
•CBP binds to the protein calmodulin in the presence of Ca 2+
•Protein A binds to the F
C region of IgG immunoglobulins
•The CBP and protein A sequences are separated by a TEV protease site
(ENLYFQE)
•This sequence is cleaved by TEV protease
•The protein is first bound to Sepharose beads covered with IgG
•Adsorbed protein cleaved with TEV protease and eluted
•Binding to calmodulin column
•Elution with Ca 2+
•This allow two-step ultra-pure protein preparation
•Very mild conditions allows isolation of protein complexes
IgG sepharose column
Ca 2+
38