Download Chapter 2 - University of the Free State

Chapter 2
Strategies for Protein Separation
•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)
technology
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)
Proteins differ by size
myoglobin
myosin
insulin (β
β-chain)
2
Proteins have different shapes
Human insulin (β
β-chain)
Gal4 homodimer
Proteins have charges
Ribbon
Electrostatic surface
3
Molecular movement in an electric field
-
+
+
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
Aliphatic amino acids (VAGLIP)
Glycine, Gly, G
no charge
Hydrophobicity = 0.67
MW 57Da
pKa COOH = 2.35
pKa NH2 = 9.6
pI=5.97
Alanine, Ala, A
no charge
Hydrophobicity = 1.0
MW 71Da
pKa COOH = 2.34
pKa NH2 = 9.69
pI = 6.01
Valine, Val, V
no charge
Hydrophobicity = 2.3
MW 99Da
pKa COOH = 2.32
pKa NH2 = 9.62
pI = 5.97
Leucine, Leu, L
no charge
Hydrophobicity = 2.2
MW 113Da
pKa COOH = 2.36
pKa NH2 = 9.60
pI = 5.98
Isoleucine, Ile, I
no charge
Hydrophobicity = 3.1
MW 113Da
pKa COOH = 2.36
pKa NH2 = 9.68
pI = 6.02
Proline, Pro, P
no charge
Hydrophobicity = -0.29
MW 97Da
pKa COOH = 1.99
pKa NH2 = 10.96
pI = 6.48
4
Aromatic amino acids (FYW)
Phenylalanine, Phe, F
no charge
Hydrophobicity = 2.5
Absorbs UV
MW 147Da
pKa COOH = 1.83
pKa NH2 = 9.13
pI=5.48
Tyrosine, Tyr, Y
weak charge
Hydrophobicity = 0.08
Absorbs UV
MW 163Da
pKa COOH = 2.20
pKa NH2 = 9.11
pI=5.66
Tryptophan, Trp, W
no charge
Hydrophobicity = 1.5
Absorbs UV
MW 186Da
pKa COOH = 2.38
pKa NH2 = 9.39
pI=5.89
Polar but uncharged (SNQT)
Serine, Ser, S
no charge
Hydrophobicity = -1.1
MW 87Da
pKa COOH = 2.21
pKa NH2 = 9.15
pI = 5.68
Threonine, Thr, T
no charge
Hydrophobicity = -0.75
MW 101Da
pKa COOH = 2.11
pKa NH2 = 9.62
pI = 5.87
Asparagine, Asn, N
no charge
Hydrophobicity = -2.7
MW 114Da
pKa COOH = 2.02
pKa NH2 = 8.08
pI = 5.41
Glutamine, Gln, Q
no charge
Hydrophobicity = -2.9
MW 128Da
pKa COOH = 2.17
pKa NH2 = 9.13
pI = 5.65
5
Sulphur containing (CM)
Cysteine, Cys, C
weak charge
Hydrophobicity = 0.17
MW 103Da
pKa COOH = 1.96
pKa NH2 = 8.18
pI = 5.07
Methionine, Met, M
no charge
Hydrophobicity = 1.1
MW 131Da
pKa COOH = 2.28
pKa NH2 = 9.21
pI = 5.74
Charged (DEHKR)
Acidic
Aspartic acid, Asp, D
negative charge
Hydrophobicity = -3.0
MW 115Da
pKa COOH = 2.19
pKa NH2 = 9.60
pI = 2.77
Glutamic acid, Glu, E
negative charge
Hydrophobicity = -2.6
MW 129Da
pKa COOH = 2.19
pKa NH2 = 9.67
pI = 3.22
Basic
Histidine, His, H
Weak positive charge
Hydrophobicity = -1.7
MW 137Da
pKa COOH = 1.82
pKa NH2 = 9.17
pI = 7.59
Lysine, Lys, K
positive charge
Hydrophobicity = -4.6
MW 128Da
pKa COOH = 2.18
pKa NH2 = 8.95
pI = 9.47
Arginine, Arg, R
positive charge
Hydrophobicity = -7.5
MW 156Da
pKa COOH = 2.17
pKa NH2 = 9.04
pI = 10.76
6
Amino acid pKa values
A.A.
Carboxylic
acid
Amine
Side Chain
A
2.3
9.9
-
C
1.8
10.8
8.6
D
2.0
10.0
4.5
E
2.2
9.7
4.5
F
1.8
9.1
-
G
2.4
9.8
6.8
H
1.8
9.2
I
2.4
9.7
-
K
2.2
9.2
11.1
L
2.4
9.6
-
M
2.3
9.2
-
N
2.0
8.8
-
P
2.0
10.6
-
Q
2.2
9.1
-
R
1.8
9.0
12.5
S
2.1
9.2
-
T
2.6
10.4
-
V
2.3
9.6
-
W
2.4
9.4
-
Y
2.2
9.1
9.8
The Henderson-Hasselbalch equation
HA
H+ + A-
(1)
Ka = [H+][A-]/[HA]
(2)
[H+] = Ka[HA]/[A-]
(3)
- log[H+] = - logKa - log[HA]/[A-]
(4)
pH = pKa + log[A-]/[HA]
(5)
pH = pKa + log(R)
(6)
pH - pKa = log(R)
(7)
10(pH - pKa) = R
(8)
7
Acid and base fractions in a titration
[A-]/[HA] = R
(1)
[A-] = [HA]R
(2)
AT = [A-] + [HA]
(3)
Substitute eq. 2 in eq. 3
AT = [HA]R + [HA]
= [HA](1 + R)
[HA] = AT
1
(1 + R)
Substitute rearranged eq. 2 in eq. 6
[A-] = AT
R
(1 + R)
(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-NH2, an α-COOH and an ε-NH2 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 α–NH2 will carry a charge in the protonated –NH3+ form, we are interested
in the HA species
[HA] = AT(1/(1+R))
Assume AT = 1
∴[HA] = 1/(1+ 10-2) = 1/1.01 = 0.99
Thus the α–NH2 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) = 105
The dissociated –COO- is the charged species
We are therefore interested in the A- species and not the HA
[A-] = AT(R/(1+R))
Assume AT = 1
∴ -] = 105/(1+ 105) = 100,000/100,001 = 0.999
∴[A
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–NH2 next
•The pKa value of the ε–NH2 is 11.1
10(pH-pKa) = R
∴10(7.2-11.1) = 10-3.9
The protonated ε–NH3+ is the charged species
We are therefore interested in the HA species and not the A[HA-] = AT(1/(1+R))
Assume AT = 1
∴[HA] = 1/(1+ 10-3.9) = 1/1.0001259 = 0.999
Thus the ε–NH2 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-NH2 + α-COOH + ε–NH2
+ 0.99 - 0.999 + 0.999 = + 0.99
9
Charge versus solution pH of cysteine
1. Calculate R for each dissociable group
2. Calculate [A-] or [HA]
3. Calculate fractional charge
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
COOH
R
1.7
0.019952623
0.199526231
1.995262315
19.95262315
199.5262315
1995.262315
19952.62315
199526.2315
1995262.315
19952623.15
199526231.5
1995262315
19952623150
1.99526E+11
1.99526E+12
SH
[A-]
charge
0.019562304
0.166337531
0.666139425
0.952273279
0.995013121
0.999499064
0.999949884
0.999994988
0.999999499
0.99999995
0.999999995
0.999999999
1
1
1
-0.019562304
-0.166337531
-0.666139425
-0.952273279
-0.995013121
-0.999499064
-0.999949884
-0.999994988
-0.999999499
-0.99999995
-0.999999995
-0.999999999
-1
-1
-1
R
8.3
5.01187E-09
5.01187E-08
5.01187E-07
5.01187E-06
5.01187E-05
0.000501187
0.005011872
0.050118723
0.501187234
5.011872336
50.11872336
501.1872336
5011.872336
50118.72336
501187.2336
NH2
[A-]
charge
5.01187E-09
5.01187E-08
5.01187E-07
5.01185E-06
5.01162E-05
0.000500936
0.004986879
0.047726721
0.333860575
0.833662469
0.980437696
0.998008711
0.999800514
0.999980048
0.999998005
-5.01187E-09
-5.01187E-08
-5.01187E-07
-5.01185E-06
-5.01162E-05
-0.000500936
-0.004986879
-0.047726721
-0.333860575
-0.833662469
-0.980437696
-0.998008711
-0.999800514
-0.999980048
-0.999998005
R
10.8
1.58489E-11
1.58489E-10
1.58489E-09
1.58489E-08
1.58489E-07
1.58489E-06
1.58489E-05
0.000158489
0.001584893
0.015848932
0.158489319
1.584893192
15.84893192
158.4893192
1584.893192
!
[HA]
1
1
0.999999998
0.999999984
0.999999842
0.999998415
0.999984151
0.999841536
0.998417615
0.984398338
0.863193111
0.38686318
0.059350943
0.006270012
0.000630559
charge
1
1
0.999999998
0.999999984
0.999999842
0.999998415
0.999984151
0.999841536
0.998417615
0.984398338
0.863193111
0.38686318
0.059350943
0.006270012
0.000630559
Total charge
0.980437691
0.833662419
0.333860073
0.047721693
0.004936604
-1.58489E-06
-0.004952611
-0.047880173
-0.33544246
-0.849264081
-1.11724458
-1.61114553
-1.94044957
-1.993710035
-1.999367445
"
Titration curve of cysteine
1.0
0.5
0.0
Charge
pH
-0.5
-1.0
-1.5
-2.0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14
pH
10
Calculate the pH versus charge for a peptide
The peptide DKG
pKa = 9.8 H3N+
H O
α
C C
H O
H O
α
α
N C C N C C O- pKa = 2.4
CH2
H CH2
COO-
CH2
pKa = 3.9
CH2
H CH3
CH2
NH3+
pKa = 10.5
Charge versus solution pH of peptide DKG
pH
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
N terminus
pKa = 9.8
F charge
R
1
1
0.999999998 0.999999998
0.999999984 0.999999984
0.999999842 0.999999842
0.999998415 0.999998415
0.999984151 0.999984151
0.999841536 0.999841536
0.998417615 0.998417615
0.984398338 0.984398338
0.863193111 0.863193111
0.38686318
0.38686318
0.059350943 0.059350943
0.006270012 0.006270012
0.000630559 0.000630559
6.30918E-05 6.30918E-05
Aspartic acid R-group
Pka=3.9
F charge
R
0.000125877 -0.000125877
0.001257343 -0.001257343
0.012432735 -0.012432735
0.11181577
-0.11181577
0.557311634 -0.557311634
0.926412444 -0.926412444
0.992119316 -0.992119316
0.999206302 -0.999206302
0.999920573 -0.999920573
0.999992057 -0.999992057
0.999999206 -0.999999206
0.999999921 -0.999999921
0.999999992 -0.999999992
0.999999999 -0.999999999
1
-1
Lysine R-group
pKa=10.5
F charge
R
1
1
1
1
0.999999997 0.999999997
0.999999968 0.999999968
0.999999684 0.999999684
0.999996838 0.999996838
0.999968378 0.999968378
0.999683872 0.999683872
0.996847691 0.996847691
0.96934657
0.96934657
0.759746927 0.759746927
0.240253073 0.240253073
0.03065343
0.03065343
0.003152309 0.003152309
0.000316128 0.000316128
C terminus
pKa=2.9
F charge
R
0.001257343 -0.001257343
0.012432735 -0.012432735
0.11181577
-0.11181577
0.557311634 -0.557311634
0.926412444 -0.926412444
0.992119316 -0.992119316
0.999206302 -0.999206302
0.999920573 -0.999920573
0.999992057 -0.999992057
0.999999206 -0.999999206
0.999999921 -0.999999921
0.999999992 -0.999999992
0.999999999 -0.999999999
1
-1
1
-1
Total charge
0.998616781
0.986309922
0.875751492
0.330872565
-0.483724394
-0.918534922
-0.99135724
-0.999443004
-1.003064939
-1.030644692
-1.2402522
-1.759746839
-1.969346561
-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
Titration curve of peptide DKG
1.0
0.5
Charge
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
pH
SDS Page electrophoresis
-
-
-
-
-
-
-
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
Vertical slab gel apparatus
Polyacrylamide percentage
•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
Discontinuous SDS-PAGE
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 Cland 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-
Iso-electric focussing (IEF)
•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
1st dimension
IPG strip
SDS-PAGE
Resolved by mass
2nd dimension
Resolved by pI
Example of a 2D Gel
240µg 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
Use non-linear IPG stips, since most of the proteome has pI values between 4 and 7
Sensitivity of 2DGE
•Proteins levels differ by 105-109 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
Protein solubility
Factors affecting protein solubility
•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
+
+
+
+
+
+
-
-
-
-
+
-
-
-
+
+
+
+ +
+
+
+ +
+
+
-
+
+
+
-
+
+
-
+
+
-
+
-
+
+
+
++ +
+
-
+
+
+ +
+
+
+
δ-
+
+
δ+
18
Iso-electric precipitation
pH < pI
+
+ +
+
+
+
- +
+
-
pH
+
+
+
+ -
•Repulsion
+
- +
-
-
+
- +
+
pH > pI
pI
-
+
-
-
+
-
+
+
-
+ -
-
-
•Little repulsion
•Hydrophobic
interaction
predominates
•Also electrostatic
attraction between
opposite charges
- -
+
+ -
•Repulsion
•A protein is least soluble close to its pI
“Salting in” proteins
Attracting electrostatic charges
+
+
+
+
aggregated
+
+
+
+
+ + - +
+ salt
+ -+
+
+
- +
+
+
+ - +
+
+ +
+ +
-
-
-
+
+ -
soluble
- + -+
Ions screen electrostatic charge
Attractive forces weaken
19
“Salting out” proteins
-
+
+
+
-
-
Water molecules ordered next to
exposed hydrophobic patches
-
+ + salt
-
+
+
+
+
-
+
-
-
+ -
-
+
-
-
+
Ions compete for water molecules
Hydrophobic patches exposed
Aggregation results
-
Hydrophobic association minimizes water molecule order
Ring of ordered water
molecules
Hydrophobic droplet
•Volume of sphere = 4πr3/3
•Surface of sphere = 4πr2
•1ml sphere has surface area of 4.8cm2
•2 spheres of 1ml will have combined area of 9.6mm2
•If the two droplets fuse, the combined volume is 2ml
•The total surface area of the 2ml droplet is 7.7cm2
∴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
Increased temperature increases hydrophobic association
temp
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
Increased salt increases hydrophobic association
ClNa+
salt
ClCl-
Na+
ClNa+
Na+
Cl-
More disordered
water molecules
•At higher salt concentrations, weak interactions between the disordered 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
The Hofmeister series
Salting out described by:
log10(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
Finding the best salting out protocol for your protein
% Saturation
First trial
% Enzyme
precipitated
% Protein
precipitated
Purification
factor
0-40
4
25
40-60
62
22
2.8
60-80
32
32
1.0
80 supernatant
2
21
Conclusion: Enzyme precipitated more in 40-60 than 60-80; try 45-70
Second trial
0-45
6
32
45-70
90
38
70 supernatant
4
30
2.4
Conclusion: Good recovery, but poorer purification; try 48-65
Third trial
0-48
10
35
48-65
75
25
65 supernatant
15
40
3.0
22
Effect of organic solvents on protein solubility
•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
Solvents used to precipitate proteins
•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
Organic solvents and elevated temperatures
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
Gel filtration chromatography
24
Molecules are separated by size
Sample of 3 different
sized molecules
(Stokes radius)
Excluded
Partially included
Totally included
Stationary
phase
Elution profile
A280
Mobile
phase
Time
Parameters of a gel filtration run
Vo
Ve
Vt
Ve > Vt ???
Vt = Vo + Vi
For a column, Vo can be determined by eluting blue dextran (Mr
= 2,000,000)
Vi is internal volume plus particle volume (inaccessible to all),
and is difficult to measure
Vi can be calculated from Vt-Vo
Excluded molecules elute at Vo
Molecules that have access to full Vi elute at Vt
What if elution volume Ve > Vt ? Inert stationary phase?
25
Partition coefficient
The partition coefficient (Kd) for a molecule is given by
Total volume accessible to molecule
Ve - Vo
Vt - Vo
Total internal volume
•If Kd = 0, the molecule is totally excluded
•If Kd = 1, molecule has full access
•If Kd > 1, there is adsorption to the column matrix
•Kd is not the “true” partition coefficient, since Vi
includes gel particle volume
Resolution
Ve1
Ve2
W2
W1
R=2
Ve2 – Ve1
W1 + W2
∴The difference in elution volume divided by the average peak width, W
If R = 1, we have baseline resolution
26
Theoretical plates
•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
•number of theorical plates (N) is given by
5.55 Ve2
W2
•Where W is peak width, and Ve is the elution volume
Height equivalent of a theoretical plate
•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
LW2
5.55 Ve2
•Where L is the column height, W is peak width, and Ve is the
elution volume
27
Van Deemter equation
•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
shape and size distribution
H=
A
+ Bυ + C
υ
Finding the optimum flow rate
van Deemter relation
A (diffusion)
B (non-equilibrium)
C (column quality)
A+B+C terms
5
Plate Height (H)
4
3
Optimum flow rate
2
1
0
0
5
10
15
20
Elution rate (ν)
•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
Gel matrices
Trade name
Supplier
Composition
Comment
Sephadex
Pharmacia
Cross-linked dextran
Cannot be manufactured with
exclusion limit > 600,000Da
(particle not rigid, and collapses)
Sephacryl
Pharmacia
N,N’methylenebisacrylamide
cross-linked dextran
Higher exclusion limits than
Sephadex
Polyacrylamide
(Bio-Gel P)
Bio-Rad
N,N’methylenebisacrylamide
cross-linked acrylamide
Similar to Sephadex
Sepharose
Bio-Gel A
Pharmacia
Bio-Rad
Agarose
Very high exclusion limits
Combined
polyacrylamide-agarose
gels
Higher flow-rates than agarose
Ultragel
Gel matrix has optimum ~linear range
Elution volume
V0
Vi
poor
good
poor
Log10(Mr)
29
Ion-exchange chromatography
Proteins have charges
Ribbon
Electrostatic surface
30
Charged (DEHKR)
Acidic
Aspartic acid, Asp, D
negative charge
Hydrophobicity = -3.0
MW 115Da
pKa COOH = 2.19
pKa NH2 = 9.60
pI = 2.77
Glutamic acid, Glu, E
negative charge
Hydrophobicity = -2.6
MW 129Da
pKa COOH = 2.19
pKa NH2 = 9.67
pI = 3.22
Basic
Histidine, His, H
Weak positive charge
Hydrophobicity = -1.7
MW 137Da
pKa COOH = 1.82
pKa NH2 = 9.17
pI = 7.59
Lysine, Lys, K
positive charge
Hydrophobicity = -4.6
MW 128Da
pKa COOH = 2.18
pKa NH2 = 8.95
pI = 9.47
Arginine, Arg, R
positive charge
Hydrophobicity = -7.5
MW 156Da
pKa COOH = 2.17
pKa NH2 = 9.04
pI = 10.76
The charge of protein depends on pKa
31
Principle of ion-exchange
•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 z1 and IEX resin
charged group z2, is given by
F=
z1z2
Dr2
•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
IEX bead matrices
Resin matrices are composed of different materials
•Cellulose
•Agarose
•Dextran
•Synthetic polymers
•Polyacrylamide
IEX matrices will also act as gel filtration media
32
Functional groups
+1
Q
Anion
exchangers
DEAE
0
Cation
exchangers
CM
S
-1
3
4
5
6
7
8
9
10
pH
•DEAE (diethylaminoethyl) and CM (carboxymethyl) are weak exchangers
•Sulfonyl (S) and quaternary ammonium ions (Q) are strong exchangers
Strong and weak exchangers
Anion exchangers
Strength
Functional group
Quaternary ammonium
strong
-O-CH2-N+-(CH3)3
Diethylaminoethyl
weak
-O-CH2-CH2-NH+-(CH2-CH3)2
Diethylaminopropyl
weak
O-CH2-CHOH-CH2-NH+-(CH2-CH3)2
Cation exchangers
Functional group
Sulfopropyl (S)
strong
O-CH2-CHOH-CH2-O-CH2-CH2-CH2-SO3-
Methyl sulfonate (SP)
strong
O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-SO3-
Carboxymethyl (CM)
weak
-O-CH2-COO-
33
A280
A280
A280
A280
v
v
v
v
charge
Cation exchangers
+
0
-
A280
A280
A280
v
v
A280
pH
Anion exchangers
v
v
Affinity chromatography
34
Attachment of the ligand to the matrix
The covalent attachment of the ligand to the matrix requires
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
Attachment to CNBr-activated agarose
OH
gel
OH
CNBr
O
gel
C
NH
O
NH2R
OH
gel
O
C
NHR
NH2+
35
Tagged recombinant proteins
Tag
Sequence
Histidine
HHHHHH
C-myc
EQKLISEEDL
HA
YPYDVPDYA
VSV-G
YTDIEMNRLGK
HSV
QPELAPEDPED
V5
GKPIPNPLLGLDST
FLAG
DYKDDDDKG
Glutathion-S-transferase
23kDa; 210 residues
VSV-G: vesicular stomatitis virus G protein
HSV: Herpes simplex virus
Immobilized metal affinity chromatography (IMAC)
•Based on the affinity of metal ion for basic amino acid residues
•Ions mostly used include Ni2+, Co2+, Cu2+, Zn2+ and Fe2+
•Metal ion is immobilized by partial coordination binding by IDA, NTA
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 Ni2+-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 Ni2+
36
Metal chelators used in IMAC
OH
CH2CH
CH2COOCH2
CH2
OH
CH2CH
N
CH2
Imido diacetic acid (IDA)
N
CH
COO-
CH2COOCH2COO-
Nitrilo triacetic acid (NTA)
CH2COO-
OH
CH2CH
CH2CH2N
CH2
CH2COOCH2COO-
N
Tris(carboxymethyl)
ethylene diamine (TED)
CH2COO-
Imidazole is structural analog of histidine
C NH
CH2
HC
Histidine
NH
Imidazole
37
Tandem affinity purification (TAP)
•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 Ca2+
•Protein A binds to the FC 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 Ca2+
•This allow two-step ultra-pure protein preparation
•Very mild conditions allows isolation of protein complexes
TAP tag purification scheme
IgG sepharose
column
E. coli cell
TAP-tagged protein
Elute by TEV
protease cleavage
calmodulin column
Ca2+
Ca2+ EDTA
Pure protein
Ca2+ EDTA
Elute with EDTA
or EGTA
Ca2+
Ca2+
CBP binds to calmodulin
only in presence of Ca2+
38