CH339K
Physical Methods: How to Purify and
Sequence a Weapons-Grade Protein
First Question
How do I measure the amount
of protein I have?
UV Absorption Spectrophotometry
Second Question
How can I spot my protein in the
great mass of different proteins?
Electrophoresis
+
Gel matrix
V
F = qV/d
+
-
d
d
-
-
-
Charged
Molecule
(Charge q)
V
F f  q  qE
d
q  charge
E  field strength
Fb  fv
f  frictionalcoefficient
v  velocity
At equilibrium :
fv  qE
or
v q

E f
v q

E f
The frictional coefficient f depends on the size of the
molecule, which in turn depends upon the molecular mass,
so:
q
v
M
i.e. the velocity depends on the charge/mass ratio, which
varies from protein to protein
Polyacrylamide Gels
Polyacrylamide gel electrophoresis of whole cell proteins of
three strains of lactic acid bacteria.
Agarose
Gelidium sp.
SDS PAGE
Sodium Dodecyl (Lauryl) Sulfate
O
+
Na O
H2
C
O
S
O
C
H2
H2
C
C
H2
H2
C
C
H2
H2
C
C
H2
H2
C
C
H2
C
H2
CH 3
SDS binds to proteins at a constant ratio of 1.4 g SDS/g protein
Constant q/M ratio
Disulfide cleavage
Disulfide cleavage and chain separation
+ bME
Isoelectric Point
Abrin A - Predicted Charge
30
20
Predicted pI
5.088
Charge on Protein
10
0
-10
-20
-30
-40
0.0
2.0
4.0
6.0
8.0
pH
10.0
12.0
14.0
Isoelectric Focusing
pH
Carrier Ampholytes
• Amphoteric Electrolytes
• Mixture of molecules containing multiple
amino- and carboxyl- groups with
closely spaced pIs
• Partition into a smooth, buffered pH
gradient
Separation by pI
Isoelectric Focusing
Below the pI, a protein has a positive charge and migrates
toward the cathode
Above the pI, a protein has a negative charge and migrates
toward the anode
Isoelectric Focusing
Foot Flesh Extracts from Pomacea flagellata and Pomacea patula
catemacensis
Protein Purification Steps
1 unit = amount of enzyme that catalyzes
conversion of 1 mmol of substrate to product in 1
minute
Purification visualized
Example:
Purification of Ricin
Georgi Markov
1929-1978
Ricinus communis – castor oil
plant
Ricin
Ricin B chain
(the attachment bit)
Ricin Action
• Ricin and
related
enzymes
remove an
adenine base
from the large
ribosomal RNA
• Shut down
protein
synthesis
The possibility that
ricin might be used
as an asymmetric
warfare weapon has
not escaped the
attention of the
armed services.
The last time I was
qualified to know for
sure, there were no
effective antidotes.
Raw
Extract
(NH4)2SO4
Cut
Affinity
Gel
Filtration
Salting In – Salting out
n
1 2
IonicStrength: I   ci zi
i 1 2
ci  concentration of ion i
zi  chargeon ion i
• salting in: Increasing ionic strength increases protein
solubility
• salting out: Increasing further leads to a loss of solubility
Salting in – salting out
The solubility of haemoglobin in different electrolytes as a
function of ionic strength.
Derived from original data by Green, A.A. J. Biol. Chem. 1932, 95, 47
Salting in: Counterions
help prevent formation of
interchain salt links
Solubility reaches
minimum at pI
Salting out: there’s simply less water available to solubilize
the protein.
Different proteins have different
solubilities in (NH4)2SO4
Lyotropic  ChaotropicSeries
Cations: N(CH3)3H+> NH4+> K+> Na+> Li+> Mg2+>Ca2+> Al3+>
guanidinium / urea
Anions: SO42−> HPO42−> CH3COO−> citrate > tartrate > F−> Cl−>
Br−> I−> NO3−> ClO4−> SCN−
1) Bring to 37% Saturation – ricin still soluble, many other
proteins ppt
2) Collect supernatant
3) Bring to 67% Saturation – ricin ppt, many remaining
proteins still soluble
4) Collect pellet
5) Redissolve in buffer
Dialysis and Ultrafiltration
(How do you get the %@$&#! salt out?)
Raw
Extract
(NH4)2SO4
Cut
Affinity
Gel
Filtration
Separation by chromatography
Basic Idea:
You have a stationary phase
You have a mobile phase
Your material partitions out
between the phases.
Affinity Chromatography
Structure of Agarose
Agarose is a polymer of agarobiose, which in turn consists of one
unit each of galactose and 3,6-anhydro-a-L-galactose.
Ricin sticks to galactose, so store-bought agarose acts as an
affinity column right out of the bottle, with ricin binding the beads
while other proteins wash through.
Begin adding 0.2 M
Lactose
Raw
Extract
(NH4)2SO4
Cut
Affinity
Gel
Filtration
Castor Beans contain two proteins that
bind galactose
B
SS
A
A
SS
Ricinus communis Agglutinin (RCA)
MW = 120,000
B
SS
A
Ricin
MW = 60,000
B
Gel Filtration
Gel Filtration
Gel Filtration (aka Size
Exclusion)
Note: smaller = slower,
whereas in SDS-PAGE,
smaller = faster.
Note
Fig. 3. Measurement of molecular weight of native NAGase enzyme of green crab by gel
filtration on Sephadex G-200: standard proteins (empty circles); green crab NAGase (filled
circle).
From Zhang, J.P., Chen, Q.X., Wang, Q., and Xie, J.J. (2006) Biochemistry (Moscow) 71(Supp. 1)
855-859.
Gel Filtration Separation of Ricin
Ricin
RCA
Raw
Extract
(NH4)2SO4
Cut
Affinity
Gel
Filtration
Okay, Now Let’s Sequence
the A-Chain
Bovine Insulin
21 residue A chain
31 residue B chain
Connected by disulfides
In order to sequence the protein, the
chains have to be separated
Chain Separation
• Interchain disulfide broken by high
concentrations of bME
• Chains are about the same size – but
can take advantage of different pIs
– B-Chain
– A-Chain
pI ~ 5.3
pI ~ 7.2
Ion Exchangers
•Apply bME – treated ricin to DEAE-cellulose at pH 7
•At pH 7:
•A chain (pKa 7.2) is essentially uncharged,
•B chain (pKa 4.8) is highly negative
•A chain washes through the column
•B chain sticks, eluted with gradient of NaCl
2-D Electrophoresis (an aside)
• Can use two different properties of a
protein to separate electrophoretically
• For analysis of cellular protein content,
often use 2-dimensional
electrophoresis:
• 1st dimension is isoelectric focusing
• 2nd dimension is SDS PAGE
2-D Electrophoresis (cont.)
• Can use other protein properties to
separate
– Simple PAGE at 2 different pHs
– PAGE and SDS PAGE
Sequencing with Phenylisothiocyanate
• Applied Biosystems 492 Procise Protein
Sequencer
Chain Cleavage: Cyanogen Bromide
C-Terminal Sequencing
• Carboxypeptidases are enzymes that
chew proteins from the carboxy
terminus
• Can incubate a protein (preferably
denatured – more later) with a
carboxypeptidase
• Remove aliquot at intervals (time
course)
• Run amino acid analysis of aliquots
C-Terminal Sequencing of Rat Plasma
Selenoprotein
From Himeno et al (1996) J. Biol. Chem. 271: 15769-15775.
Tandem Mass Spectrometry can also be used to
determine peptide sequences