CH339K

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CH339K

• Physical Methods How to Purify and Sequence a
  Weapons-Grade Protein

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First Question

• How do I measure the amount of protein I have?

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UV Absorption Spectrophotometry
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Second Question

• How can I spot my protein in the great mass of
  different proteins?

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Electrophoresis
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The frictional coefficient f depends on the size
of the molecule, which in turn depends upon the
molecular mass, so
i.e. the velocity depends on the charge/mass
ratio, which varies from protein to protein
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Polyacrylamide Gels
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Polyacrylamide gel electrophoresis of whole cell
proteins of three strains of lactic acid
bacteria.
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Agarose
Gelidium sp.
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SDS PAGE
Sodium Dodecyl (Lauryl) Sulfate
SDS binds to proteins at a constant ratio of 1.4
g SDS/g protein
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Constant q/M ratio
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Disulfide cleavage
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Disulfide cleavage and chain separation
bME
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Isoelectric Point
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Isoelectric Focusing
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pH
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Carrier Ampholytes

• Amphoteric Electrolytes
• Mixture of molecules containing multiple amino-
  and carboxyl- groups with closely spaced pIs
• Partition into a smooth, buffered pH gradient

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Separation by pI
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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
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Isoelectric Focusing Foot Flesh Extracts from
Pomacea flagellata and Pomacea patula catemacensis
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Protein Purification Steps
1 unit amount of enzyme that catalyzes
conversion of 1 mmol of substrate to product in 1
minute
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Purification visualized
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ExamplePurification of Ricin
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Georgi Markov

• 1929-1978

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Ricinus communis castor oil plant
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Ricin
Ricin B chain (the attachment bit)
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Ricin Action

• Ricin and related enzymes remove an adenine base
  from the large ribosomal RNA
• Shut down protein synthesis

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• 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.

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Raw Extract
(NH4)2SO4 Cut
Affinity
Gel Filtration
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Salting In Salting out

• salting in Increasing ionic strength increases
  protein solubility
• salting out Increasing further leads to a loss
  of solubility

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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
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Salting in Counterions help prevent formation of
interchain salt links
Solubility reaches minimum at pI
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Salting out theres simply less water available
to solubilize the protein.
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Different proteins have different solubilities in
(NH4)2SO4
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Lyotropic ? ChaotropicSeries Cations N(CH3)3Hgt
NH4gt Kgt Nagt Ligt Mg2gtCa2gt Al3gt guanidinium
/ urea Anions SO42-gt HPO42-gt CH3COO-gt citrate gt
tartrate gt F-gt Cl-gt Br-gt I-gt NO3-gt ClO4-gt SCN-
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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

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Dialysis and Ultrafiltration(How do you get the
_at_! salt out?)
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Raw Extract
(NH4)2SO4 Cut
Affinity
Gel Filtration
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Separation by chromatography Basic Idea You have
a stationary phase You have a mobile phase Your
material partitions out between the phases.
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Affinity Chromatography
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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.
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Begin adding 0.2 M Lactose
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Raw Extract
(NH4)2SO4 Cut
Affinity
Gel Filtration
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Castor Beans contain two proteins that bind
galactose
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Gel Filtration
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Gel Filtration
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Gel Filtration (aka Size Exclusion)
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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.
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Gel Filtration Separation of Ricin
Ricin
RCA
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Raw Extract
(NH4)2SO4 Cut
Affinity
Gel Filtration
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Okay, Now Lets Sequence the A-Chain
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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
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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 pI 5.3
• A-Chain pI 7.2

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Ion Exchangers
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• 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

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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

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2-D Electrophoresis (cont.)

• Can use other protein properties to separate
• Simple PAGE at 2 different pHs
• PAGE and SDS PAGE

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Sequencing with Phenylisothiocyanate
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• Applied Biosystems 492 Procise Protein Sequencer

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Chain Cleavage Cyanogen Bromide
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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

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C-Terminal Sequencing of Rat Plasma Selenoprotein
From Himeno et al (1996) J. Biol. Chem. 271
15769-15775.
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Tandem Mass Spectrometry can also be used to
determine peptide sequences