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

d

- - -

-

+

-

-

+

ChargedMolecule

(Charge q)

dV

F = qV/d

Gel matrix

f

q

E

v

qEfv

v

f

fvF

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q

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b

f

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:mequilibriuAt

velocity

tcoefficien frictional

strength field

charge

f

q

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M

qv

The frictional coefficient The frictional coefficient f f depends on the size of the depends on the size of the molecule, which in turn depends upon the molecular mass, molecule, which in turn depends upon the molecular mass, so:so:

i.e. the velocity depends on the charge/mass ratio, which i.e. the velocity depends on the charge/mass ratio, which varies from protein to proteinvaries from protein to protein

Polyacrylamide Gels

Polyacrylamide gel electrophoresis of whole cell proteins of three strains of lactic acid bacteria.

Agarose

Gelidium sp.Gelidium sp.

SDS binds to proteins at a constant ratio of 1.4 g SDS/g proteinSDS binds to proteins at a constant ratio of 1.4 g SDS/g protein

Na+

OS

OCH2

CH

2

CH2

O

O

CH

2

CH2

CH

2

CH2

CH

2

CH2

CH

2

CH2

CH3

SDS PAGESodium Dodecyl (Lauryl) Sulfate

Constant q/M ratioConstant q/M ratio

Disulfide cleavage

Disulfide cleavage and chain separation

+ ME

Isoelectric Point

Abrin A - Predicted Charge

-40

-30

-20

-10

0

10

20

30

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

pH

Ch

arg

e o

n P

rote

in

Predicted pI5.088

Isoelectric FocusingIsoelectric 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 pISeparation by pI

Isoelectric Focusing

BelowBelow the pI, a protein has a positive charge and migrates the pI, a protein has a positive charge and migrates toward the cathodetoward the cathodeAboveAbove the pI, a protein has a negative charge and migrates the pI, a protein has a negative charge and migrates toward the anodetoward the anode

Isoelectric Focusing Foot Flesh Extracts from Pomacea flagellata and Pomacea patula

catemacensis

Protein Purification Steps1 unit = amount of enzyme that catalyzes 1 unit = amount of enzyme that catalyzes conversion of 1 conversion of 1 mol of substrate to product in 1 mol of substrate to product in 1 minuteminute

1 unit = amount of enzyme that catalyzes 1 unit = amount of enzyme that catalyzes conversion of 1 conversion of 1 mol of substrate to product in 1 mol of substrate to product in 1 minuteminute

Purification visualized

Example:Purification of Ricin

Georgi Markov1929-1978

Ricinus communisRicinus communis – castor oil – castor oil plantplant

Ricin

Ricin B chainRicin B chain(the attachment bit)(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.

RawExtract

(NH4)2SO4

Cut

Affinity GelFiltration

Salting In – Salting out

iz

ic

zcI

i

i

n

iii

ion on charge

ion ofion concentrat

2

1 :Strength Ionic

1

2

• 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

Solubility reaches minimum at pI

Salting in: Counterions help prevent formation of interchain salt links

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−> HPO4

2−> CH3COO−> citrate > tartrate > F−> Cl−> Br−> I−> NO3

−> ClO4−> SCN−

1) Bring to 37% Saturation – ricin still soluble, many other proteins ppt

2) Collect supernatant3) Bring to 67% Saturation – ricin ppt, many remaining

proteins still soluble4) Collect pellet5) Redissolve in buffer

Dialysis and Ultrafiltration(How do you get the %@$&#! salt out?)

RawExtract

(NH4)2SO4

Cut

Affinity GelFiltration

Separation by chromatographySeparation by chromatographyBasic Idea:Basic Idea:You have a You have a stationary phasestationary phaseYou have a You have a mobile phasemobile phaseYour material partitions out between Your material partitions out between the phases.the phases.

Affinity Chromatography

Structure of AgaroseAgarose 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 Begin adding 0.2 M LactoseLactose

RawExtract

(NH4)2SO4

Cut

Affinity GelFiltration

B

AB

BAASS SS

SS

Ricinus communis Agglutinin (RCA)MW = 120,000

RicinMW = 60,000

Castor Beans contain two proteins that bind galactose

Gel Filtration

Gel Filtration

Gel Filtration (aka Size Exclusion)

Fig. 3. Fig. 3. Measurement of molecular weight of native NAGase enzyme of green crab by gel 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 filtration on Sephadex G-200: standard proteins (empty circles); green crab NAGase (filled circle). (filled circle).

From Zhang, J.P., Chen, Q.X., Wang, Q., and Xie, J.J. (2006) From Zhang, J.P., Chen, Q.X., Wang, Q., and Xie, J.J. (2006) Biochemistry (Moscow)Biochemistry (Moscow) 7171(Supp. 1) (Supp. 1) 855-859.855-859.

Note:Note: smaller = slowersmaller = slower, , whereas in SDS-PAGE, whereas in SDS-PAGE, smaller = fastersmaller = faster..

NoteNote

RCARCA

RicinRicin

Gel Filtration Separation of RicinGel Filtration Separation of Ricin

RawExtract

(NH4)2SO4

Cut

Affinity GelFiltration

Okay, Now Let’s Sequence the A-Chain

Bovine InsulinBovine Insulin21 residue A chain21 residue A chain31 residue B chain31 residue B chainConnected by disulfidesConnected by disulfides

In order to sequence the protein, the In order to sequence the protein, the chains have to be separatedchains have to be separated

Chain Separation

• Interchain disulfide broken by high concentrations of ME

• Chains are about the same size – but can take advantage of different pIs– B-Chain pI ~ 5.3– A-Chain pI ~ 7.2

Ion Exchangers

•Apply ME – 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. From Himeno et al (1996) J. Biol. Chem. 271271: 15769-15775.: 15769-15775.

Tandem Mass Spectrometry can also be used to determine peptide sequences