Protein Purification

February 5 2003

Abasiccomprehensionofthe
methodsdescribedhereis
necessaryforanappreciationof
thesignificanceandthe
limitationsoftheinformation
presentedinthetext

ProteinIsolation
Musthavesensitivemethodfordetection.
Selectagoodsourcefortheprotein.
a.Richsourceofmaterial.
i.e.HeartmitochondriaforcytochromeC

b.bakersyeast(Saccharomycescerevisiae)
c.Escherichiacoli(recombinantexpression)
Tissuespecificity:Brainvs.kidneyvs.eye.
Chickens,cows,pigsorratsareoftenused.
Molecular cloning techniques have allowed
biochemists to overexpress desired proteins in bacteria
orC.H.O.(ChineseHamsterOvary)cellsbyisolatingthe
geneandplacingitintoahostsystem.

Methodsofsolubilizationanimalcells
Cellscanbelysedbyhypotonicshock.
Cellswithhighsaltinsideandnosaltoutsidewill
swellandrupture
Bacteriaoutermembranesmustbedigested.
Gramnegativebacteria
Heneggwhitelysozymedigests(14)linkagesin
the (glycosidicbonds)ofpolysaccharides.
Mechanicalbreakageblendershomogenizers
Frenchpresshighpressure20,000lbs/in2forced
throughasmallholedisruptscells
ultrasoundorsonicationdisruptscells.

Centrifugation
Lysatebroken(lysed)cellscanbeseparatedusing
differentialcentrifugation

RPMspundown
separatesbydensitydifferencesorbysize(MW)ofparticles.
Cellularfractionationcanseparate:
mitochondria
microsomes
ribosomes
solubleproteins

Centrifugation:Units

v
1 d ln r M 1 V
s 2 2

r dt
Nf
Where:
= angular velocity
v = velocity of particle
R = distance from center of rotation
M = molecular weight
V = partial specific volume of particle
= density of solvent
Sedimentation velocity (Svedberg
Coefficient)
S = s x 10-13

Stability:proteinscandenature!!
Hbonds,ionicbonds,VanderWaalsinteractions,and
Hydrophobicinteractionscanbedisrupted.
Denaturationistheprocessbywhichaproteinlosesits
nativeoractiveshapeorconformation.
Temperaturecanplayarole
coldlabile
heatlabile
ProtectagainstProteases,Inhibitors,ChangesinpH,
Protein can be airdenatured egg white meringue
absorptiontosurfaces
Damagedbyoxidation02
Heavy and transition metals damage proteins they bind to
proteinCu+Hg+
Bacterialcontaminationcandestroytheprotein

ActivityMeasurements
Inordertofollowthepurityofanenzyme,you
needamethodtomeasureitsactivity.
Spectraphotometricanalysisisonecommonmethodto
measureactivity.
Substrate[S]Product[P]achangeof[S]withtime
ifSiscoloredabsorbslightwecanuseBeersLaw.

A=bc

A=log%T
cconcentration
-millimolarextinctioncoefficient
Aabsorbance
bpathlength
ifAthencat
Tpercenttransmittance
max

enzyme

Forthereaction:NADHNAD++H
Absorbance

NADH

A
NAD+

300 nm

350 nm

A
millimolar NADH oxidized

T
mg

Max = 340 nm

min
mg of protein

Volume is 1 ml so micromoles
NADH oxidized

= Specific activity

Startwithoneliteroflysedcells.
We measure the rate of .01 ml of cells at at concentration of 20
mg/ml.i.e.theamountofenzymewewillassayis0.01ml
WegetarateofA=0.5A/min
1millimolar=6.22A=mM
0.5/6.22=.008millmolar/minandourassayvolume=1ml
1millimolarinavolumeofoneml=1micromole/ml=mole
C=.008molesin1ml/min=.04moles
0.2mg
min/mg

Totalactivity:.04molesx20mg/ml=0.8moles/ml
0.8molesx1000ml=800molesin1literofcells
ml min

Red=isourenzyme
Ifweremovegreens&bluesthespecificactivityincreases,
however,ourtotalactivityremainsthesame.
If
Weloseredthetotalactivitydecreases.

Weusuallymonitorboththetotalactivityandspecificactivity
foreachpurificationstep.
UntiltheSpecificActivityreachesamaximalvalue.
Howdoweknowifitispure?UsuallySDSPage
SeeTable54inVoetandVoet
Someenzymeshavenoeasyassaybuttheproductofthereactioncanbe
usedinanotherreaction:

enz2
ABC
enz1

NADHNAD+
CoupledReactions:Wecoupleenz2toenz1andmeasureNADHtoget
A

Useofradioactivity
NH2

ATPADP+Pi
O
-O

P
O-

O
O
O-O

P
O-

O
O
-O

O-

OH

SeparateATP+Pi+ADPonTLCmeasureradioactivity
Pi
ATP
Phosphoimagermakesthiseasyelsecutspotsandcountin
scintillationcounter.

StrategyofPurification
Fractionationproceduresorstepstoisolateproteinbasedon
physicalcharacteristics.
Characteristic Procedure
Charge
1. Ionexchange
2. Electrophoresis
3. Isoelectricfocusing
Polarity

1. Adsorptionchromatography
2. Paperchromatography
3. Reversephasechromatography
4. Hydrophobicinteraction

Characteristic Procedure
Size

1.Dialysisandultrafiltration
2.Gelelectrophoresis
3.Gelfiltration
4.Ultracentrifugation

Specificity

1.Affinitychromatography
2.Immunopurification

Solubility

1.Saltprecipitation
2.Detergentsolubilization

IonicStrength

1
2
I c i Zi
2

Ci=themolarconcentrationoftheithspecies
Zi=itsioniccharge
1MNa+Cl

Z=1Na+
Z=1Cl

1=(1Mx1)Na+(1Mx1)Cl
2

Fordiortrivalentions,whereIisdifferentthanM
1MMgCl2
while

Mg++=1M,andZ=2
Cl=2M,andZ=1

I=(1x22)Mg+(2x12)Cl=4+2=3
2
2

Saltingout
Use(NH4)2SO4:itisaVerySolublesaltthatdoesnot
harmproteins.
RefertotheHofmiesterSeries

Solubility of carboxy-hemoglobin at its

isoelectric point

Solubility of -lactoglobulin as a function of

pH

Chromatography
Analytical methods used to separate molecules.
Involves a mobile and a stationary phase.
Mobile phase is what the material to be separated is
dissolved in.
Stationary phase is a porous solid matrix which the
mobile phase surrounds.
Separation occurs because of the differing
chemistries each molecule has with both the mobile
and stationary phase.
Chemistries are different depending on the specific
method.

Types of chromatography
Gas - Solid: Mobile phase is gaseous, stationary phase is
a solid matrix.
Liquid - Solid: Mobile phase is liquid, stationary phase is
a solid matrix.
If separation is based on ionic interaction the method is called
Ion Exchange chromatography.
If separation is based on solubility differences between the
phases the method is called adsorption chromatography.
If the separation is base on size of molecule the method is
called gel filtration or size exclusion.
If the separation is base on ligand affinity the method is called
Affinity chromatography.

IonExchangeChromatography
Asolidmatrixwithapositivechargei.e.R+canbind
differentanionswithdifferentaffinities.
Wecanswaponecounterionforanother
(R+A)+B(R+B)+A
R=ResinandexchangesAnions()
Thisisananionexchangeresin.
Therearealsocationexchangeresins.ThetypeofanRgroup
candeterminethestrengthofinteractionbetweenthematrix,R
andthecounterion.

IfRisR

(RA+)+B+(RB+)+A

Proteins have a net charge.

ThechargeispositivebelowpI,
whilethechargeisnegativeabovepI
Thechoiceofexchangeresindependsonthechargeof
theproteinandthepHatwhichyouwanttodothe
purification.
Oncetheproteinbinds,allunboundproteinsare
washedoffthecolumn.Boundproteinsareelutedby
increasingtheionicstrength,changingthecounterion
orchangingthepHalteringthechargeontheprotein
orthecolumn.

Paper chromatography
Stationaryphasevs..theMobilephase
Partitioningbetweenthetwophases

A in stationary phase
Kp
A in mobile phase
Partitioncoefficient

ThemoreH2Osolublethesloweritmigrates.
Themoreorganicsolublethemoreitmigrates.
The aqueous component of the solvent combines with
the cellulose of the paper and becomes the stationary
phase.

distance traveled by the substance

Rf
distance traveled by the solvent front
Materials can be visualized by:
Radioactivity
Fluorescence
UV absorbency
Stained with one of several dyes
Ninhydrin
Iodine
Sulfuric acid

Ninhydrin visualizes amino acids

Two dimensional separation of

Amino acids

GelFiltration
Sizeexclusion
Amatrixwithholesinit.

Vt=Vx+Vo
Vo=voidvolume=volumeoutsidethecavesorknooks
andcrannies
Vxoccupiedbygelbeads
Vo35%ofVt

Gel filtration can be used to determine

the molecular mass of proteins

Ve=elutionvolumeVo=exclusionvolume
Commonmatrix:dextran,agarose,orpolyacrylamide
alsodesaltsproteins

Beforeswellingthedrybeadsize5%ofVt
60%areholes
Holesizescanbemadedifferent
Smallmoleculesseealargercolumnvolume
thanbigmoleculesandtheygethungupinthe
caves.
Largeproteinsareexcluded,whilesmallprotein
areincluded.
Separationonsizeandshape.

Dialysis is a process that separates molecules according to size

through the use of semipermeable membranes containing
pores of less than macromolecular dimensions

AffinityChromatography
Basedonmolecularcomplementarybetweenanenzymeand
substrate.
Thesubstrate(R)islinkedtoamatrixwithaspacerarm

OnlyproteinthatbindsRwillsticktocolumn.putcitrateoncolumn
citratedehydrogenasewillspecificallybind.Addexcesscitrateand
theenzymewillbereleased.

The purification of Staphylococcal nuclease using

the ligand, diphosphothymadine

Electrophoresis

The migration of ions in an electric field

Fele = qE where q is the charge
and E is the electric Field strength
Opposing this is Ffriction = vf where v =
velocity of migration f is the frictional
v q
force.

E f
qE = vf

Paper electrophoresis

Acrylamide gel electrophoresis

Disc gel using a glass tube

Separates on charge and size

pH matters as well as the pI of the protein.
Can be run at several pH values depending
on proteins.
DNA can also be separated on agarose
gels. Genomic sized DNA can also be
separated but requires more sophisticated
equipment.

Proteins can be visualized by several methods

Stained with a Dye:

Coomassie blue

Fluorescamine stain for

fluorescence
Silver staining very sensitive
proteins can be labeled with
radioactivity
and visualized by exposure to Xray film

SDS-PAGE
Add sodium dodecyl sulfate, a 12 carbon detergent to give
a negative charge to the protein.
SDS also denatures the protein and collapses into a
globular ball.

The proteins are separated by molecular mass