Professional Documents
Culture Documents
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
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
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.
GelFiltration
Sizeexclusion
Amatrixwithholesinit.
Vt=Vx+Vo
Vo=voidvolume=volumeoutsidethecavesorknooks
andcrannies
Vxoccupiedbygelbeads
Vo35%ofVt
Ve=elutionvolumeVo=exclusionvolume
Commonmatrix:dextran,agarose,orpolyacrylamide
alsodesaltsproteins
Beforeswellingthedrybeadsize5%ofVt
60%areholes
Holesizescanbemadedifferent
Smallmoleculesseealargercolumnvolume
thanbigmoleculesandtheygethungupinthe
caves.
Largeproteinsareexcluded,whilesmallprotein
areincluded.
Separationonsizeandshape.
AffinityChromatography
Basedonmolecularcomplementarybetweenanenzymeand
substrate.
Thesubstrate(R)islinkedtoamatrixwithaspacerarm
OnlyproteinthatbindsRwillsticktocolumn.putcitrateoncolumn
citratedehydrogenasewillspecificallybind.Addexcesscitrateand
theenzymewillbereleased.
Electrophoresis
Paper electrophoresis
Coomassie blue
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.