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QUIZ 2
1. List 2 advantages of using microbial cellsas an enzyme source.
2. Distinguish extracellular and intracellularenzyme.
3. List three (3)ways of how to imrove the
roduction yield of enzyme.
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!eneral strategies for enzyme
urification "uestions should be as#ed before embar#ing on the
rotein urification are$
%hat is the rotein&enzyme re'uired for %hat source should be used %hat is #nown about the rotein& enzyme ow should the rotein be assayed&detected
*ac#ground #nowledge re'uired to lan a suitable strategy
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What is the protein/enzyme required for?
+or economic reasons cost , time are veryimortant in the manufacture of enzyme
-he amount re'uired , urity levels deend on
the end use of the manufactured rotein.
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xamles$ /f the rotein&enz is sold for research used (i.e. sulied by 0igma
*L etc) the 'uantities re'uired are small whilst in terms of urity
the removal of interfering activity will be essential. +or industrial alications such as in food industry (i.e. enzroduced by ovo , 0turge for degrading starch into glucose ,maltose or for use in domestic detergents ) large 'uantities arere'uired , urity is usually of imortant to cost.
+or theraeutic alications (i.e. tissue lasminogen activator fortreating mycocardial infection or monoclonal antibodies for cancer
imaging) urity is of the utmost imortance , 'uantities re'uiredare relatively small
-he amount of urified rotein will not only deend on the amountof starting material but also on yield.
4rotein is lost at each ste of a urification rocedure.
/n order to maximize yield the minimum no. of stes should beused. owever the final urity will be lower if minimizing the no. of stes
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What source should be used?
Defined by the final alication hoose the most stable , abundant 0hould also consider the availability ,
'uantity of the source.
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xamles$5ouse liver for urifying small 'uantities of an
enzyme *ovine liver would be more suitable for larger
amount 6nimals that be able to #ee in the laboratory are
referable to animals from the wild ells which can be grown in culture i.e. E.coli,
S.cerevisiae or mammalian cells are referable to
animals
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7easons to use cell culture$8Large 'uantities of cell can be obtainedulture conditions can be carefully
controlled giving more batch reroducibilityeed not to #ill animals
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What is known about the
protein/ enzyme? 9nowledge of the chemical , hysical roerties of the
rotein , its cellular localization will aid the design of aurification rocess.
/f the rotein has been reviously isolated from a differentsource the #nowledge can be alied to the rotein fromthe new source. -hus it:s localization is li#ely to remainthe same as is whether it is a glycorotein or liorotein
;ther characters$8 0ize < often remain similar / (isoelectric oint)$ the at which a articular molecule or
surface carries no net electrical charge. ydrohobicity
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/ntracellular localization can be #nown by$ 6ssaying subcellular fractionations5icroscoic examination using a lable secific for
the rotein eg. 0uitable labelled ligand or antibody
6lternatively the activity of the rotein mayidentify its localization. g. nzymes involved in transcrition will be located
in the nucleus 7ecetors for extracellular growth factors will be
found in the lasma membrane
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4roerties of glycorotein or liorotein
can be exloited during urificatione.g.glycorotein can be urified by lectin
affinity chromatograhyLiorotein < hearin affinity
chromatograhy
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-he choice of extraction method and buffer
comosition used will be determined by$ /ntracellular xtracellular 0oluble /nsoluble or membrane bound Located in subcellular organelle
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xtracellular rotein a high degree of urification isachieved by removal of the cells 5embrane8bound roteins will re'uire detergents or
organic solvents to solubilize them /solation of the subcellular organelle rior to extraction
will give a high degree of urification. nz or recetor:s activity can be exloited for affinityurification on a substrate or ligand or an analogue.
9nowledge of the size , / of the rotein will be usefulfor the selection of suitable matrices , conditions for
gel filtration , ion exchange chromatograhy. 6 rior #nowledge of the stability of the rotein , itssensitivity to temerature extreme roteases air, metal ions will also aid the design of a urificationrocedure
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ow should the protein be
assayed/detected?
1. 'uiments
2. *uffers3. 6ssays
=. Determination of total roteins
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1. 'uiments
old cabinet& cold room /ce ma#er +ridges 0ectrohotometer (2>? < @>?nm)
;ther e'uiments for assaying$ scintillation counter forradiolabelled samle L/06 (immunolabelled samle) and4L
lectrohoresis 'uiments for cell disrution entrifuge (refrigerated) olumn chromatograhy
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2. *uffers
ssential for enz stability ontrol the of the solution to avoid denaturation or
inactivation of enz&rotein AbufferB will deend on the alication i.e C roton A B C. sually 2>8?> m5 are ade'uate 7efer to EData fo *iochemical 7esearch Dawson
7.5.. lliot D.. lliot %.. , Fones 9.5.G for buffer
rearation
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+actors influence the choice of
buffer Desired 6nionic&cationic buffer secies
.g. for ionic exchange chromatograhy a cationicbuffer should be used (i.e. -ris H hoshate x)
Iariation of with ionic strength or temerature hemical reactivity
.g. 1Jamine -ris can interfere with rotein analysis..g. Lowry method or amino acid analysis
*iological activity .g. hoshate is a articiant in biological reaction
, may either inhibit or inactivate an enzyme
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+actors influence the choice of
buffer /nteraction with other comonents .g. hoshate comlexes with di8 , olyvalent metal ions -hus inhibiting meal ion < deendant enzymes
*orate comlexes with many organics , hydroxyl grous eseciallythose on ;
4enetration of biological membranes 8e.g. -ris
-oxicity .g. barbitone ,cacodylate buffers
6bsortion at 2@>nm or less .g. maleate
xenses if used on a large scale solubility
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K!ood: buffers
Develoed by !ood , his colleagues *iologically ,chemically non8reactive non8toxic
do not absorb in the I region their 9a showminimum deendence on temerature or ionicstrength
5ore exensive xamle
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!reparation of "uffers for !rotein
#$traction 4roteins are extremely heterogeneous
biological macromolecules. -heir roerties can be severely affected
by small changes in hydrogen ionconcentration and thus a stable of the
rotein environment is necessary
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-heory of buffering
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4reventing buffer contamination
-o revent bacterial or fungal growth$a. buffer can be filtered through a sterile
ultrafiltration deviceb. can be mixed with >.>2 sodium azidec. can be stored at = .℃
5icrobial contamination is common tohoshate buffered saline (4*0) but this maybe avoided at 15 hoshate stoc# solution.
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3. 6ssays
6fter each stage of urification assays for therotein of interest , its urity are re'uired to assessthe efficiency of the urification.
0ecific assays for enz is needed to identify whichfractions contain the enz rior to ooling for the nextste
/n addition the yield for each ste can bedetermined.
7esults from total rotein assays when combinedwith secific assays rovide information on thedegree of urification achieved by each ste , thesecific activity of the enz of interest.
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nzyme activity#$ample% 6lcohol dehydrogenase$
6DM M t; 8888N 6D M acetaldehyde
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&alculation enzyme acti'ity 6ssay mix$ >.OO ml >.1 5 -ris&l O.> 1>> m5 t; >.? m5
6DM 6ssay started by addition of >.>1 ml 6D (enzyme) ?>x diluted from
stoc# solution /ncrease of absorbance at 3=> nm followed$
P63=> Q >.R&min.
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!urification table
5easure for each urification ste$ -he volume of the enzyme solution (ml) -he rotein content of the solution (mg.ml81) -he activity of the enzyme solution (.ml81)
(otal amount of enzyme )U*%
6ctivity (.ml81) x volume (ml)
+pecific acti'ity )U,m-.*%
6ctivity (.ml81) & rotein content (mg.ml81)
0ield )1*%
-otal amount of enzyme after a urification ste & total amount of enzyme beforethat ste
!urification factor% 0ecific activity of enzyme after a urification ste & secific activity before that
ste
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!urification table )e$ample*
+tep olume (otal (otal +pecific 0ield !urification
acti'ity protein acti'ity factor
)ml* )U* )m-* )U/m-* )1*3333333333333
(1) ?>> 3>>> 1?>>> >.2 1>> 88 60 (2) 1>> 2=>> =>>> >.R @> 3.>/ (3) =? 1==> ?>> 2.O =@ 1=.?!+ (=) ?> 1>>> 12? @.> 33 =>.>
SSSSSS SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS
0tes$ (1) rude cell extractT (2) ammonium sulfate fractionationT(3) ion exchange chromatograhyT (=) gel filtration.
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Degree of urification Q secific activity at ste2 secific activity at ste1
ach ste can be assessed by yield , degree of urification for its efficiency
/deally an assay should be simle highly secific , raid in order to allowmany fractions to be screened for activity rior to the next stage of theurification
7aid assays minimize storage times btw stes thus minimizingroteolysis (rotein degradation).
5any enz can be raidly assayed (U1> min) sectrohotometrically)
0ecific activity Q rotein&enzyme (mg&unit) -otal rotein (mg)
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/mmunological assays
can be used for many roteins rovided anaroriate antibody is available. 5ost fre'uently used $
7adioimmunoassays (7/6) nzyme8lin#ed assays (L/06)
ot raid as enz activity assays an detect denature rotein ighly secific , multile samles can be screened 4urity is achieved when further urification stes do
not remove any bands in electrohoresis gelsubse'uent urification ste do not increase thesecific activity.
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=. Determination of total rotein
ssential for the estimation of the degree ofurification
+ew techni'ues direct or indirect ltraviolet sectrohotometry *iuret method Lowry rocedure
*icinchomimic acid rotocol Dye8binding rocedure
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600/!5- ?
1. ltraviolet sectrohotometry2. *iuret method
3. Lowry rocedure
=. *icinchomimic acid rotocol
?. Dye8binding rocedure +or the above techni'ues of rotein
determination
Discuss the rincile of each techni'ue xlain the rocedure of each techni'ue
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4reserving activity
/ntracellular roteins will be subVected to many inactivatingconditions once released from its native environment.
%ithin the cell the is maintained at WR.?8X.? theAroteinB is high (W1>>mg&ml) , the reducing otential is
high. ;n disrution into buffer the AroteinB is reduced , the
rotein occur in an oxidizing environment. /n addition tissue disrution will lead to the brea#down of
comartmentalization.
4articularly is the disrution of lysosomes which willrelease rotease into the environment , causes anacidification of solution.
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Fudicious choice of buffers must thereforebe made to minimize denaturation
inactivation ,&or roteolysis of intracellularroteins xtracellular roteins are not usually as
sensitive < have evolved to maintainactivity in the less controlled environmentoutside the cell.
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ow to reserve activity of
enzyme1. 5inimizing denaturation
2. 5inimizing inactivation
3. 5inimizing roteolysis=. ;ther recautions
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1. 5inimizing denaturation 4rincial causes of denaturation$
xtremes of xtremes of temerature ;rganic solvents
5ost roteins stable over a broader range W ?8O or greater arry out the initial stes of urification at =J igh tem (N=>J) should be avoid unless the rotein is #nown to
be heat8stable ;rganic solvents (i.e. acetone , alcohol) and chaotroic agents
(i.e. urea , guanidine hydrochloride) should be avoided
0ome roteins resistant to these denaturants , they can beexloited during the urification to denature contaminant roteins. ;rganic solvents can be used at low tem to reciitate rotein.
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2. 5inimizing inactivation
nz containing a free sulhyryl grou inthe active site may be raidly oxidized
after disrution of the cell. D-6 should not be included in thebuffers used for metal ion deendentenz urification.
5any enz can be stabilized by includingcofactors or substrates in buffers.
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3. 5inimizing roteolysis 0eed , low tem (W=>J) during the initial stages. 6 coctail of rotease inhibitors should be included to the
extraction buffer , other buffers used in the initial stes. /nclusion of sucrose&maltose in the extraction buffer can
stabilize the lysosomal membane , minimize disrution. 7educing reagents i.e. 28mercato8ethanol or dithiothreitol
(D--) should be added to all buffers. 28mercato8ethanol$ ?82>m5 added to the buffer immediately
rior to use. /t oxidize very raid , lost its rotective action within2=h.
D--$ last longer suitable for use in storage buffers less odorons
18?m5 usually ade'uate should add to buffers immediately riorto use 5etal ions can inactivate sulhydryl grous chelating agent
i.e. D-6 (>.181.>m5) should be added to all buffers
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=. ;ther recautions Dilute rotein solutions are often unstable due to adsortion to
surface , dissociation of subunits. 0ome roteins are more suscetible to adsortion to surface i.e.
glass. eed to use siliconized containers or olyroylene container.
5ost rotein will bind irreversibly to olystyrene 6ddition of *06 (Y>.1) non8ionnic detergents (i.e. -ween 2> or-riton Z81>> >.1) may also minimize adsortion losses.
!lycerol is often included in buffers at 1>82> to minimize activitylosses , ?> for storage 5ay interfere with subse'uent urification stes.
6lternative to glycerol are sugars i.e. glucose or sucrose. *acteriostatic agents i.e. sodium azide >.>2 8 >.>? alsousually added to buffer.
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!U4I5I&6(I78 75 #8Z09#+
o fix methods or rules in rotein&enzyme urification
Disrution
xtraction
larification
4reciitation
hromatograhy methods
5etal chelate/on exchange
hromatofocusing
6ffinity
!el filtration covalent
ydrohobicinteraction
!rimary
separationtechniques
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9ain types of purification methods
-he following main tyes of urification methods for roteins can be distinguished$1. 4reciitation methods2. 0earation based on molecular size3. 0earation based on charge=. 0earation based on secific interaction with other biomolecules?. 0earation based on other rinciles
xamles$1. 6mmonium sulfate reciitation2. !el filtration3. /on8exchange chromatograhy=. *io8affinity chromatograhy?. ydrohobic interaction chromatograhyT hydroxyaatite chromatograhy
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