8/20/2019 Chapter 2c Jan2010

1/47

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.

8/20/2019 Chapter 2c Jan2010

2/47

!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

8/20/2019 Chapter 2c Jan2010

3/47

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.

8/20/2019 Chapter 2c Jan2010

4/47

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

8/20/2019 Chapter 2c Jan2010

5/47

What source should be used?

Defined by the final alication hoose the most stable , abundant 0hould also consider the availability ,

'uantity of the source.

8/20/2019 Chapter 2c Jan2010

6/47

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

8/20/2019 Chapter 2c Jan2010

7/47

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

8/20/2019 Chapter 2c Jan2010

8/47

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

8/20/2019 Chapter 2c Jan2010

9/47

/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

8/20/2019 Chapter 2c Jan2010

10/47

4roerties of glycorotein or liorotein

can be exloited during urificatione.g.glycorotein can be urified by lectin

affinity chromatograhyLiorotein < hearin affinity

chromatograhy

8/20/2019 Chapter 2c Jan2010

11/47

-he choice of extraction method and buffer

comosition used will be determined by$ /ntracellular  xtracellular  0oluble /nsoluble or membrane bound Located in subcellular organelle

8/20/2019 Chapter 2c Jan2010

12/47

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

8/20/2019 Chapter 2c Jan2010

13/47

ow should the protein be

assayed/detected?

1. 'uiments

2. *uffers3.  6ssays

=. Determination of total roteins

8/20/2019 Chapter 2c Jan2010

14/47

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

8/20/2019 Chapter 2c Jan2010

15/47

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

8/20/2019 Chapter 2c Jan2010

16/47

+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

8/20/2019 Chapter 2c Jan2010

17/47

+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

8/20/2019 Chapter 2c Jan2010

18/47

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

8/20/2019 Chapter 2c Jan2010

19/47

!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

8/20/2019 Chapter 2c Jan2010

20/47

-heory of buffering

8/20/2019 Chapter 2c Jan2010

21/47

8/20/2019 Chapter 2c Jan2010

22/47

8/20/2019 Chapter 2c Jan2010

23/47

8/20/2019 Chapter 2c Jan2010

24/47

8/20/2019 Chapter 2c Jan2010

25/47

8/20/2019 Chapter 2c Jan2010

26/47

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.

8/20/2019 Chapter 2c Jan2010

27/47

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.

8/20/2019 Chapter 2c Jan2010

28/47

nzyme activity#$ample% 6lcohol dehydrogenase$

6DM M t; 8888N 6D M acetaldehyde

8/20/2019 Chapter 2c Jan2010

29/47

&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.

8/20/2019 Chapter 2c Jan2010

30/47

!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

8/20/2019 Chapter 2c Jan2010

31/47

!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.

8/20/2019 Chapter 2c Jan2010

32/47

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)

8/20/2019 Chapter 2c Jan2010

33/47

/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.

8/20/2019 Chapter 2c Jan2010

34/47

=. 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

8/20/2019 Chapter 2c Jan2010

35/47

 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

8/20/2019 Chapter 2c Jan2010

36/47

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.

8/20/2019 Chapter 2c Jan2010

37/47

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.

8/20/2019 Chapter 2c Jan2010

38/47

ow to reserve activity of

enzyme1. 5inimizing denaturation

2. 5inimizing inactivation

3. 5inimizing roteolysis=. ;ther recautions

8/20/2019 Chapter 2c Jan2010

39/47

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.

8/20/2019 Chapter 2c Jan2010

40/47

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.

8/20/2019 Chapter 2c Jan2010

41/47

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

8/20/2019 Chapter 2c Jan2010

42/47

=. ;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.

8/20/2019 Chapter 2c Jan2010

43/47

!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

8/20/2019 Chapter 2c Jan2010

44/47

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

8/20/2019 Chapter 2c Jan2010

45/47

8/20/2019 Chapter 2c Jan2010

46/47

8/20/2019 Chapter 2c Jan2010

47/47