Kuliah-6 Pemurnian Enzim Translate

7/21/2019 Kuliah-6 Pemurnian Enzim Translate

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PEMURNIAN

ENZIM

Mata Kuliah Biokimia- MKK4213

STF MUHAMMADIAH



!h" i#olat$ $%&"m$#''

To u%($)#ta%( $%&"m$ *)o*$)ti$#:Enzymes work in a complex system

(inside cell), == we must try frst ina #im*l$ #"#t$m ++

U#$( i% ,oo( i%(u#t)iala**li.atio%#- Pectinase : Juice production

- Chymosin : cheese production

- Glucose isomerase : H! production



Mengapa Ezim Terisolasi ??

• Untuk memahami sifat enzim:

Enzim bekera dalam sistem !ang kompleks

"dalam sel#$ %% kita harus mencoba pertama

dalam sistem sederhana %%

• &igunakan dalam makanan ' aplikasi industri

( Pektinase: produksi Juice

( )h!mosin: produksi keu

( Glukosa isomerase: produksi *+,



!h" i#olat$ $%&"m$#''

"he isolated enzyme can #echaracterized:o optimum acti$ity in $arious conditions

(pH, temperatur, ionic stren%h etc)o specifcity

o kinetic parameter

o mechanism o& catalysiso re%ulation

o !tructure

U%($)#ta%( th$ )ol$ o, $%&"m$#



Enzim terisolasi dapat dicirikan:• -egiatan !ang optimal dalam berbagai kondisi "p*$

temperatur$ strengh ionik dll#

• kekhususan• parameter kinetik• mekanisme katalisis• peraturan• struktur

• Memahami peran enzim dalam sistem !ang lebih

kompleks

Mengapa enzim terisolasi??



H ow t o ! e p ar

at e" h e s e ' # ( e c t s

1 2 3

9 10 11 12

6

4 8

5

7

4

5

8

wood stone cotton wood wood cotton stone wood stone cotton stone cotton

cotton

wood

stone

Shape

Size

Density

Shape

Density

Size

,ie.ing different sizes &ifferent sedimentation

&ifferent rolling speed

4 6 7 85

1 3 4 6 7 8 9 10 11 122 5

Juang /* "0112# 3)basics



0$.ti$# a%( St)at$"

0$.ti$#:to isolate a particular enzyme &rom

all other proteins (enzyme) and cellcomponents with- maximum possi#le "i$l(- maximum catalytic a.tiit"- maximum possi#le *u)it"



Tuua% a%( St)at$i

tuua%5u%tuk m$%i#ola#i $%&im t$)t$%tu

(a)i #$mua *)ot$i% lai% 6$%&im7(a% kom*o%$% #$l ($%a%- Mu%ki% ha#il mak#imum- Aktiita# katalitik mak#imum- K$mu)%ia% mak#imum "a%mu%ki%



St)at$"5

0$.ti$# a%( St)at$"

*nity!eparatio

n

+aterial hole

extraction

Crude extractHomo%enisation ar%e scale separation

.ureenzyme

!eries o&smallscaleseparatio

n



St)at$"

.rotein are di$erse in compositionstructure #eha$iour, you should knowa#out their ori%in/ s your purifcationstrate%y depends on it/

0/ here is this enzyme or protein present inthe cell1 (intracellular, extracellular,

mem#ranous)/2/ How you can puri&y this protein in as &ew

steps as possi#le without the loss o&acti$ity/

3/ 4eepin% in consideration o& temperature and



St)at$i

.rotein yan% #era%am dalam perilakustruktur komposisi, nda harus tahu tentan%asal-usul mereka/ !e#a%ai strate%i

pemurnian nda ter%antun% padanya/0/ 5imana enzim ini atau hadir protein dalam

sel1 (intraseluler, ekstraseluler, mem#ran)/

2/ 6a%aimana nda dapat memurnikan proteinini se#a%ai #e#erapa lan%kah mun%kintanpa kehilan%an akti$itas/

3/ +ena%a dalam pertim#an%an suhu dan

waktu/



Sou).$# o, E%&"m$#

Mi.)oo)a%i#m8 a%imal8 *la%t 6ti##u$a%( .$ll7 7ennin &rom Mucor miehei

7ennin &rom stomach o& cal&

H$t$)oloou# $/*)$##io%

8enetically en%ineered #acteria, yeastto produce enzyme

- 7ennin 9 chymosin &rom %/e/ yeast

- mylo%lucosidase &rom %/e/ 6acillus

licheni&ormis



Sum$) (a)i $%&im

+ikroor%anisme, hewan, tum#uhan(arin%an dan sel)

-7ennin dari +ucor miehei

-7ennin dari lam#un% anak sapi

ekspresi heterolo%6akteri rekayasa %enetika, ra%i untuk

memproduksi enzim - 7ennin 9 chymosin dari %/e/ ra%i- milo%lukosidase dari %/e/ 6acillus

licheni&ormis



I%(u#t)ial $%&"m$# may #eextracted &rom any li$in% or%anism:

&un%i (o$er a hal&)

#acteria (o$er 0 9 3)

animal (;<)

plant (<)

Sou).$# o, E%&"m$#



Sum$) (a)i $%&im

Enzim industri dapat diekstraksi dari

setiap organisme hidup:

• amur "lebih dari setengah#• bakteri "lebih 4'5#• he6an "78#• tanaman "28#



Sou).$# o, E%&"m$#

Mi.)o$# a)$ *)$,$))$( to plants andanimals as sources o& enzymes #ecause:

"hey are %enerally cheaper to produce "heir enzyme contents are more predicta#le and

controlla#le .lant and animal tissues contain more potentially

harm&ul materials than micro#es, includin%phenolic compounds (&rom plants)



Sum$) (a)i $%&im

+ikro#a yan% le#ih suka tanaman dan hewanse#a%ai sum#er enzim karena:

+ereka umumnya le#ih murah untukmemproduksi

>si enzim mereka le#ih dapat diprediksi dandikontrol

?arin%an tanaman dan hewan men%andun% le#ih#anyak #ahan yan% #erpotensi #er#ahayadaripada mikro#a, termasuk senyawa &enolik (daritanaman)



Enzyme EC Sources 9pplication

α(9m!lase 4055 Aspergillus E 3aking

)atalase 5555; Aspergillus < +ood

)ellulase 4052 Trichoderma E =aste

&e>tranase 40555 Penicillium E +ood

Glucose o>idase 5542 Aspergillus < +ood

actase 40504 Aspergillus E &air!

ipase 4554 Rhizopus E +ood

/ennet 4204; Mucor miehei E )heese

Pectinase 4055@ Aspergillus E &rinks

Protease 4204; Aspergillus E 3aking

E: extracellular enzyme@ >: intracellular enzyme

Fu%al E%&"m$#



Enzyme Sources Application

α(9m!lase 4055 Bacillus E ,tarch

β(9m!lase 4050 Bacillus E ,tarch

9sparaginase 4@55 Escherichia coli < *ealth

Glucose isomerase @45@ Bacillus < +ructose s!rup

Penicillin amidase 4@555 Bacillus < Pharmaceutical

Protease 420552 Bacillus E &etergent

Ba.t$)ial E%&"m$#



3asic Principles of Protein "Enz!me# Purification

)ell Organelle

*omogenization

Macromolecule

Nucleic

acid

Carbohydrate "ipid#

,ize )harge ,olubilit! 9ffinit!

Small moleculeCell

DebrisProteinAmino acid, Sugar,

Nucleotides, etc

Gel filtration,

SDS-PAG,

!ltrafiltration

"on e#change,

"soelectric focusingSalting-out

$Ammonium

sulfate )

Affinitychromatography



9$%$)al *)oto.ol ,o) $%&"m$*u)i:.atio%

0/ "akin% the intact cell tissue/2/ Homo%enisation3/ !eparation o& cell de#ris and insolu#le stuA

/ .recipitation o& protein with the saltB/ 8ettin% rid o& salt #y dialysis/ urther purifcation #y column and ion

exchan%e chromato%raphy ,

D/ Each a#o$e step is &ollowed #y enzyme assayacti$ity(in case you lost your enzyme )

;/ indin% out the exact molecular wei%ht #ycolumn chromato%raphy and #y !5!-8el-

electrophoresis



P)otokol umum u%tuk*$mu)%ia% $%&im

0/ +en%am#il arin%an sel utuh/2/ homo%enisasi3/ .emisahan puin% sel dan hal-hal yan% tidak

larut/ .en%endapan protein den%an %aramB/ +enyin%kirkan %aram den%an dialisis/ .emurnian le#ih lanut den%an kolom dan

kromato%raf pertukaran ion,D/ !etiap lan%kah di atas diikuti oleh akti$itas

enzim assay (dalam kasus nda kehilan%anenzim nda)

;/ +encari tahu #erat molekul yan% tepat



!kema khas pemurnian enzim



D$$lo*$ E%&"m$ A##a"

Ho; (o ;$ )$.o%i&$ th$ $%&"m$that ;$ a)$ looki% ,o)'

D$$lo* a%al"ti.al a##a" usually #ased on the reaction that the enzyme

catalyzes in the cell



P$%uia%*$%$ma%a% $%&im

6a%aimana kita men%enali enzim yan% kitacari1

+en%em#an%kan ui analisis- #iasanya didasarkan pada reaksi #ahwa

enzim men%katalisis dalam sel



O

O

OH

OH

OH

HO

N+O

O-

OH

N+O

O-

O

OH

OH

OH

OH

HO

'.8 8alactose '.

F

β %alactosidase

D$$lo*$ E%&"m$ A##a"

Mo%ito)i% P)o)$## o,



Mo%ito)i% P)o)$## o,Pu)i:.atio% P)oto.ol

StepTotal protein

(mg)Total activity

(units)

Specificactivity

(units/mg)

Yield(%)

Purificationlevel (Purity

factor)

nitial e!tract 5@$111 5@1$111 51 511 5

("#$)S&$

precipitation2$;11 547$111 41 A0 4

on'e!cange 5$0B7 55@$@11 A1 BB A

Sizee!clusion

;7; B@$111 5$511 @1 551

Affinitycolumn

5B@ @0$@11 41$111 4@ 4$111

(6er%, "ymoczko, G !tryer/ (22) Biochemistry , Bth ed/ /H/ reeman G Co/, ew Iork, I,p/ ;)

Pemantauan -emauan Pemurnian Protocol



PURIFI<ATI0N METH0DS

H i ti ,



Homo$%i#atio% o,#am*l$

Homo%enisation 5th$ *)o.$## o, )$aki% o*$%

.$ll#

5epend on the source- +ammalian tissue- .lant, &un%al, #acterial material- Extraction o& mem#rane-#oundenzymes

H i i ( i



Homo$%i#a#i (a)i#am*$l

homo%enisasi:proses melan%%ar sel ter#uka

"er%antun% pada sum#er- ?arin%an mamalia- "anaman, amur, #ahan #akteri- Ekstraksi enzim mem#ran-terikat



Homo$%i#atio%M$tho(#

Ph"#i.al

rench pressure

cell

!onication

8lass #eads

<h$mi.al D$t$)$%t# 6SDS7

E%&"m$#



Homo$%i#atio%M$tho(#

fsik -!el tekanan

.erancis -sonication -manik-manik kaca

kimia-5eteren (!5!)-Enzim (lyzozyme)-penyan%%a

hipotonik

< ll F ti ti



<$ll F)a.tio%atio% S$*a)atio%

!eparation o& cell de#ris and insolu#lestuA can #e &ractionated #y.$%t)i,uatio%

>n centri&u%ation, ($%#$) mat$)ial ;ill.oll$.t at th$ ottom o& the tu#e in a *$ll$t whereas material with lo;$)($%#it" ;ill )$mai% i% th$ #olul$,)a.tio% called the #u*$)%ata%t

'nly the &raction that contains the protein

F k i i l



F)ak#i%a#i #$l P$mi#aha%

.emisahan puin% sel dan hal-hal yan%tidak larut dapat di&raksinasi den%an

sentri&u%asi.ada sentri&u%asi, #ahan padat akanmen%umpulkan di #a%ian #awahta#un% di pelet sedan%kan #ahanden%an densitas yan% le#ih rendahakan tetap dalam &raksi larut dise#utsupernatan

Hanya se#a%ian kecil yan% #erisi



<$ll F)a.tio%atio%

Centri&u%ation

!upernatant

.elletiltration

(other

methods)



Staili&$ Sam*l$ 6 #taili#a#i#am*$l7

<o%t)ol *H Jse appropriate #uAer

<o%t)ol t$m*$)atu)$ 4eep samples on ice orwork in cold room ( K oC)

.rechill instruments

P)$$%t,)othi%,oami% Handle %ently/

Mai%tai%

• kontrol p*

(Gunakan pen!angga !ang tepat

• kontrol suhu

(Terus sampel di atas es atau

bekera di ruangan !ang dingin "1 (2o)#

(instrumen Prechill

• Mencegah buih ' busa

(Menangani dengan lembut

• Menaga sampel terkonsentrasi



Staili&$ Sam*l$

P)ot$a#$ i%hiito)#

.henylmethylsul&onylLuoride (.+!)

S

O

O

F

.+!



P)ot$i% Soluilit"

Salti% i% t a low salt

concentration, ionsprotect char%es and

allow proteins to &old/

Salti% out >ons compete with water

to interact with side%roups/

hen MsaltN is hi%henou h salt wins

MsaltN

s o l u # i l i

t y

Osaltin% inPOsaltin%outP



Soluili#a#i *)ot$i%

pen%%araman di-.ada konsentrasi rendah%aram, ion melindun%i#iaya dan memun%kinkan

protein melipat/

pen%%araman->on #ersain% den%an air

untuk #erinteraksi den%ankelompok sisi/

-4etika M%aramN cukup

tin%%i, %aram menan%

MsaltN

s o l u # i l i

t y

Osaltin% inPOsaltin%outP



Salti% i% #alti% out

S lti i ( S lti



Salti%-i% a%( Salti%-out

5iAerent proteins precipitate atdiAerent salt concentrations, so th$

#alt .o%.$%t)atio% ma" $a(u#t$( to *)$.i*itat$ th$($#i)$( *)ot$i%

Ammo%ium #ul,at$ is the mostcommonly used rea%ent &or saltin%out enzymes (proteins)

Salti% i% a%( Salti%



Salti%-i% a%( Salti%-out

.rotein yan% #er#eda endapan padakonsentrasi %aram yan% #er#eda,

sehin%%a konsentrasi %aram dapatdisesuaikan untuk men%endapkanprotein yan% diin%inkan

monium sul&at adalah di%unakanrea%en yan% palin% umum untukpen%%araman enzim (protein)



Dial"#i#

&orm o& size exclusionchromato%raphy/

Jsed to desalt andconcentrate proteinsamples/

5ialysis tu#in% has setmol$.ula) ;$iht .uto= 6M!<07/ 'nlymolecules that smallerthan M!<0 will mo$e

out o& the dialysis #a%/



Dial"#i#

!uatu #entukkromato%raf eksklusiukuran/

5i%unakan untukmen%hilan%kan %aramdan #erkonsentrasisampel protein/

6erat molekul dialisistu#in% telah menetapkandipoton% (+C')/ Hanyamolekul yan% le#ih kecildari +C' akan



Dial"#i#



<olum% <h)omato)a*h"

+ost common method &or separatin%enzymes and other proteins

4e#anyakan metode umum untuk

memisahkan enzim dan protein lain

S ti P t i



S$*a)ati% P)ot$i%#6m$mi#ahka% *)ot$i%

<h)omato)a*h"

+o#ile phase

C .hase that carriessample throu%houtprocedure/

C iQuid

C 8as

!tationary phase

C +atrix that retardsthe mo$ement o&sample #ein% carried#y the mo#ile phase/

• -romatografi

(fase gerak

(+ase !ang memba6a

sampel seluruhprosedur

(cair

(gas

• fase stasioner (Matriks !ang

menghambat pergerakan

sampel !ang diba6a oleh

fase gerak

9$l :lt)atio%



Enzymes are separated onthe #asis o& #i&$/

ar%e molecules exit frst/

+o#ile phase iQuid

!tationary phase >nsolu#le, porous

car#ohydrate #eads

9$l-:lt)atio%<h)omato)a*h"

lso called #i&$ $/.lu#io%.h)omato)a*h"

9$l :lt)atio%



Enzim dipisahkan atasdasar ukuran/

+olekul #esar keluarpertama/

&ase %erak

-cair

&ase stasioner-arut, manik-manik

kar#ohidrat #erpori


• ?u%a dise#ut kromato%raf eksklusi ukuran

9$l :lt)atio%




9$l :lt)atio%



9$l-:lt)atio%<h)omato)a*h" !ample is applied to the top o& a column

containin% porous #eads 9 matrix

!mall molecules will pass throu%h the pores o&the #eads (while lar%er ones cannot)

ar%e molecules Low more rapidly throu%h thecolumn and elute frst

>ntermediate size molecules will elute at anintermediate position (occasionally enter the#eads)

"he small molecules will elute last, #ecause

take lon%er path

9$l :lt)atio%



9$l-:lt)atio%<h)omato)a*h" !ampel diterapkan ke atas kolom yan% #erisi

#erpori manik 9 matriks +olekul kecil akan melewati pori-pori manik-

manik (sementara yan% le#ih #esar tidak #isa) +olekul #esar men%alir le#ih cepat melalui

kolom dan terelusi pertama +olekul ukuran menen%ah akan men%elusi di

posisi menen%ah (kadan%-kadan% memasukimanik-manik)

+olekul-molekul kecil akan men%elusi lalu,karena men%am#il alan la%i

Io% E/.ha%$



Io% E/.ha%$<h)omato)a*h"

!eparatesmolecules #ased

on .ha)$/ +o#ile phase

8enerally liQuid !tationary phase

Electrostaticallychar%ed ions#ound toinsolu#le,

chemically inertmatrix/ Elution o&

protein dd salt to

compete with#indin% o& sample

H2C

C

O

O-

carboxymethyl (CM)

cellulose

H2C

CH2

N

CH2

CH3

H2C

CH3

H

diethylaminoethyl (DEAE)

cellulose

(nion

(Cationexchan%e)

• Memisahkanmolekul

berdasarkan bia!a• fase gerak

(umumn!a cairan

• fase stasioner (Elektrostatis

dibebankan ion terikat

larut$ matriks kimia

inert• Elusi protein

(Tambahkan garam

untuk bersaing dengan

pengikatan sampel

untuk fase diam

(Perubahan p* "alter

bia!a protein#

Io% E/.ha%$



>on exchan%e resins contain .ha)$( )ou*#/ 7esin pertukaran ion men%andun% %u%us

dikenakan/

>& these %roups are acidic in nature theyinteract with positi$ely char%ed proteins arecalled .atio% exchan%ers/

?ika kelompok-kelompok ini #ersi&at asam di

alam mereka #erinteraksi den%an protein#ermuatan positi& dise#ut kation exchan%er/

C+ cellulosecation exchan%er

CH2

-C''-

CH2-C''-

FFF

F

.ositi$ely

char%ed (#asic)protein orenzyme


Io% E/.ha%$



>& these %roups are basic in nature, theyinteract with ne%ati$ely char%ed molecules arecalled a%io% exchan%ers/

?ika kelompok-kelompok ini dasar di alam,mereka #erinteraksi den%an molekul#ermuatan ne%ati& dise#ut penukar anion/

5EE celluloseanion exchanger 5EE selulosaanion exchan%er

CH2-CH2 -HF(CH2CH2)-

-

--

e%ati$elychar%ed

(acidic) proteinor enzyme6ermuatanne%ati& (asam)protein atauenzim

CH2-CH2 -HF

(CH2CH2)


Io% E/.ha%$



or protein #indin%, the pH is fxed (usually near neutral)under low salt conditions/ Example cation exchan%ecolumnRJntuk protein yan% men%ikat, pH adalah tetap (#iasanya

mendekati netral) dalam kondisi rendah %aram/ +isalnyatukar kation kolom ///

C+ cellulose

cation exchan%er

CH2-C''-

CH2-C''-

FF

FF

.ositi$ely char%edprotein or enzyme #indto the column6ermuatan positi&protein atau enzim

men%ikat ke kolom--

--

--

--

e%ati$ely char%edproteins pass throu%hthe column.rotein #ermuatan

ne%ati& melewatikolom


Io% E/.ha%$



"o elute enzyme o& interest, add increasin%ly hi%her amount o& salt(increase the ionic stren%th)/ aF will interact with the cation resinand Cl- will interact with our positi$ely char%ed protein to elute oAthe column/Jntuk men%elusi enzim yan% menarik, tam#ahkan umlah yan%semakin tin%%i %aram (menin%katkan kekuatan ion)/ a F akan#erinteraksi den%an resin kation dan Cl- akan #erinteraksi den%anprotein kami #ermuatan positi& untuk men%elusi dari kolom/

C+ cellulose

cation exchan%er

CH2-C''-

CH2-C''-

FFF

F

C+ cellulose

cation exchan%er

CH2-C''-

CH2-C''-

F

F

F

F

aF

aF

Cl-

aF2

Cl-

Cl-

Cl-

aF2

F >ncreasin%MaClN o& theelution #uAer




Io% E/.ha%$



ow salt Hi%h salt




E/am*l$#

"ame onizale group Type

&E9E(,ephade> &ieth!laminoeth!l =eakl! basic

,P(,epharose Meth!lsulfonate ,trongl! acidic

3io(/e> B1 )arbo>!lic acid =eakl! acidic

P cellulose Phosphate,trongl! D 6eakl!

acidic



A>%it" <h)omato)a*h"

+o#ile phase

Jsually liQuid !tationary

phase nti#ody9su#stra

te9li%and #ound

Immu%oa>%

it"

Affinity cromatograpy is a method of separating enz!me ' protein

mi>tures based on a highl! specific interaction bet6een : antibod! and

antigen

substrate and enz!me

ligand and receptor

-romatografi afinitas adalah metode memisahkan campuran enzim ' proteindidasarkan pada interaksi !ang sangat spesifik antara: antibodi dan antigen

substrat dan enzim

ligan dan reseptor

fase gerak

biasan!a cairan

fase stasioner

9ntibodi ' substrat '

ligan terikat manik

lembam



A>%it" .h)omato)a*h"



Po##il$ $lutio%#t)at$i$#5 pH

>on stren%h

5enature

Competitor li%and oranalo%

A>%it" .h)omato)a*h"

-emungkinan

Pengembangan strategi

elusi:p*

ion strengh

mengubah ,ifat ,esuatu

3endaPesaing ligan 9T9U

analog



A a>%it" .olum%





Total *)ot$i% 6m7 Suantity o& protein

present in &raction

Total a.tiit" 6u%it#o, a.tiit"7 Jse a portion o& sample

to determine acti$ity/

+ultiply acti$ity #y total$olume to determine

total acti$ity/

Jumlah protein "mg#

Jumlah protein hadir dalam

fraksi

Jumlah akti.itas "unit

kegiatan#

Menggunakan sebagian

dari sampel untukmenentukan akti.itas

-egiatan kalikan dengan

total .olume untuk

menentukan akti.itas total





S*$.i:. a.tiit" 6u%it# o, a.tiit"m7

? "i$l(5 measure o& acti$ity retaineda&ter each step in procedureukuran akti$itas ditahan setelah setiaplan%kah dalam prosedur/< yield = "otal acti$ity at particular

step

"otal acti$ity o& initial extract

!// = "otal acti$ity

"otal protein





Pu)i:.atio% l$$l5 +easure o&increase in purity o& protein throu%houtprocedure/

"in%kat pemurnian: +en%ukur penin%katan

kemurnian protein seluruh prosedur/

.urifcationle$el =

!pecifc acti$ity at particular step

!pecifc acti$ity o& initial extract





StepTotal protein

(mg)Total activity

(units)

Specificactivity

(units/mg) Yield (%)

Purificationlevel

nitial e!tract 5@$111 5@1$111 51 511 5

("#$)S&$

precipitation2$;11 547$111 41 A0 4

on'e!cange 5$0B7 55@$@11 A1$4 BB A$14

Sizee!clusion

;7; B@$111 51A4$4 @1 51A$4

Affinitycolumn

5B@ @0$@11 41111 4@ 4111

(6er%, "ymoczko, G !tryer/ (22) Biochemistry , Bth ed/ /H/ reeman G Co/, ew Iork, I,

p/ ;)



Purification Summary



Purification Summary

STEP

Total

Activity

(Unit)

Total

Protein

(mg)

Specific

Activity

(Unit/mg)

Yield

(%)

Purification

factor (Fold)

Culture Filtrate !"# $ "&! & &

Colloidal C'itin Affinity " " &"&$ $" *"!

+EAE,Toyopearl - .* ."#* #"&& &"* &." "$&

utyl,Toyopearl - .* &"&* "$ #"$& $"!& $$"&*

Ra'CiA

* SP SP

!

(+,a)

.

#

S+S,PA0E

1Y-203A-

A + P Y 4 5 6 A Y Y P

A + S Y 5 7 6 + Y Y P

Ra,C'iA

Ba,C'iA&

8,terminal amino acid 9e:uence9

alignment of Ra,C'iA and Bc,C'iA&

<ha)a.t$)i&atio% o,



o +olecular masso 'ptimum acti$ityin $arious conditions

(pH, temperatur,ionic stren%h etc)o Enzymespecifcityo

4ineticparametero +echanism o&catalysis

o !tructure (T-ray

<ha)a.t$)i&atio% o,E%&"m$

• massa molekul• -egiatan optimal

dalam berbagai kondisi

• "p*$ temperatur$ ionstrengh dll#

• spesifisitas enzim• parameter kinetik

• Mekanisme katalisis• ,truktur "(ra!

kristalografi#• peraturan

El h i



El$.t)o*ho)$#i# !eparates

molecules #asedon molecularmass and9orchar%e/

+emisahkanmolekul#erdasarkanmassa molekul

dan 9 atau #iaya

http:99www/science/&au/edu9chemistry9+ari9#iochemla#9manual/html



SDS-PA9E

!odium dodecylsul&atepolyacrylamide%elelectrophoresis

!eparation#ased onmolecular mass/

Coat sampleswith !5! to %i$euni&orm char%eto mass ratio/

+akes all

,odium dodec!lsulfat poliakrilamida

gel elektroforesis

Pemisahan

berdasarkan massamolekul

,ampel mantel

dengan ,&, untuk

memberikan bia!aseragam untuk

rasio massa Membuat semua

protein bermuatan

negatif

Protein separation using S,S'PA-E(.aemmli system) un te electroporesis until dye



(.aemmli system)

Stac+ing

gel

esolving

gel

01 Apply protein/dye samples

into polyacrylamide gel

2ells

Terap+an sampel protein / dye3

dalam sumur gel polia+rilamida

1 un te electroporesis until dye

reaces te end of te gel

4alan+an ele+troforesis sampai dye3

mencapai a+ir gel

51 emove te gel from

te apparatus and stain

for proteins

.epas+an gel dari3 alat

dan noda untu+3 protein

I l t i F i



I#o$l$.t)i. Fo.u#i%

!eparation #ased on char%e/ .olyampholite (small

multichar%ed polymers) used toprepare pH %radient in the %el

Can #e used to experimentallydetermine the p> $alues/

• Pemisahan berdasarkan bia!a

• Pol!ampholite "polimermulticharged kecil# digunakan untuk

mempersiapkan p* gradien dalam

gel• &apat digunakan untuk eksperimen

menentukan nilai p<



"H4 I'J

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