EnzAssay CLC

7/23/2019 EnzAssay CLC

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Chia-Lin Chung

R. Nelson Lab

Cornell University

Enzyme Activities Assay

Reference: Venisse, J.-S., Gullner, G., and Brisset, M.-N. !!". #viden$e %or the

involve&ent o% an o'idative stress in the initiation o% in%e$tion o% (ear by Erwinia

amylovora. )lant )hysiology "*+ "-".

Enzyme Extraction:

Bu%%er+

*! &M sodiu& (hos(hate bu%%er /(0 .*1

" &M (olyethylenegly$ol" &M (henyl&ethylsul%onyl %luoride

2 3 /45v1 (olyvinyl(yrolydone

!.!" 3 /v5v1 6riton 7-"!!

Enzyme Assay:

Ascorbate Peroxidase (AsPOX)

8bsorban$e+ 9! n&, o'idation o% as$orbi$ a$id

Rea$tion Mi'ture+ "! :l lea% e'tra$t ; " &l rea$tion &i'

!. M 6ris50CL bu%%er /(0 .21!.* &M as$orbi$ a$id

!.* &M 0<

Glutathione Reductase (GR)

8bsorban$e+ =! n&, o'idation o% N8>)0

Rea$tion Mi'ture+ *! :l lea% e'tra$t ; " &l rea$tion &i'!. M 6ris50CL bu%%er /(0 .21

= &M #>68

!. &M N8>)0

!.* &M o'idi?ed glutathione

Glutathione-S-rans!erase (GS)

8bsorban$e+ =! n&Rea$tion Mi'ture+ *! :l lea% e'tra$t ; " &l rea$tion &i'

!." M (otassiu& (hos(hate /(0 .*1

=. &M redu$ed glutathione" &M "-$hloro-,-dinitroben?ene

Peroxidase (POX)

8bsorban$e+ ! n&, %or&ation o% tetraguaia$olRea$tion Mi'ture+ *! :l lea% e'tra$t /*7 or "!7 dilution1 ; &l rea$tion &i'

*! &M sodiu& a$etate bu%%er /(0 1

* &M guaia$ol* &M 0<



Chia-Lin Chung

R. Nelson Lab

Cornell University

Enzyme Activities Assay by Chia-Lin Chung

". 4@ seedlings are ino$ulated by s(raying .* &l o% "!5&l s(ore sus(ension /!.!3

64een !1 on lea% sur%a$e

. or days a%ter ino$ulation, &ove the (lants %ro& G0 to the lab.=. Cut one in%e$ted lea% /ino$ulated area1 %ro& the (lant. A&&ediately 4eight and grind

!.* g o% lea% tissue 4ith liuid nitrogen. Use a s(atula to $olle$t all the ground

(o4der into a ".* &l or &l &i$rotube, add " &l e'tra$tion bu%%er, then vorte' %or a%e4 se$onds.

. Centri%uge at "2!!! r(& %or !&in at o C. /Move the $entri%uge into the $old roo&

%or at least overnight.1*. 6rans%er the su(ernatant to a ne4 &i$rotube, and @ee( the $rude e'tra$t on i$e.

8naly?e the $rude e'tra$t right a4ay. 6he a$tivity dro(s do4n i% the sa&(le is %ree?e-

tha4ed.

. >e(ends on the target en?y&e, (i(ette " &l rea$tion &i'ture into ".* &l &i$rotubes.

8dd "*! ul o% the $rude e'tra$t to the rea$tion &i'ture, vorte', and re$ord the start (oint o% ti&e.

. 8%ter $ertain a&ount o% ti&e /eg. ! &in %or GS6, * &in %or (ero'idase1, trans%er therea$tion sa&(les to the $uvettes. 6o (revent the o'idation in the air, $over the o(ening

o% $uvettes 4ith (ara%il&.

2. Use the s(e$tro&eter to read the absorban$e at the s(e$i%i$ 4avelength. Re$ord e'a$tti&e o% reading. 6i&e $ourse study is needed, be$ause

#n?y&e a$tivity D E/8 F 8"1 5 /6 F 6"1 5 &g (rotein D $hange o% absorban$e 5 (er

&g (rotein (er &in. />eter&ine (rotein $on$entration o% the sa&(les usingCo&&assie /Brad%ord1 )rotein 8ssay Hit.1

Pre"aration o! the Rea#ents

Enzyme Extraction:

Extraction $u!!er (!reshly "re"ared)

%& ml '&& ml && ml

*! &M sodiu& (hos(hate bu%%er

/(0 .*1

!. &l M &onobasi$

/Na0)< 0<I M

"=.99 1

." &l M dibasi$

/Na0)<, anhydrousM "".91

". &l M &onobasi$

/Na0)< 0<I M

"=.99 1

2. &l M dibasi$

/Na0)<, anhydrousM "".91

" &M (olyethylenegly$ol /M..

2!!!1

!. g !.2 g =. g

" &M (henyl&ethylsul%onyl

%luoride /M.. "."91

- !." M )MSK sto$@+ !.2 g

)MSK ; *! &l iso(ro(anol,

*!! ul !." M

)MSK sto$@

/add right

be%ore use1

" &l !." M )MSK

sto$@

/add right be%ore use1

&l !." M )MSK sto$@

/add right be%ore use1



Chia-Lin Chung

R. Nelson Lab

Cornell University

store at oC1

2 3 /45v1 (olyvinyl(yrolydone

/M.. !!!!1

g 2 g = g

!.!" 3 /v5v1 6riton 7-"!! *! :l "!! :l !! :l

Enzyme Assay:

Ascorbate Peroxidase (AsPOX)

8bsorban$e+ 9! n&, o'idation o% as$orbi$ a$id /de$rease o% absorban$e at 9! n&1

Rea$tion Mi'ture+ "! :l lea% e'tra$t ; " &l rea$tion &i'

Rea$tion Mi'%& ml '&& ml && ml

!. M 6ris50CL bu%%er /(0 .21 ! &l "M 6ris-

0Cl

2! &l "M 6ris-

0Cl

!.* &M as$orbi$ a$id /M.. "."1

- *! &M as$orbi$ a$id sto$@+ !.!22 g

as$orbi$ a$id ; "! &l 0<, store atoC, only %or " 4@1 /$an be

o'idi?ed in the air in " day1

*! ul *! &M

as$orbi$ a$id

sto$@

*!! ul *! &M

as$orbi$ a$id

sto$@

&l *! &M

as$orbi$ a$id

sto$@

!.* &M 0< /=!3, M.. =1 .2* :l *. :l

Glutathione Reductase (GR)

8bsorban$e+ =! n&, o'idation o% N8>)0

N8>)0 ; 0; ; GSSR→ GS0 ; N8>);I dete$t the o'idation o% N8>)0 /de$rease

o% absorban$e at =! n&1Rea$tion Mi'ture+ *! :l lea% e'tra$t ; " &l rea$tion &i'

Rea$tion Mi' %& ml '&& ml && ml

!. M 6ris50CL bu%%er /(0 .21 ! &l "M 6ris-

0Cl

2! &l "M 6ris-0Cl

= &M #>68 !. :l !.*M #>68 . :l !.*M #>68

!. &M N8>)0 /M.. 2==1

- 2. &M N8>)0 sto$@+!.!9 g N8>)0 ; . &l

0<, aliuot then store at -!oC1

"."9 &l 2. &M

N8>)0 sto$@

.=2 &l 2. &M

N8>)0 sto$@

!.* &M o'idi?ed glutathione

/M.. ".1

- *! &M GSSG sto$@+ !.=! go'idi?ed glutathione ; "! &l

0<, aliuot then store at -!oC1

!.* &l *! &M

GSSG sto$@

" &l *! &M

GSSG sto$@

Glutathione-S-rans!erase (GS)

8bsorban$e+ =! n&



Chia-Lin Chung

R. Nelson Lab

Cornell University

GS0 ; C>NB→ GS->NB $onugate ; 0Cl, dete$t the %or&ation o% GS->NB

$onugate /in$rease o% absorban$e at =! n&1Rea$tion Mi'ture+ *! :l lea% e'tra$t ; " &l rea$tion &i'

!." M (otassiu& (hos(hate /(0 .*1

=. &M redu$ed glutathione" &M "-$hloro-,-dinitroben?ene


!." M (otassiu& (hos(hate /(0

.*1

. &l M &onobasi$

/H0)<1

=.9* &l M dibasi$

/H 0)<1

.2 &l M &onobasi$

/H0)<1

"=."2 &l M dibasi$

/H 0)<1

=. &M redu$ed glutathione

/M.. =!.=1- =! &M sto$@+ "."! g redu$ed

glutathione ; "! &l 0<,

aliuot then store at -!o

C1

*!! ul =!

&M redu$edglutathione

sto$@

" &l =! &M

redu$ed glutathionesto$@

" &M "-$hloro-,-

dinitroben?ene /M.. !.**1

- "!! &M sto$@+ !.!* g C>NB; ! &l #t<0, store at -! oC1

*!! ul "!!

&M C>NB

sto$@

" &l "!! &M C>NB

sto$@

Peroxidase (POX) Guaiacol smells really bad* he +hole "rocess has to be

conducted , .E .OO/*

8bsorban$e+ ! n&, %or&ation o% tetraguaia$ol

guaia$ol ; 0< → tetraguaia$ol ; 2 0<%or&ation o% tetraguaia$ol /in$rease o% absorban$e at ! n&1

Rea$tion Mi'ture+ " :l lea% e'tra$t ; " &l rea$tion &i'

*! &M sodiu& a$etate bu%%er /(0 1* &M guaia$ol

* &M 0<


*! &M sodiu& a$etate bu%%er /(0 1 !." g sodiu& a$etate

/M.. 2.!=1

". g sodiu& a$etate

/M.. 2.!=1

* &M guaia$ol /M.. "."1 "** :l ="! :l

* &M 0< /=!3, M.. =1 ".* :l 2* :l

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EnzAssay CLC