A Novel FluorimetricAssay for the Detection of TACE (α ...
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l e v o N A l e v o N A c i r t e m i r o u l F c i r t e m i r o u l F ( E C A T f o n o i t c e t e D e h t r o f y a s s A ( E C A T f o n o i t c e t e D e h t r o f y a s s A α α - - e s a t e r c e S e s a t e r c e S ) ) e l e v a W g n o L a g n i s U y t i v i t c A e t a r t s b u S e d i t p e P T E R F h t g n e l e v a W g n o L a g n i s U y t i v i t c A e t a r t s b u S e d i t p e P T E R F h t g n a r e V a r e V a v o n a m h k a R a v o n a m h k a R , , g n o d u X g n o d u X , u h Z , u h Z g n i y g n e F g n i y g n e F , i L , i L n e f o a i X n e f o a i X g n o h Z g n o h Z d n a g n o H a t i n A , d n a g n o H a t i n A , e h o a i X e h o a i X g n o T g n o T O 9 4 1 2 , . c n I c e p S a n A O 9 4 1 2 , . c n I c e p S a n A ’ ’ A S U , 1 3 1 5 9 A C , e s o J n a S , e v A e l o o T A S U , 1 3 1 5 9 A C , e s o J n a S , e v A e l o o T n o i t c u d o r t n I - F N T ( E C A T α r o 7 1 M A D A d e l l a c o s l a , ) e m y z n e g n i t r e v n o c α- h g u o r h t n o i t a z i l i t r e f d n a , s i s e n e g o r u e n , s i s e n e g o y m n i d e v l o v n i s i , e s a t e r c e s c a f r u s l l e c f o g n i d d e h s f o s s e c o r p e h t t n a n i m o d e r p e h t s i E C A T . s n i e t o r p e . F N T e r u t a m e l b u l o s f o n o i t a r e n e g e h t r o f e l b i s n o p s e r ' e s a d d e h s ' 1 f o t n e m p o l e v e d d n a h c r a e s e r e h t r o f e d a m n e e b e v a h s t r o f f e e l b a r e d i s n o C - F N T - i t n a α n i s e s n o p s e r y r o t a m m a l f n i f o y t i r e v e s e h t e c u d e r o t s t n e g a y a m t n e g a l a c i g o l o c a m r a h p a y b E C A T f o n o i t i b i h n i e h T . s e t a t s e s a e s i d - F N T f o t c e f f e e h t e t a l u d o m o t h c a o r p p a e v i t a n r e t l a n a t n e s e r p e r α . 2 e w , s r o t i b i h n i E C A T f o g n i n e e r c s t u p h g u o r h t h g i h e t a t i l i c a f o T L X Q g n i s u E C A T r o f e t a r t s b u s e d i t p e p l e v o n a d e z i s e h t n y s M T M A F - 5 / 0 2 5 e h t – t i k w e n a d e p o l e v e d e w , e t a r t s b u s T E R F s i h t g n i s U . r i a p T E R F e h t t c e t e d o t d e s u e b n a c t i k s i h T . y a s s A y t i v i t c A E C A T 0 2 5 ™ e t y L o s n e S y l h g i h s i t I . s r o t i b i h n i E C A T f o g n i n e e r c s r o f d n a e m y z n e e h t f o y t i v i t c a . e m y z n e f o s t n u o m a m a r g o n a n b u s t c e t e d n a c d n a e v i t i s n e s s n o i s u l c n o C h c i h w , t i k y a s s a E C A T 0 2 5 ™ e t y L o s n e S e h t – t i k y a s s a E C A T w e n a d e p o l e v e d e v a h e W L X Q e v i t i s n e s y l h g i h a s e s u M T . e t a r t s b u s T E R F M A F - 5 / 0 2 5 s h t g n e l e v a w n o i s s i m e d n a n o i t a t i c x e r e g n o l e h T m o r f e c n e r e f r e t n i e h t e z i m i n i m M A F - 5 f o . s t n e n o p m o c l l e c d n a s d n u o p m o c t s e t f o e c n a b r o s b a d n a e c n e c s e r o u l f o t u a f o g n i r o t i n o m s u o e n e g o m o h , s u o u n i t n o c f o e l b a p a c s i t i k y a s s a E C A T 0 2 5 ™ e t y L o s n e S s i h T . n o i t c a e r c i t a m y z n e e h t C I 0 5 t n e t s i s n o c e r a t i k y a s s a E C A T 0 2 5 ™ e t y L o s n e S h t i w d e n i m r e t e d r o t i b i h n i n a r o f e u l a v . a t a d d e h s i l b u p h t i w : s e c n e r e f e R . ) 7 9 9 1 ( 3 3 7 , 5 8 3 e r u t a N . l a t e . L M , s s o M . 1 . ) 3 0 0 2 ( 9 9 7 2 , 3 1 . t t e L . m e h C . d e M . r o o i B . l a t e . I J , n i v e L . 2 s d o h t e M d n a s l a i r e t a M ) 5 8 0 2 7 # t a C ( t i K y a s s A y t i v i t c A E C A T 0 2 5 ™ e t y L o s n e S L X Q ( E C A T M T d n a d e n g i s e d - e t a r t s b u S T E R F ) M A F - 5 ( - e d i t p e p - ) 0 2 5 . d o h t e m s i s e h t n y s e s a h p d i l o s c o m F y b d e z i s e h t n y s r e f f u B n o i t c a e R E C A T n o i t a r o p r o C h c r a e s e R f o t c u d o r p d e t n e t a p a s i 0 - I P A T ( r o t i b i h n I E C A T ) s e i g o l o n h c e T ) A C , o g e i D n a S , m e h c o i b l a C ( E C A T n a m u H t n a n i b m o c e R H N - a p D - R S S S R V A Q A L - z b A ( a p D / z b A E C A T 2 - a c M ( a p D / a c M d n a ) H N - R A - a p D - L G L P K 2 d i l o s c o m F y b d e z i s e h t n y s - s e t a r t s b u s T E R F ) . ] ) p n D ( p a D = a p D [ d o h t e m s i s e h t n y s e s a h p e h t y b d e d n e m m o c e r s a d e s u s a w t i K y a s s A E C A T 0 2 5 ™ e t y L o s n e S 0 4 e r a t i k s i h t r o f s e m u l o v n o i t c a e r e h T . l o c o t o r p µ 0 1 , e m y z n e f o l µ f o l 0 5 , r e f f u b / d n u o p m o c t s e t µ d e m r o f r e p e r e w s n o i t a b u c n i l l A . e t a r t s b u s f o l 7 3 t a o . s e t a l p k c a l b l l e w - 6 9 n i d e r u g i f n o c e r e w s y a s s A . C 0 2 5 ™ e t y L o s n e S f o y t i v i t i s n e S . 4 e r u g i F . t i K y a s s A E C A T d e r u s a e m s a w e c n e c s e r o u l F h t i w e t a r t s b u s T E R F f o n o i t a b u c n i r e t f a n i m 0 6 t a y a s s a f o y t i v i t i s n e S . E C A T f o s n o i t u l i d l a i r e s . e m y z n e f o g n 1 3 . 0 s a w s n o i t i d n o c e s e h t . t o l p n e t n e M - s i l e a h c i M . 2 e r u g i F L X Q e h t r o F M T E C A T M A F - 5 / 0 2 5 s e i t i c o l e v l a i t i n i , e t a r t s b u s T E R F e r e w e m y z n e y b s i s y l o r d y h f o ) o V ( g n 0 1 f o n o i t a b u c n i r e t f a d e n i m r e t e d f o e g n a r a h t i w E C A T e h T . s e t a r t s b u s f o s n o i t a r t n e c n o c y b d e z y l a n a e r e w a t a d g n i t l u s e r K e h T . n o i s s e r g e r r a e n i l - n o n m e u l a v 2 3 s a w d e n i a t b o µ . M o i t a r d n u o r g k c a b / l a n g i s f o n o s i r a p m o C . 5 e r u g i F . s e t a r t s b u s E C A T t n e r e f f i d r o f s e t a r t s b u s E C A T L X Q d n a a p D / a c M , a p D / z b A ( M T a t a ) M A F - 5 / 0 2 5 5 f o n o i t a r t n e c n o c l a n i f µ 0 1 h t i w d e t a b u c n i e r e w M r o f o i t a r d n u o r g k c a b / l a n g i S . r u o h 1 r o f e m y z n e f o g n L X Q M T s a d e t n e s e r p s i e t a r t s b u s d e l e b a l M A F - 5 / 0 2 5 . % 0 0 1 e l p i c n i r P y a s s A T E R F n o d e s a b s i y a s s A y t i v i t c A E C A T 0 2 5 ™ e t y L o s n e S e h T d n a M A F - 5 . e l p i c n i r p ) r e f s n a r t y g r e n e e c n a n o s e r r e t s r ö F r o e c n e c s e r o u l F ( n o i t p r o s b a e h T . s e d i t p e p T E R F r o f r i a p r o t p e c c a - r o n o d l e v o n a s i 0 2 5 ™ L X Q s p a l r e v o y l t c e f r e p 0 2 5 ™ L X Q f o m u r t c e p s - 5 f o m u r t c e p s n o i s s i m e e h t h t i w i b o h p o r d y h e h t e k i l n U . ) 1 e r u g i F ( M A F c i l i h p o r d y h a s i 0 2 5 ™ L X Q , p n D c e d i t p e p e h t f o y t i l i b u l o s e h t s e s a e r c n i 0 2 5 ™ L X Q f o y t r e p o r p s i h T . d n u o p m o c y b d e s u a c m e l b o r p e h t s u h t ; e t a r t s b u s y n a m f o e r u t a n c i b o h p o r d y h e h t . d e t a i v e l l a s i s r e h c n e u q d n a s r o n o d t n e c s e r o u l f . 1 e r u g i F L X Q f o e g a v a e l c c i t y l o e t o r P M T 0 2 5 ™ L X Q . E C A T y b e d i t p e p T E R F M A F - 5 / 0 2 5 e t a r a p e s o w t o t n i e g a v a e l c n o p U . M A F - 5 h t i w d e r i a p n e h w r e h c n e u q t n e l l e c x e n a s i t a d e r o t i n o m e b n a c d n a d e r e v o c e r s i M A F - 5 f o e c n e c s e r o u l f e h t , e s a e t o r p y b s t n e m g a r f . m n 0 2 5 / m n 0 9 4 = m E / x E s e i d u t s r o t i b i h n I . 6 e r u g i F r o f y a s s a e t a d i l a v o T . L X Q e h t , g n i n e e r c s r o t i b i h n i M T M A F - 5 / 0 2 5 n i e m y z n e f o g n 0 1 h t i w d e t a b u c n i s a w e t a r t s b u s 0 1 ( 0 - I P A T r o t i b i h n i E C A T f o e c n e s e r p e h t 2 - o t 0 1 3 n i m 5 y r e v e n e k a t e r e w s g n i d a e r c i t e n i K . ) M n C I d e t a l u c l a c e h T . n i m 0 6 r o f 0 5 . M n 9 1 s a w s t l u s e R s t l u s e R . s c i t e n i k y a s s A . 3 e r u g i F L X Q M T s a w e t a r t s b u s M A F - 5 / 0 2 5 . E C A T f o g n 0 1 h t i w d e t a b u c n i s a w l a n g i s t n e c s e r o u l F = m E / x E t a d e r o t i n o m y l s u o u n i t n o c . n i m 0 6 r o f m n 0 2 5 / m n 0 9 4 0 0 0 0 5 0 0 0 0 1 0 0 0 5 1 0 0 0 0 2 0 0 0 5 2 0 8 0 6 0 4 0 2 0 n i m , e m i T RFU d n u o r g k c a b e m y z n e 0 0 2 0 4 0 6 0 8 0 0 1 S/B, % M A F - 5 a c M z b A 0 0 0 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 0 1 0 0 0 2 1 0 0 0 4 1 RFU 0 1 5 5 . 2 5 2 . 1 5 2 6 . 0 3 1 3 . 0 6 5 1 . 0 l l e w / g n , e m y z n E 0 0 5 0 0 1 0 5 1 0 0 2 0 5 0 1 5 1 0 2 5 2 , S µM V 0 1 0 0 . 0 1 0 . 0 1 . 0 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 2 0 4 0 6 0 8 0 0 1 0 2 1 M n , 0 - I P A T Rel. Activity, % 5-FAM FRET Peptide TACE Cleavage 5-FAM QXL 520 TM QXL 520 TM
Transcript of A Novel FluorimetricAssay for the Detection of TACE (α ...
levoN A levoN A cirtemiroulF cirtemiroulF ( ECAT fo noitceteD eht rof yassA ( ECAT fo noitceteD eht rof yassA αααα-- esaterceS esaterceS ))elevaW gnoL a gnisU ytivitcA etartsbuS editpeP TERF htgnelevaW gnoL a gnisU ytivitcA etartsbuS editpeP TERF htgn
areV areV avonamhkaR avonamhkaR ,, gnoduX gnoduX ,uhZ ,uhZ gniygneF gniygneF ,iL ,iL nefoaiX nefoaiX gnohZ gnohZ dna gnoH atinA , dna gnoH atinA , ehoaiX ehoaiX gnoT gnoTO 9412 ,.cnI cepSanA O 9412 ,.cnI cepSanA ’’ ASU ,13159 AC ,esoJ naS ,evA elooT ASU ,13159 AC ,esoJ naS ,evA elooT
noitcudortnI-FNT( ECAT α ro 71MADA dellac osla ,)emyzne gnitrevnoc α-
hguorht noitazilitref dna ,sisenegoruen ,sisenegoym ni devlovni si ,esatercescafrus llec fo gniddehs fo ssecorp eht tnanimoderp eht si ECAT .snietorp e
.FNT erutam elbulos fo noitareneg eht rof elbisnopser 'esaddehs' 1
fo tnempoleved dna hcraeser eht rof edam neeb evah stroffe elbaredisnoC-FNT-itna α ni sesnopser yrotammalfni fo ytireves eht ecuder ot stnega
yam tnega lacigolocamrahp a yb ECAT fo noitibihni ehT .setats esaesid-FNT fo tceffe eht etaludom ot hcaorppa evitanretla na tneserper α .2
ew ,srotibihni ECAT fo gnineercs tuphguorht hgih etatilicaf oTLXQ gnisu ECAT rof etartsbus editpep levon a dezisehtnys MT MAF-5/025
eht– tik wen a depoleved ew ,etartsbus TERF siht gnisU .riap TERF eht tceted ot desu eb nac tik sihT .yassA ytivitcA ECAT 025™etyLosneS ylhgih si tI .srotibihni ECAT fo gnineercs rof dna emyzne eht fo ytivitca
.emyzne fo stnuomamargonanbus tceted nac dna evitisnes
snoisulcnoC hcihw ,tik yassa ECAT 025™etyLosneS eht– tik yassa ECAT wen a depoleved evah eW
LXQ evitisnes ylhgih asesu MT .etartsbus TERF MAF-5/025
shtgnelevaw noissime dna noitaticxe regnol ehT morf ecnerefretni eht eziminim MAF-5 fo .stnenopmoc llec dna sdnuopmoc tset fo ecnabrosba dna ecnecseroulfotua
fo gnirotinom suoenegomoh ,suounitnoc fo elbapac si tik yassa ECAT 025™etyLosneS sihT.noitcaer citamyzne eht
CI 05 tnetsisnoceratik yassa ECAT 025™etyLosneS htiw denimreted rotibihni na rof eulav.atad dehsilbup htiw
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.)3002( 9972 ,31 .tteL .mehC .deM .rooiB .la te .IJ ,niveL .2
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LXQ( ECAT MT dna dengised- etartsbuS TERF )MAF-5(-editpep-)025.dohtem sisehtnys esahp diloscomF yb dezisehtnys
reffuB noitcaeR ECAT noitaroproC hcraeseR fo tcudorp detnetap a si 0-IPAT( rotibihnI ECAT
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HN-apD-RSSSRVAQAL-zbA(apD/zbA ECAT 2 -acM(apD/acM dna )HN-RA-apD-LGLPK 2 diloscomF yb dezisehtnys- setartsbus TERF )
.])pnD(paD=apD[ dohtem sisehtnys esahp eht yb dednemmocer sa desu saw tiK yassA ECAT 025™etyLosneS
04 era tik siht rof semulov noitcaer ehT .locotorp µ 01 ,emyzne fo l µ fo l 05 ,reffub/dnuopmoc tset µ demrofrep erew snoitabucni llA .etartsbus fo l
73 ta o .setalp kcalb llew-69 ni derugifnoc erew syassA .C
025™etyLosneS fo ytivitisneS.4 erugiF.tiK yassA ECAT derusaem saw ecnecseroulF
htiw etartsbus TERF fo noitabucni retfa nim 06 ta yassa fo ytivitisneS .ECAT fo snoitulid laires
.emyzne fo gn 13.0 saw snoitidnoc eseht
.tolp netneM-sileahciM .2 erugiFLXQ eht roF MT ECAT MAF-5/025
seiticolev laitini ,etartsbus TERF erew emyzne yb sisylordyh fo )oV( gn 01 fo noitabucni retfa denimreted fo egnar a htiw ECAT ehT.setartsbus fo snoitartnecnoc yb dezylana erew atad gnitluser
K ehT .noisserger raenil-non m eulav 23 saw deniatbo µ .M
oitar dnuorgkcab/langis fo nosirapmoC.5 erugiF .setartsbus ECAT tnereffid rof setartsbus ECAT
LXQ dna apD/acM ,apD/zbA( MT a ta )MAF-5/025 5 fo noitartnecnoc lanif µ 01 htiw detabucni erew M
rof oitar dnuorgkcab/langiS .ruoh 1 rof emyzne fo gnLXQ MT sa detneserp si etartsbus delebal MAF-5/025
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elpicnirP yassA TERF no desab si yassA ytivitcA ECAT 025™etyLosneS ehT dna MAF-5 .elpicnirp )refsnart ygrene ecnanoserretsröF ro ecnecseroulF( noitprosba ehT .seditpep TERF rof riap rotpecca- ronod levon a si 025™LXQ
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