A C T A U N I V E R S I T A T I S L O D Z I E N S I S _______________FOLIA BIOCHIMICA ET BIOPHYSICA 4, 1986 ________
Anna J e n i o r o u e k a , Wanda Leyko
CHARACTERIZATION OF HUMAN PLACENTAL SUPEROXIDE DISMUTASE (SOD-1) ACTIVITY AS MEASURED BY THE POLAROGRAPHIC METHOD*
Human p l a c e n t a l s u p e ro x id e d is m u ta s e (SOD-1 ) i s o l a t i o n p ro c e d u re and p r o p e r t i e s a r e r e p o r t e d . The i n f lu e n c e upon th e SOD-1 e n s y m a tic a c t i v i t y o f th e d e n a t u r a t i n g a g e n ts ( u r e a , SDS, i n c re a s e d te m p e r at u -r e , d i f f e -r e n t pH v a l u e « ) and i n h i b i t o -r s (a x id e -N -j, d i e t h y l d i t h i o c a r - bamate-DDC ) was d e te rm in e d by means o f th e p o la ro g r a p h i c method which p rov ed t o be th e moat s u i t a b l e f o r t h i s k in d o f i n v e s t i g a t i o n s .
INTRODUCTION
S u p e r o x i d e d i s m u t a s e s h av e b e e n i s o l a t e d from numerous mam-m a l i a n t i s s u e s . Human s u p e r o x i d e d i s m u t a s e ( E . C . 1 . 1 5 . 1 . 1 ) (SOD-1 ) was i s o l a t e d and s t u d i e d a s a c o p p e r p r o t e i n from e r y t h r o c y t e s , b r a i n and l i v e r 2 2 , 2 6 , 27. Bov ine Cu-Zn SOD-1 i s t h e most s t u -d i e -d p r o t e i n among t h i s l a r g e c l a s s o f enzymes w h i ch h av e s i m i -l a r p r o p e r t i e s w i t h r e s p e c t t o e a c h o t h e r . SOD-1 fro m c y t o s o l o f e u c a r y o t i c o r g a n is m s c o n t a i n s 2 ato m s o f c o p p e r p e r m o le c u l e o f n o l e c u l a r w e i g h t 32 000 d a l t o n s [ 1 2 ] . C opp er i s e s s e n t i a l f o r t h e a c t i v i t y o f t h e enzyme, w hich ca n be sum m ar ise d b y t h e e q u a -t i o n s [ 5] :
c u 2+ + o 2 - o 2 + c u + j c u + + o 2 - c u 2+ + h 2o 2
♦ T h i s work was p a r t l y r e p o r t e d a t th e I l l r d Brno Symposium on M o le c u la r B io p h y s ic s ! E l e c t r o a n a l y s i s o f B io p o ly m ers, Brno 1980.
Z in c seems t o h av e r a t h e r a s t r u c t u r a l r o l e [ 5 , 7, 8 , 1 0, 1 4 ]. The p r i m a r y s t r u c t u r e o f t h e human e r y t h r o c y t e s SOD-1 h a s b e en r e c e n t l y r e p o r t e d by B a r r a e t a l . , [ 2 ] , B r i g g a , F e e [ 6 ] an d H i l l e t a l . [ 2 0 ] . The human p l a c e n t a l SOD-1 h a s b e en i s o l a t e d and r o u g h l y c h a r a c t e r i s e d f o r t h e f i r s t t i m e by P h a n van H i e n e t a l . [ 4 1 ] . The p r o p e r t i e s o f t h e human enzyme a r e o b v i o u s l y i n t e r e s t i n g i n v iew o f p o t e n t i a l a p -p l i c a t i o n s t o m e d i c a l p r o b l e m s . T h i s p a p e r d e s c r i b e s some o f t h e p r o p e r t i e s o f t h e human p l a c e n t a l SOD-1 as w e l l a s t h e , e f f e c t o f some d e n a t u r a t i n g a g e n t s and i n h i b i t o r s upon i t s a c t i v i t y m easu-r e d by t h e p o l a r o g r a p h i c m et ho d .
MATERIAL AND METHODS
F r e s h p l a c e n t a e w ere p u r i f i e d fr om t h e c o n n e c t i v e and o t h e r a c c e s s o r y t i s s u e s and t h e b l o o d was t h r o u g h l y w ash ed o u t f i r s t w i t h t a p and t h e n w i t h d i s t i l l e d w a t e r . SOD-1 was p r e p a r e d a c -c o r d i n g t o t h e m ethod o f F r i e d e t a l . [ .1 3] . The sam ple o f human SOD-1 was s u b s e q u e n t l y c h r o m a t o g r a p h e d on C e l l u l o s e D E-32. F u r t h e r p u r i f i c a t i o n was p e r f o r m e d by g e l f i l t r a t i o n on B i o - g e l P - 6 0 ( P o l i s h p a t e n t P - 2 0 0 2 5 7 ) . The p u r i t y o f t h e enzyme w a s . t e s t e d by means o f a n a l y t i c a l 7.5ft p o l y a c r y l a m i d e g e l e l e c -t r o p h o r e s i s (PAGE) i n v e r o n a l b u f f e r pH 8 . 6 an d 3 . 5 mA p e r g e l . The SOD-1 a c t i v i t y on t h e g e l s was l o c a l i s e d by t h e p h o t o c h e m i
-c a l method (B e s u c h a m p an d F r i d o v i c h [5]).
P r o t e i n b and s w ere s t a i n e d w i t h C oo m assie B l u e . S u b u n i t m o le cu -l a r w e i g h t was d e t e r m i n e d by t h e m e t h o d ' o f W e b e r a nd O s -b o r n [36] . I s o e l e c t r i c f o c u s i n g was p e r f o r m e d a c c o r d i n g t o S v e n s s o n [31] . A b s o r p t i o n s p e c t r a w er e r e c o r d e d on a Beckman S p e c t r o p h o t o m e t e r . The SOD-1 a c t i v i t y was a s s a y e d by t h e p o l a r o g r a p h i c m ethod o f c a t a l y t i c c u r r e n t s a p p l i e d t o a wave o f o xy g en by R i g o e t a l . [ 2 8 ] on a R a d e l k i s 0H-102 p o l a r o - g r a p h .
RESULTS AND DISCUSSION
P u r i f i c a t i o n and p r o p e r t i e s o f t h e human SOD-1
The e x a m i n a t i o n o f t h e p u r i t y o f t h e enzyme p r e p a r a t i o n s by means o f PAGE r e v e a l e d t h a t t h e f i n a l p r e p a r a t i o n s o b t a i n e d from human p l a c e n t a w e re homogenous e l e c t r o p h o r e t i c a l l y . The homoge-n e i t y o f t h e p r e p a r a t i o n s was a l s o c o n f i r m e d by t h e m ethod o f i s o e l e c t r i c f o c u s i n g . The v a l u e o f t h e i s o e l e c t r i c p o i n t o f SOD-1 i s o l a t e d from human p l a c e n t a i s 4 . 7 and i t i s s i m i l a r t o t h e v a l u e s r e p o r t e d e l s e w h e r e f o r t h e human enzyme [ 7 , 1 6 ] . The me-t h o d o f F r i e d e t a l . [ l 3 ] a p p l i e d f o r t h e i s o l a t i o n o f SOD-1 from human p l a c e n t a w i t h s u b s e q u e n t c h r o m a t o g r a p h y and g e l f i l t r a t i o n p r o v e d t o be t h e p r o p e r method o f o b t a i n i n g a p u r e and homogenous p r e p a r a t i o n o f SOD-1. I n a l l c a s e s o f e l e c t r o -p h o r e t i c -p r o c e d u r e s a s i n g l e p r o t e i n band was s e e n w hich e x h i -b i t e d s p e c i f i c e n z y m a t i c a c t i v i t y o f 3 . 7 ± 0 . 1 1 • 10 M ( n = 6 ) . S u b u n i t m o l e c u l a r w e i g h t o f SOD-1 from human p l a c e n t a was d e t e r -m ined by SDS-PAGE. The s a m p le s o f SOD1 w er e s u b m i t t e d t o e l e c -t r o p h o r e s i s a f -t e r i n c u b a -t i o n w i t h d i f f e r e n t r e a g e n t s i n o r d e r t o e s t a b l i s h t h e d i s s o c i a t i o n c o n d i t i o n s o f t h e p r o t e i n m o l e c u l e i n t o s u b u n i t s .
T a b l e 1
The human p l a c e n t a l SOD-1 s u b u n it m o le c u la r w eig h t d e t e r m i n a t io n by means o f th e SDS PACE
O zn ac ze nie masy c z ą s tec z k o w ej p o d je d n o s te k SOD-1 z ło ż y s k a lu d z k ie g o m etodą e l e k t r o f o r e z y w ż e lu po liakryloam ido w y m * SDS
I n c u b a ti o n c o n d i t io n s 1 h , 45°C 12 SDS 2 m in , 100°C 1Z SDS 4 M u r e a 5 h , 37°C 1% SDS 6 M u r e a I t ( 2Z) 2- m e r- c a p t o e th a n o l M o le c u la r w e ig h t ( d a l t o n s ) MM +S.D. (n « 6 ) 64 000 +1 900 16 000 +1 800 16 000 +1 C00
5 ° _ Anna J e z io r o w s k a , Wanda T,eyko
T a b l e 1 su m m ar is e s t h e r e s u l t s o f t h e s u b u n i t w e i g h t e s t i m a -t i o n - f o r d i f f e r e n -t SOD-1 s a m p l e s . A c c o r d i n g t o t h e l i t e r a t u r e d a t a t h e mammalian c y t o s o l i c d i s m u t a s e s a r e composed o f two i d e n -t i c a l s u b u n i -t s , e a c h o f t h e m o l e c u l a r w e i g h t 16 OOO [ 3 , 9 , 1 7 ] , The p r o t e i n b an d s o f a b o u t 16 0 00 d a l t o n s o b t a i n e d a f t e r t h e s i -m u l ta n e o u s a c t i o n o f SDS, u r e a , 2 - m e r c a p t o e t h a n o l and h e a t t o t h e enzyme a r e i d e n t i f i e d a s s u b u n i t s . S i m i l a r r e s u l t s w er e r e p o r t e d f o r t n e b o v i n e [ l , 3 8 ] an d ho g enzyme [ 4 ] , a s w e l l a s f o r t h e p l a c e n t a l SOD-1 o b t a i n e d an d p u r i f i e d by S u g i u r a e t a l . [ 3 o ]. 2 - m e r c a p t o e t h a n o l seems n e c e s s a r y f o r t h e c o m p l e t e d i s s o -c i a t i o n o f t h e SOD-1 m o l e c u l e i n t o s u b u n i t s . I t a c t s p r o b a b l y on d i s u l f i d e b r i d g e s l o c a l i s e d w i t h i n e a c h s u b u n i t , r e s u l t i n g i n con-f o r m a t i o n a l c h a n g e s o con-f t h e p r o t e i n m o l e c u l e and a t o t a l b r e a k -d o w n i n t o s u b u n i t s , w h ic h a r e l i n k e d by s t r o n g n o n - c o v a l e n t i n -t e r a c -t i o n s . High m o l e c u l a r f r a c t i o n s (m.w. a b o u t 64 OOO) o b t a i n -ed i n t h e e x p e r i m e n t s a r e p r o b a b l y t h e r e s u l t o f t h e f o r m a t i o n o f a g g r e g a t e d SOD-1 m o l e c u l e s , du e t o i n t e r a c t i o n s b e tw e e n whole enzyme m o l e c u l e s i n t h e p r e s e n c e o f SDS. T h i s k i n d o f a g g r e g a t e s o f t h e human enzyme m o l e c u l e s h a v e b ee n a l r e a d y r e p o r t e d [ 1 7 ] . F i g . 1
U l t r a v i o l e t and v i s i b l e a b s o r p t io n s p e c t r a o f SOD-1 from human p l a c e n t a . 0 .5 mg/ml SOD-1 i n 50 bM p h o sp h a te b u f f e r (pH 7 . 4 ) was exam ined i n 1 cm c e l l i n
a Beckman s p e c tr o p h o to m e te r
Widmo S0D-1 z ło ż y s k a lu d z k ie g o w z a k r e s i e u l t r a f i o l e t u i w id zia ln y m . Widmo u - zy sk an o d l a p r ó b k i 0 , 5 mg/ml SOD-1 w 50 nM b u f o rz e fosforanow ym ( pH 7 , 4 ) w
The u l t r a v i o l e t and v i s i b l e r a n g e a b s o r p t i o n s p e c t r a o f t h e human p l a c e n t a l SOD-1 a r e shown i n F i g . 1. The a b s o r p t i o n p r o -f i l e s a r e s i m i l a r t o t h o s e r e p o r t e d -f o r SOD-1 fro m o t h e r human t i s s u e s [ 7 , 1 6, 30, 4 l ] .
E f f e c t o f d e n a t u r a t l n g a g e n t s and i n h i b i t o r s
The r e s u l t s o f t h e i n f l u e n c e o f h i g h c o n c e n t r a t i o n s o f u r e a ( 6 M and 10 M) and t h e a c t i o n o f 1% and 4% SDS upon t h e enzyma-t i c a c enzyma-t i v i enzyma-t y o f SOD-1 a r e shown i n T a b l e 2.
T a b l e 2
The i n f l u e n c e o f 8 M and 10 M u re a and 12 and 42 SDS upon th e human p l a c e n t a l SOD-1 a c t i v i t y and d i s s o c i a t i o n
o f i t s m o lec u le i n t o s u b u n i ts
Wpływ 8 M i 10 M mocznika o r a z IX i 42 SDS na akty w no ść SOD-1 z ło ż y s k a lu d z k ie g o i d y s o c j a c j ą c z ą s te c z k i enzymu na p o d je d n o s tk i N a t iv e SOD-1 a c t i v i t y SOD-1 a c t i v i t y a f t e r in c u b a t io n 1 . 4 • 10" 6 M w it h 8 M u rea w it h 10 M u r ea 1 . 0 6 • 10‘ 6 M 0 . 9 2 • 10"6 M ( 7 5 2 ) (65%) w it h 1% SDS w it h 42 SDS 1 . 4 • 10- 6 M 0 . 8 4 • 10“ 6 M ( 1 0 0 2 ) ( 6 0 2 ) ' The l o s s o f a c t i v i t y a f t e r t h e t r e a t m e n t w i t h 8 M and 10 M u r e a , 25% an d 35% r e s p e c t i v e l y was i m m e d i a t e l y r e v e r s e d a f t e r d i l u t i n g o f t h e i n c u b a t e d sam pl e w i t h p h o s p h a t e b u f f e r pH 7 . 4 . The i n a c t i v a t i o n c a u s e d by t h e a c t i o n o f 4% SDS was a l s o r e v e r -s i b l e . A f t e r p r e c i p i t a t i o n w i t h c o n c e n t r a t e d KC1 an d c e n t r i f u g a -t i o n a f -t e r 48 h i n room t e n p e r a t u r e t h e enzyme g a i n e d i t s 100% a c t i v i t y . I n b o t h c a s e s , t h e SDS PAGE o f t h e i n c u b a t e d sa m p l e s r e v e a l e d two p r o t e i n b a n d s o f m o l e c u l a r w e i g h l a r w e i g h t s 64 OOO and 16 OOO w i t h t h e p r o p o r t i o n o f 75% and 25% r e s p e c t i v e l y .
T e s t s o f SOD-1 s t a b i l i t y w e re c a r r i e d o u t a c c o r d i n g t o t h e
-F i g . 2
T em perature s t a b i l i t y o f human p la c e n t a l SOD-1 a t d i f f e r e n t pH v a lu e s o f th e in c u b a t io n m ed ia. A liq u o t s w ere w ithdraw n a t i n t e r v a ls and a ss a y e d f o r th e
re m a in in g a c t i v i t y
T e s t s t a b i l n o ś c i te m p era tu ro w ej SOD-1 z ło ż y s k a lu d z k ie g o w ró żn y c h w a r to ś c i a c h pH. P ró b k i p o b ie ra n o w różn ych p r z e d z i a ła c h czasowych i ozn acza no p o z o s t a ł a
aktyw n ość enzym atyczny
[ 3 4 , 3 7 ] . At pH 7 . 4 SOD was m ost s t a b l e - 50% i n a c t i v a t i o n was o b s e r v e d a f t e r 50 min i n c u b a t i o n a t 70°C. T o t a l l o s s o f a c t i v i -t y was fo u nd a -t -t h e same pH v a l u e a f -t e r 30 min i n c u b a t i o n a t 90°C. However, a t pH l o t h e enzyme r e t a i n s 50% o f t h e n a t i v e a c t i v i t y f l u e n c e o f t h e i n c r e a s e d t e m p e r a t u r e and d i f f e r e n t pH v a l u e s upon human p l a c e n t a l SOD-1 a c t i v i t y a r e g i v e n i n F i g . 2 . The a c t i v i
-t y o f -t h e enzyme a f -t e r i n c u b a -t i o n was m e asur ed p o l a r o g r a p h i c a l l y an d c a l c u l a t e d a s a p e r c e n t a g e o f t h e i n i t i a l a c t i v i t y o f t h e i n -t a c -t enzym e. S u p e r o x i d e d i s m u -t a s e s a r e e x t r e m e l y s t a b l e enzymes
7.4
a o
10
a f t e r 60 min i n c u b a t i o n a t 70°C, b u t a t t h e low pH v a l u e s i t i s r e a d i l y i n a c t i v a t e d . The a c t i v i t y was fo und u n a f f e c t e d by b u f -f e r i n g t h e p r o t e i n f o r 12 h w i t h i n t h e r a n g e o f pH 4 . 8 - 1 0 . \ The e n z y m a t i c a c t i v i t y d e c r e a s e s s i g n i f i c a n t l y w i t h t h e i n c r e a s e d v a l u e s o f pH abo ve 10. A lso a f t e r i n c u b a t i o n o f t h e p r o t e i n a t pH 3 t h e a c t i v i t y was r e d u c e d by 40%. The r e s u l t s ab ov e i n d i c a t e t h a t t h e human p l a c e n t a l S0D-1 i s e s s e n t i a l l y i n d e p e n d e n t o f pH o v e r a w ide r a n g e . T h i s p r o p e r t y h a s b ee n e s t a b l i s h e d b e f o r e f o r t h e b o v i n e [ 3 3 ] , hog [4] an d hum- an p l a c e n t a l SOD-1 [ 3 0 ] . The o b s e r v a t i o n s p e r f o r m e d by V a l e n -t i n e e t a l . [4 0] o f t h e pH d e p e n d e n c e o f t h e v i s i b l e s p e c -t r a an d EPR s p e c -t r a i n d i c a -t e d t h a t t h e pH d e p e n d e n t c h a n g e s b e t -ween 3 and 4 . 5 c o u l d be a c c o u n t e d f o r by c o n f o r m a t i o n a l c h a n g e s a t t h e c o p p e r b i n d i n g s i t e . I t i s c l e a r f rom t h e r e s u l t s ab ove and p r e v i o u s s t u d i e s t h a t t h e m e t a l b i n d i n g p r o p e r t i e s o f c o p p e r - z i n c SOD-1 a r e s t r o n g l y pH d e p e n d e n t . I n t h e s t u d i e s o f t h e i n h i b i t o r s a c t i o n upon t h e human p l a -c e n t a l a -c t i v i t y two d i f f e r e n t i n h i b i t o r s w ere c h o s e n i n t h e p r e -s e n t w o rk . Th ese a r e s a z i d e (N^) and d i e t h y l d i t h i o c a r b a m a t e (DDC). The p o l a r o g r a p h i c method o f t h e SOD a c t i v i t y a s s a y a p -p e a r e d e s -p e c i a l l y s u i t a b l e i n t h i s k i n d o f d e t e r m i n a t i o n s , b e -c a u s e i t was n o t a f f e c t e d by n o r by DDC, i n c o n t r a r y t o t h e s p e c t r o p h o t o m e t r i c a s s a y s y st e m s [ 1 5 , 3 5 ] . I t h a s b e en r e c e n t l y r e p o r t e d t h a t a z i d e [ 2 9 ] co m b ines w i t h t h e p r o s t h e t i c m e t a l o f t h e i r o n c o n t a i n i n g SOD o f E. c o l i a s w e l l a s w i t h t h e Cu-Zn SOD-1 and Mn-SOD from human l i v e r [ 2 3 ] . The i r o n SOD i s most s e n s i t i v e t o w a r d i n h i b i t i o n by a z i d e . The Cu-Zn SOD-1 i s l e a s t s e n s i t i v e w h i l e t h e Mn-SOD e x h i b i t s m o d er a t e s u s c e p t i b i l i t y . Thus i n h i b i t i o n by a z i d e , w h ich i s i m p a i r e d an d r e v e r s e d r a p i d l y , a p -p e a r s t o p r o v i d e t h e u s e f u l c r i t e r i o n f o r d i s t i n g u i s h i n g among c l a s s e s o f t h e s e enz ym es.
I n t h i s work 50% i n h i b i t i o n o f t h e SOD-1 a c t i v i t y was o b -t a i n e d a -t -t h e c o n c e n t r a t i o n e q u a l 30 mM o f . The am ount o f 10 mM o f a z i d e i n h i b i t e d t h e enzyme by 10% ( P i g . 3 ) . R e v e r s i b i l i t y o f t h e SOD-1 i n h i b i t i o n - w a s g a i n e d by d i a l y s i s a g a i n s t p h o s p h a -t e b u f f e r pH 7 . 4 . The c o p p e r c h e l a t i n g a g e n t d i e t h y l d i t h i o c a r b a -m ate (DDC) h a s b ee n shown t o c a u s e m arked i n a c t i v a t i o n o f SOD-1 b o t h i n v i t r o an d i n v i v o [ 1 8 , 1 9 , 2 4 ] , I n t h e p r e s e n t work t o
-F ig . 4
I n h i b i t i o n o f human p l a c e n t a l SOD-1 a c t i v i t y by d i e th y l d it h i o c a r b a m a t e (DDC): A - as a f u n c t i o n o f th e DDC c o n c e n t r a t i o n ; B as a f u n c t i o n o f th e in c u b a
-t i o n -tim e w i-t h 3 nM of DDC
Hamowanie ak ty w n o ś ci SOD-1 z ło ż y s k a lu d z k ie g o p rz e z d w u e ty lo d w u tio k arb a m i- n ia n (DDC): A - ja k o f u n k c ja s t ę ż e n i a DDC; B - ja k o fu n k c ja c z a s u in k u b a c ji
z 3 ®M DDC F i g . 3 OD-1 ac t c e n t r a t j
Hamowanie ak ty w n o ści SOD-1 * ło ż y sk a lu d z k ie g o p r z e z azydek ( N j ) o ozn acz o-ny ch s t ę ż e n ia c h
I n h i b i t i o n o f th e human p l a c e n t a l SOD-1 a c t i v i t y by a z id e ( N j ) a t th e in d ic a t e d c o n c e n t r a ti o n s
t a l i n h i b i t i o n o f t h e human p l a c e n t a l s u p e r o x i d e d i a m u t a s a was o b s e r v e d a t t h e c o n c e n t r a t i o n o f 3 mM o f DDC and a f t e r 60 min o f i n c u b a t i o n ( F i g . 4 ) . The l o s s o f e n z y m a ti c a c t i v i t y was accompa-n i e d by t h e a p p e a r a accompa-n c e o f a c o l o u r e d ( y e l l o w t o o r a accompa-n g e ) enzym e- - i n h i b i t o r c o m p le x . As i t h as b een o b s e r v e d e a r l i e r [1 9 ] t h e loss i n e n z y m a t i c a c t i v i t y c o u l d n o t be r e v e r s e d by d i a l y s i s a g a i n s t d i s t i l l e d w a t e r ( 5 0 0 v o l ) i n 4°C f o r 24 h , b u t a f t e r d i a l y s i s , -4 t h e n a t i v e a c t i v i t y was r e s t o r e d by i n c u b a t i o n w i t h 5 . 10 M CuSO^ d u r i n g 1 h a t 37°C.
DDC by i n h i b i t i n g t h e Cu c o n t a i n i n g for m o f SOD may be a u s e -f u l p h a r m a c o l o g i c a l t o o l i n e s t a b l i s h i n g t h e v i t a l p r o t e c t i v e r o le o f t h i s enzyme. I t may be u sed i n o r d e r t o e v a l u a t e t h e im p o r -t a n c e o f SOD a s a p r o t e c t i v e enzyme i n e u c a r y o t e s d u r i n g e x p o -s u r e t o h i g h c o n t e n t o f 0 2 [ l i ] . DDC may a l s o be a p o t e n t i a l l y p o w e r f u l s e n s i t i z i n g a g e n t i n tum o r t h e r a p y , s i n c e t h e r e i s i n -c r e a s i n g e v i d e n -c e t h a t tum or c e l l s h av e l o w e r l e v e l s o f SOD [ 2 1 , 25, 32, 3 9 ] . REFERENCES [ 1 ] A b e r n a t h y J . L . , S t c i n m a n H . li . , H i l l R. L . , 1974, J . B i o l . C h e « ., 24 9, 7339-7342. [ 2 ] B a r r a D. , M a r t i n i F . , B o 8 s a F. , R o t i 1 i o G. , B a n n i s t e r J . V. # B a n n i s t e r W. H ., 1978, B iochim . B io ph ys. R es. Commun., JB_1, 1195-1201.
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Anna J e z io r o w s k a , Wanda L eyk o
CHARAKTERYSTYKA AKTYWNOŚCI DYSMUTAZY PONADTLENKOWEJ (S0D-1 ) Z ŁOŻYSKA LUDZKIEGO MF.TOD4 POLAROGRAFICZNĄ
P rz ed sta w io n o spo só b iz o lo w a n ia i o c z y s z c z a n ia dysm utazy p o n ad tlen k o w ej EC 1 .1 5 .1 . 1 ( SOD-1 ) z ło ż y s k a l u d z k ie g o . Badano wpływ czynników d e n a t u r u ją
-cych b i a ł k o (m o c z n ik a , SDS, podw yższonej te m p e r a tu ry , zmian pH) o r a z i n -h i b i to r ó w (a z y d k u , dw uety lod w utio karbam inianu -D D C ) s t o s u j ą c do o z n a c z a n ia zm ian a kty w n o śc i enzymu metodą p o l a r o g r a f i c z n ą , k t ó r a o k a z a ła s i ą byó n a j -b a r d z i e j o d p ow ied nią do te g o ty p u -b a d a ó .
