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1/6

[ 3] CATALASEm Vitro 2

H i g h c o n c e n t r a t i o n s o f p o l y v a l e n t a n io n s , w h i c h m a y b e c o n t a i n e d i n

t e s t s a m p l e s o r i n t h e b a t c h o f g lu t a t h i o n e r e d u c t a s e u s e d , c a n i n h ib i t

g l u t a t h i o n e p e r o x i d a s e .

A s d i f f e r e n c e s in e n z y m i c p r o p e r t i e s , e . g . , i n k i n e ti c s , o f g l u t a t h i o n e

p e r o x i d a s e c a n n o t b e e x c l u d e d i n d i f f e r e n t t i s s u e s o r s p e c i e s , o p t i m a l

s u b s t r a t e c o n c e n t r a t i o n s , i f in d o u b t , s h o u l d b e r e a s s e s s e d .

I f t h e a c t i v i t y m e a s u r e d i n c r u d e s a m p l e s i s t o b e a s s i g n e d t o g lu -

t a t h f o n e p e r o x i d a s e u n e q u i v o c a l l y , a s e p a r a t i o n s t e p a n d c h a r a c t e r i z a -

t io n , e . g . , b y c o r r e l a t in g s e l e n i u m c o n t e n t a n d e n z y m i c a c t i v it y d u r i n g

f r a c t i o n a t i o n , s h o u l d b e c o n s i d e r e d .

[ 3] Catalase i n V i t r o

y HUGO AEB

2H202 eatal,s~ 2H 20 + 02 (1)

ROOH + AH2 catalascH20 + ROH + A (2)

C a t a l a s e e x e r t s a d u a l f u n c t i o n : ( 1 ) d e c o m p o s i t i o n o f

H 2 2 t o g i v e H 2

and 02 [ ca t a l y t i c ac t i v i t y , E q . (1 )] and (2 ) ox i da t i on o f H don or s , e . g . ,

m e t h a n o l , e t h a n o l , f o r m i c a c id , p h e n o l s , w i t h t h e c o n s u m p t i o n o f I m o l o f

pe r ox i de [ pe r ox i d i c ac t i v i t y , E q . ( 2 ) ] .

K i n e t ic P r o p e r t i e s

T h e p r e d o m i n a t i n g r e a c t io n d e p e n d s o n t h e c o n c e n t r a t io n o f H d o n o r

a n d t h e s t e a d y - s t a t e c o n c e n t r a t i o n o r r a te o f p r o d u c t io n o f

H2 2

i n t he

s y s t e m . I n b o t h c a s e s t h e a c t i v e c a t a l a s e - H 2 0 2 c o m p l e x I is f o r m e d fir st .

T h e d e c o m p o s i t io n o f H 20 2 , i n w h i c h a s e c o n d m o l e c u l e o f H 202 s e r v e s

a s H d o n o r f o r c o m p l e x I , p r o c e e d s e x c e e d i n g l y r a p i d ly ( ra t e c o n s t a n t k

1 7

l it e rs m o l - t s e c - t ) w h e r e a s p e r o x i d a t i v e r e a c t i o n s p r o c e e d r e l a ti v e l y

slo w ly (k - 102-103). t

T h e k i n e ti c s o f c a t a l a s e d o n o t o b e y t h e n o r m a l p a t te r n . O n t h e o n e

h a n d it is n o t p o s s i b le to s a t u r a t e t h e e n z y m e w i t h s u b s t r a t e w i t h in t h e

f e a s ib l e c o n c e n t r a t i o n r a n g e ( u p t o 5 M H 20 2), a n d o n t h e o t h e r t h e r e i s a

r a p i d i n a c t iv a t i o n o f c a t a l a s e a t H 2 02 c o n c e n t r a t i o n s a b o v e 0 .1 M , w h e n

t h e a c t i v e e n z y m e - H 2 0 2 c o m p l e x I i s c o n v e r t e d t o t h e i n a c t i v e c o m -

p l e x e s I I o r I I I. M e a s u r e m e n t s o f e n z y m e a c ti v it y a t s u b s tr a te s a t u ra t io n

1 B . C h a n c e ,

Acta Chem Scand

1 , 2 3 6 1 9 4 7 ) .

Copyright

1984 by Acade mic P ress. Inc.

M E T H O D S I N E N Z Y M O L O G Y V O L . 105 A l l r ig h ts

of reprodue l ion in any fo rm reserved

ISBN 0.12-182005-X

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22 FORMATION OR REMOVAL OF OXYGEN RADICALS [ 3]

o r d e t e r m i n a t i o n o f t h e K s i s t h e r e f o r e i m p o s s i b l e . I n c o n t r a s t t o r e a c t i o n s

p r o c e e d i n g a t s u b s t r a t e s a t u r at i o n , t h e e n z y m i c d e c o m p o s i t i o n o f H 2 0 2 is

a f i r s t -o r d e r r e a c t i o n , t h e r a t e o f w h i c h i s a l w a y s p r o p o r t i o n a l t o t h e

p e r o x i d e c o n c e n t r a t i o n p r e s e n t . C o n s e q u e n t l y , t o a v o i d a r a p id d e c r e a s e

i n t h e i n i ti al r a t e o f t h e r e a c t i o n , t h e a s s a y m u s t b e c a r r i e d o u t w i t h

r e l a t i v e l y l o w c o n c e n t r a t i o n s o f H 2 0 2 ( a b o u t 0 .0 1 M ) . 2 A s t h e a c t i v a t i o n

e n e r g y f o r t h e d e c o m p o s i t i o n o f H 2 0 2 c a t a l y z e d b y c a t a la s e i s v e r y l o w

( 2 5 0 0 - 7 1 0 0 k J / m o l ) , t h e r e i s o n l y s l i g h t d e p e n d e n c e o n t e m p e r a t u r e

(Q i0 = 1 . 0 5 - 1 . 1 2 ) . T h e d e c o m p o s i t i o n o f H 2 0 2 i n it ia ll y ( a p p r o x . 0 - 3 0 s e c )

f o l l o w s th a t o f a f ir s t- o r d e r r e a c t io n w i t h H 2 0 2 c o n c e n t r a t i o n b e t w e e n

0 . 0 1 a n d 0 . 0 5 M . T h e r a t e c o n s t a n t ( k ) f o r t h e o v e r a l l r e a c t i o n i s g i v e n b y

k = (1/At)(ln

S I / S 2

= (2 3/A t)(log

S I / S 2

(3)

w he re At = t2 - t~ = m eas ur ed t im e in te rva l and S~ and 2 = H 202

c o n c e n t r a t i o n s a t t i m e s t l a n d t 2 . T h e c o n s t a n t k c a n b e u s e d a s a d i r e c t

m e a s u r e o f t h e c a t a l a se c o n c e n t r a t i o n . I n s tu d i e s w i t h p ur if ie d e n z y m e

p r e p a r a t i o n s t h e s p e c i f i c a c t i v i t y ( k ' 0 i s o b t a i n e d b y d i v i d i n g k b y t h e

m o l a r c o n c e n t r a t i o n o f c a ta l a s e ( e ).

k l = k / e

( l i te rs mo l - l sec - l ) (4)

T h e v a l u e f o r k'~ f o r p u r e c a t a l a s e f r o m h u m a n e r y t h r o c y t e s i s 3 . 4 x 1 7

( li te r s m o l - ~ s e c- ~ ) . T h i s v a l u e i s u s e d t o c a l c u l a t e t h e a b s o l u t e c o n t e n t o f

e n z y m e in b l o o d a n d t i s s u e s . 3

ssay M e t h o d

P r i n c i p l e

I n t h e u l t r a v i o l e t r a n g e H 2 0 2 s h o w s a c o n t i n u a l i n c r e a s e i n a b s o r p t i o n

w i t h d e c r ea s i n g w a v e l en g t h . T h e d e c o m p o s i t i o n o f H 20 2 c a n b e f o l l o w e d

di rec t ly b y the d ec rea se in abs or ba nc e a t 240 nm (e240 = 0 .00394 --- 0 .0002

l i te r s m m o l - I m m - I ) . 4 T h e d i f f e r e n c e i n a b s o r b a n c e (A A240) p e r u n i t t i m e

i s a m e a s u r e o f t h e c a t a l a s e a c t i v i t y .

T o a v o i d i n a c ti v a t io n o f th e e n z y m e d u r in g t h e a s s a y ( u s u a l ly 3 0 se c )

o r f o r m a t i o n o f b u b b l e s i n t h e c u v e t t e d u e t o t h e l ib e r a ti o n o f 0 2 , i t i s

n e c e s s a r y t o u s e a r e la t i v e ly l o w H 2 0 2 c o n c e n t r a t i o n (1 0 m M ) . T h e H ~ O 2

c o n c e n t r a t i o n i s c r i t i c a l i n a s m u c h a s t h e r e i s d i r e c t p r o p o r t i o n a l i t y b e -

t w e e n t h e s u b s t r a t e c o n c e n t r a t i o n a n d t h e r at e o f d e c o m p o s i t i o n . D u e t o

t h e s p e c i al s i t u a ti o n i n c a ta l a s e t h e d e p e n d e n c e o f t h e H 2 0 2 d e c o m p o s i -

R. K. Bonnichsen, B. Chance, and H. Theorell,

Acta Chem Scand

1, 685 1947).

R. K. Bonnichsen, this series, Vol. II, p. 781.

D. P. Nelson and L. A. Kiesow,

Anal Biochem

49, 474 1972).

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[13] CATALASE in Vitro 123

t i o n o n t h e t e m p e r a t u r e i s s m a l l (Q i0 - 1 .1 ) s o t h a t m e a s u r e m e n t s c a n b e

c a r r i e d o u t b e t w e e n 0 a n d 3 7 ; h o w e v e r , 2 0 is r e c o m m e n d e d . T h e p H

a c t i v i t y c u r v e r e l a t i v e t o V0 h a s a f a ir l y b r o a d p H o p t i m u m ( p H 6 . 8 - 7 . 5 ) :

m e a s u r e m e n t s a r e m a d e a t p H 7 .0 . 5

R e a g e n t s

P h o s p h a t e b u f f e r 5 0 m M , p H 7 .0 : d i s s o l v e ( a) 6 .8 1 g K H 2 P O a , a n d ( b )

8 . 9 0 g N a 2 H P O 4 2 H 2 0 i n d i s t il l e d w a t e r a n d m a k e u p t o 1 00 0 m l

e a c h . M i x s o l u t i o n s ( a ) a n d ( b ) i n t h e p r o p o r t i o n 1 : 1 . 5 ( v / v )

H y d r o g e n p e r o x i d e 3 0 m M : d i l u t e 0 . 3 4 m l 3 0 % h y d r o g e n p e r o x i d e

w i t h p h o s p h a t e b u f f e r t o I 0 0 m l

P r o c e d u r e

M e a s u r e m e n t in B l o o d V e n o u s b l o o d c o n t a i n in g h e p a r i n o r c it r a te i s

c e n t ri f u g ed a n d t h e p l a s m a a n d l e u k o c y t e la y e r s a re r e m o v e d . T h e e r y t h -

r o c y t e s e d i m e n t is w a s h e d t h r e e t im e s w i t h i s o to n i c N a C I . A s t o c k h e m o -

l y s a t e is p r e p a r e d c o n t a i n i n g - 5 g H b / 1 0 0 m l b y th e a d d i ti o n o f f o u r p a r ts

b y v o l u m e o f d i s ti ll e d w a t e r . A 1 5 00 d il u ti o n o f th is c o n c e n t r a t e d h e m o -

l y s a t e i s p r e p a r e d w i t h p h o s p h a t e b u f f e r i m m e d i a t e l y b e f o r e t h e a s s a y i s

p e r f o r m e d a n d H b ( h e m o g l o b i n ) c o n t e n t i s d e t e r m i n e d i n d u p l i c a t e ( e . g . ,

b y t h e m e t h o d o f D r a b k i n ). F o r c a p i l la r y b l o o d , 0 . I o r 0 .0 2 m l is h e m o -

l y z e d i n 2 5 0 o r 5 0 m l d i st il le d w a t e r . I f t h e h e m o g l o b i n c o n t e n t o f th e

b l o o d i s r e q u i r e d a s r e f e r e n c e p o i n t , i t m u s t b e d e t e r m i n e d i n a s e p a r a t e

s a m p l e o f b l o o d . 6-8

M e a s u r e m e n t in T is su e s C a t a l a s e i n t i s s u e s w i t h r e l a t iv e l y h ig h a c t i v -

i t y , s u c h a s li v e r a n d k i d n e y , c a n b e d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y i f

c o m p l e t e l y s i s o f a ll o r g a n e l l e s a n d c l e a r ( o r o n l y s l ig h t ly c o l o r e d ) s o l u -

t io n s o r e x t r a c t s c a n b e o b t a i n e d . A d e t e r g e n t ( e .g . , 1 % T r i to n X - 1 00 )

m u s t b e u s e d i n t h e p r e p a r a t i o n o f t h e s t o c k h o m o g e n a t e (1 + 9 o r 1 + 1 9),

o t h e r w i s e t o o l o w v a l u e s w i l l r e s u l t . F u r t h e r d i l u t i o n s c a n b e m a d e w i t h

p h o s p h a t e b u f f e r , p H 7 . 0 ( I : 1 00 t o 1 5 0 0 , d e p e n d i n g o n t i s s u e a n d s p e -

c i e s) . H o w e v e r , i f t h e s a m p l e a f t e r l y s is o f t h e o r g a n e l le s c a n n o t b e d i -

l u te d t o t h is e x t e n t , t h e c o n s i d e r a b l e U V a b s o r p t i o n o f T r i t o n X - I 0 0 m u s t

b e k e p t i n m i n d . A s a n a l t e r n a t i v e d i g i t o n i n ( 0 . 0 1 % ) o r s o d i u m c h o l a t e

( 0 .2 5 % ) c a n b e u s e d . N o r m a l l y , c a t a l a s e a c t i v i t y o f t i s su e s a m p l e s i s

e x p r e s s e d o n a m i l li g ra m w e t w e i g h t o r m i ll ig r am t o ta l N b a s i s . A c o n v e n -

B. Chan ce, H. S ies, and A. B overis,

Physiol Rev

59, 527 (1979).

6 H. A ebi, #~ Ex pos es A nnuels de B iochimie M ~dicale, 29ieme s6rie, p. 139. M asson,

Paris, 1969.

7 H. A ebi and H . Su ter, hi Biochem ical M ethods in Red Cell Ge netics (J. J. Y unis, ed,),

p. 25 5. Ac adem ic Pres s, New Yo rk, 1969.

8 H. Ae bi, S. R . W yss, B. S cherz, and J. G ross,

Biochem Genet

14, 791 (1976).

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1 2 4 F ORM AT ION OR RE M OVAL OF OXYGE N RADICAL S [ 1 3 ]

i e n t m e t h o d f o r t h e m e a s u r e m e n t o f c a t a l a s e a c t i v i t y in t i ss u e e x t r a c t s h a s

b e e n d e s c r ib e d b y C o h e n e t

al 9

A s s a y C o n d i t i o n s

W a v e l e n g t h , 2 4 0 n m ; li g h t p a t h , 1 0 m m ; fin al v o l u m e , 3 . 0 0 m i . R e a d

t h e s a m p l e c o n ta in in g , 2 . 00 m l e n z y m e s o l u t io n o r h e m o l y s a t e a n d 1 m l

H 2 0 2 a t 2 0 ( - r o o m t e m p e r a t u r e ) a g a i n s t a b l a n k c o n ta in i ng , 1 m l p h o s -

p h a t e b u f f e r i n s t e a d o f s u b s t r a t e a n d 2 m l e n z y m e s o l u t i o n o r h e m o l y s a t e .

T h e r e a c t i o n i s s t a r t e d b y a d d i t io n o f H 2 0 2 . T h e i ni tia l a b s o r b a n c e s h o u l d

b e a p p r o x i m a t e l y A = 0 .5 0 0 . M i x w e l l w i t h a p la s t ic p a d d l e a n d f o l l o w t h e

d e c r e a s e i n a b s o r b a n c e w i t h a r e c o r d e r f o r a b o u t 3 0 s e c .

S t a b il it y o f E n z y m e

C a t a l a s e i n i n t a c t e r y t h r o c y t e s a n d i n c o n c e n t r a t e d h e m o l y s a t e s i s

s t a b l e u p t o 6 d a y s w h e n k e p t a t 2 . H o w e v e r , t h e r e is a r e l a ti v e l y r a p id

d e c l i n e o f a c t iv i t y i n d i l u te h e m o l y s a t e s w h i c h i s m o r e l ik e l y d u e t o d e -

c o m p o s i t i o n o f t h e e n z y m e i n t o s u b u n i t s t h an t o p r o t e o l y t i c c h a n g e s . A t a

c o n c e n t r a t i o n o f 1 .2 m g H b / m l t h e a c t i v i ty d e c r e a s e s b y 1 0 - 1 5 w i t h in 2 4

h r ; at a c o n c e n t r a t i o n o f 0 . 06 m g H b / m l t h e lo s s o f a c t i v i ty is 1 0 a f t e r 1

h r a n d 8 0 - 9 0 a f t e r 2 4 h r. C o n s e q u e n t l y , h e m o l y s a t e s a m p l e s s h o u l d b e

a n a l y z e d w i t h i n 5 - 1 0 m i n a f t e r d i l u t i o n .

Def i n it ion o f U n i ts an d Sp ec i f ic Ac t i v i ty

I t is n o t p o s s i b l e t o d e f i n e i n t e r n a t i o n a l c a t a l a s e u n i t s ( U ) a c c o rd i n g, t o

I U B r e c o m m e n d a t i o n s d u e t o th e a b n o r m a l k i n e ti cs . T h e r e f o r e , th e u s e o f

a n u m b e r o f d i f f e re n t l y d e f in e d u n i t s a n d d i f fe r e n t m e t h o d s o f e v a l u a t i o n

i s a c c e p t a b l e f o r t h is e n z y m e ( a s e l e c t i o n o f m e t h o d s i s l is t ed i n t h e

t ab le ). ~ U s e o f t h e r a t e c o n s t a n t o f a f ir s t - o r d e r r e a c t i o n (k ) i s r e c o m -

m e n d e d . T h e r a te c o n s t a n t r e la t e d t o th e h e m o g l o b i n c o n t e n t

k/g,

H b ) c a n

s e r v e a s a m e a s u r e o f t h e s p e c i f ic a c t i v i t y o f e r y t h r o c y t e c a t a l a s e . E q u a -

t i o n (3 ) a p p l i e s i n t h is c a s e . I f t h e d e c r e a s e i n a b s o r b a n c e i s r e c o r d e d , t h e

v a l u e o f l o g A tlA2 f o r a m e a s u r e d t i m e i n t e rv a l o r t h e ti m e r e q u i r e d f o r a

c e r ta i n d e c r e a s e in a b s o r b a n c e c a n b e d e t e r m i n e d .

F o r a t i m e i n t e r v a l o f 15 s e c t h e f o ll ow i n g , r e l a t i o n s h i p i s o b t a i n e d

according , to Eq. (3) :

k = (2.3/15)(1og AI/A2) = 0.153(1og, ALIA2) (sec - I ) (5)

T o c a l c u l a t e

k / mi

o r

k/g,

H b p r o c e e d a s f o l l o w s :

9 G . Co h e n , D . D e m b ie c , a n d J . M a r c u s ,

Anal Biochem

34, 30 (1970).

~o H . A eb i,

in

M e t h o d s o f E n z y m a t i c A n a l y s i s ( H . U . B e r g m e y e r , e d .) , 3 rd E d . V e r l a g

Ch e m ic , W e in h e im , F . R . G . , i n p r e p a r a t i o n , 1 98 3.

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[ 3 ] CATALASE in Vio o 25

OTHER METHODS OF DETERMIN TION

Method/technique References Material

Determination of H202 removal

Titrimetric methods

Iodometric Bonnichsen

e t a l . ~

Tissues, blood

Permanganometric Bonnichsen3 Tissues, blood

Spectrophotometry

Substrate: H202 (E240) Bergmeyer Purified preparations

Cohen

e t a l . 9

Tissues, organelles

Substrate: perborate (E220) Thomson et al . ~2 Tissue fractions

Photometry

(E405-E~15)

Vanadic acid Warburg and KrippahP Cell cultures

Titanium tetrachloride Pilz and Johannb Blood

Fluorimetry

Scopoletin Perschke and Broda c Aqueous

Diacetyldichlorofluorescein Keston and Brandt a solutions

Determination of 02 production

Oxygen electrode Ogata I~ Blood

Del Rio e t a l . ' 4 Plant material

Meerhof and Roos j~ Blood

Polarography

Catalase and SOD

Immunoprecipitation

(with anti-catalase)

Screening techniques

Capillary tube

Siebtest

Automated procedure

Technicon AutoAnalyzer

Rigo and RotiliC 6 Tissue homogenate

hemolysate

Higashi e t a l . Blood, tissues

Ben-Yoseph and Shapira t~ Blood

Fung and Petrishko f Microbial cultures

Gross

et al . 18

Blood

Lamy et al . 19 Blood

Leighton e t a l . 2 Tissue fractions

O. Warburg and G. Krippahl, Z . N a t u r f o r s c h u n g ll lb, 340 (1963). b W. Pilz and J.

Johann,

Z . A n a l . C l a e m .

210, 358 (1965). ~ H. Perschke and E. Broda,

N a t u r e { L o n d o n )

190,

257 (1961). d A. S. Keston and R. Brandt, A n a l . B i o c h e m . 11, I (1965). T. Higashi, M.

Yagi, and H. Hirai, J . B i o c h e m . ( T o k y o ) 49, 707 (1961). f D. Y. C. Fung and D. T. Petrishko,

A p p l . M i c r o b i o l . 26, 631 (1973).

k / m l = k a 6 )

k / g H b = k / m l l OOO / b ) = 2 . 3 /1 5 ) a / b ) I o g A I / A 2 ) s e c - ~ ) 7 )

w h e r e A ~ i s A 2 40 a t t = 0 , A 2 i s A 2 40 a t t = 1 5 s e c , a i s t h e d i l u t i o n f a c t o r [ H b

c o n c e n t r a t i o n i n b l o o d o r e r y t h r o c y t e s e d i m e n t m g H b / m l ) /H b c o n c e n -

t r at io n i n c u v e t t e r ag H b / m l ) ] , a n d b is t h e H b c o n t e n t o f b lo o d o r e r y th -

r o c y t e s e d i m e n t g r a m s / l i te r ) .

F o r t h e d i f fe r e n c e in a b s o r b a n c e o f 0 . 4 5 0 - 0 . 4 0 0 l og A I / A 2 = 0 . 0 5 1 1 5 )

t h e f o l l o w i n g r e l a t i o n h o l d s :

k = 2 . 3 / A t ) l o g A j / A 2 ) = 0 . 1 1 7 5 / A t s e c - I ) 8 )

8/10/2019 Catalase AEBI

6/6

26 FORM ATION OR REM OVAL OF OXYGEN RADICALS [ 4]

Other Methods of Determination

This contribution deals only with the catalytic (not the peroxidic) ac-

tivity of catalase. It can be measured by following either the decomposi-

tion of H202 or the liberation of 02. Accordingly, the remaining substrate

concentration at a given moment of the reaction can be determined by UV

spectrophotometry9,~ .~2 or--at the end of the reaction period--by simple

titration. 2,3 On the other hand, O~ production can be followed with the

oxygen electrode ~3-~5 or by polarography. ~6 Alternatively, catalase can

also be measured by immunoprecipitation. ~7 The method of choice for

biological material, however, is UV spectrophotometry. Titrimetric meth-

ods are suitable for comparative studies. For large series of measure-

ments there are either simple screening tests which give a quick indication

of the approximative catalase activity, 8 or automated methods (e.g., us-

ing the Technicon AutoAnalyzer) available 19,2 (see the table).

u H . U . B e r g m e y e r , B i o c h e m . Z . 327~ 255 (1955).

t2 j . F . T h o m s o n , S . L . N a n c e , a n d S . L . T o l l a k s e n , P r o c . S o c . E x p . B i o l . M e d . 157, 33

(1978).

,3 M . O g a ta , in S y m p o s i u m o n G e n e t i c s a n d B i o c h e m i s t r y o f A c a t a l a s e m i a ( I X A n n u a l

M e e t i n g o f t h e J a p a n S o c i e t y o f H u m a n G e n e t i c s ) , at W a k a y a m a M e d i c a l C o l le g e , 1 9 64 .

14 L. A . De l Rio e t a l . A n a l . B i o c h e m . 80, 409 (1977) .

15 L . J . M e e r h o f a n d D . R o o s , J . R e t i c u l o e n d o t h e l . S o c . 28, 419 (1980) .

,6 A . R i g o a n d G . R o t i l i o , A n a l . B i o c h e m . 81, 157 (1977).

~7 y . B e n - Y o s e p h a n d E . S h a p i r a , J . L a b . C l i n . M e d . 81, 133 (1973).

,8 j . G r o s s , A . H a r t w i g , a n d A . G o l d i n g , Z . M e d . L a b o r t e c h . 16, 336 (1975) .

L9 j . N . La m y e t a l . B u l l . S o c . C h i m . B i o l. 49, 116 7 (1967).

20 F . L e i g h t o n e t a l . J . Ce l l B i o l . 37, 482 (1968) .

[14] Assays of Lipoxygenase, 1,4-Pentadiene Fatty Acids,

and 02 Concent rat ions: Che milumin escen ce Methods

By

SIMO LAAKSO, ESA-MATTI LILIUS, and PEKKA TURUNEN

Lipoxygenases are iron-containing enzymes that catalyze the dioxy-

genation, by molecular oxygen, of

cis cis-1 4-pentadiene

fatty acids. The

reaction is a source of weak chemiluminescence due to dissociation of

free radicals from the main path of hydroperoxidation.~ Under alkaline

conditions the intensity of light emission can be drastically amplified by

the presence of luminol? The kinetics of luminol chemiluminescence in

' A . B o v e r i s , E . C a d e n a s , a n d B . C h a n c e , P h o t o b i o c h e m . P h o t o b i o p h y s . 1 , 1 75 0 9 8 0 ) .

2 E . - M . L i l iu s a n d S . L a a k s o , A n a l . B i o c h e m . 119, 135 (1982).

Copyright 1984by AcademicPress, Inc.

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r e s e rv ed

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