BIOCHIMIE, 1975, 57, 657-660.
Enzymatic and non-enzymatic assay of superoxide dismutase. R u i n e r FRIED (*). Centre de N e u r o c h i m i e du C.N.R.S., 1l rue H u m a n n , 67085 Slrasbourg Cedex. (22-5-1974). Summary. - - Superoxide dismutase from breef brain and rat liver was assayed in an enzymatic system, using xanthine oxidase, and a non-enzymatie system, based on aerobic reduction of nitro-blue tetrazolium in presence of phenazine methosulphate. The non-enzymatic assay is rapid and simple and permits simultaneous analysis of many samples. Similar results are found by the two methods of assay of superoxide dismutase.
Superoxide dismutase is an enzyme w h i c h scavenges a n d inactivates oxygen radicals (.O-O-) a n d singlet oxygen [1-7]. It acts as a protective agent against attacks by these t'wo of oxygen [8] ~vhich are formed i n m a n y biological systems a n d has been s h o w n to be an essential c o m p o n e n t of aerobic m e t a b o l i s m r9]. T h e e n z y m e is generally assayed by a decrease of its substrate, the oxygen radical, w h i c h is either assayed directly, for example by pulse r a d i o l y s i s [10] or i n d i r e c t l y , such as b y decrease of a d e n o c h r o m e f o r m a t i o n [11] or tetrazolium r e d u c t i o n [3-5, 12]. W h i l e pulse radiolysis a n d s i m i l a r p r o c e d u r e s r e q u i r e complex i n s t r u m e n tation, the usual c o l o r i m e t r i c m e t h o d s for determ i n a t i o n of s u p e r o x i d e dismutase have the serious d r a w b a c k of i n v o l v i n g t w o o p p o s i n g reactions, one g e n e r a t i n g the oxygen r a d i c a l ( x a n t h i n e q- x a n t h i n e oxidase b e i n g the enzyme system m a i n l y used) a n d the other, the superoxide dismutase, w h i c h in t u r n decomposes these radicals. The disadvantages of such an e n z y m a t i c system have r e c e n t l y been discussed [13, 14]. It is e v i d e n t that e n d o g e n o u s activators or i n h i b i t o r s p r e s e n t in a given system can affect the two enzymes i n different 'ways ; the same is true for added i n h i bitors. These factors complicate the study of s u p e r o x i d e dismutase. Several n o n - e n z y m a t i c methods for g e n e r a t i n g oxygen radicals have been proposed w h i c h e l i m i n a t e these c o m p l i c a t i o n s
[11, 13, 14]. Superoxide a n i o n s are also generated by aerobic oxidation of NADH, catalysed either by p h o t o c h e m i c a l activation or b y p h e n a z i n e methosulfate [15-19, and F. Lavelle a n d M. Michelson, (*) Maitre de Recherche INSERM. Permanent address : Department of Biochemistry, Creighton University, Medical School, Omaha, NB 68178, USA.
p r i v a t e c o m m u n i c a t i o n ] a n d c a n be m a n i f e s t e d b y r e d u c t i o n of n i t r o b l u e tetrazolium due to the oxygen r a d i c a l thus formed. The p r e s e n t method of d e t e r m i n a t i o n of s u p e r o x i d e is based on a r e p o r t b y N i s h i k i m i et al. [15] who f o u n d that the aerobic r e d u c t i o n of n i t r o - b l u e tetrazolium in presence of p h e n a z i n e m e t h o s u l p h a t e can be blocked by s u p e r o x i d e dismutase.
MATERIALS AND METHODS. S u p e r o x i d e dismutase was p r e p a r e d from beef b r a i n a n d rat liver by acid p r e c i p i t a t i o n and a m m o n i u m sulphate f r a c t i o n a t i o n , followed by heat t r e a t m e n t a n d c h l o r o f o r m e x t r a c t i o n [4, 5]. X a n t h i n e oxidase (cream x a n t h i n e oxidase, Sigma) a n d s u p e r o x i d e dismutase were assayed as p r e v i o u s l y described [4, 5, 20, 21] by aerobic red u c t i o n of n i t r o - b l u e tetrazolium, at 25°C, pH 7.8 at 540 n m in a n Yvon-Jobin spectrophotometer. Superoxide dismutase is assayed b y i n h i b i t i o n of l e t r o z o l i m n r e d u c t i o n in the x a n t h i n e oxidase system. F o r purified enzyme of fractions w i t h high s u p e r o x i d e dismutase activity, x a n t h i n e oxidase was used to give about 1.0 a b s o r b a n c e change at 540 n m p e r 10 m i n (¢ high )>) ; for fractions or p r e p a r a t i o n s w i t h low dismutase activity, x a n t h i n e oxidase contro]s were set at about 0.3 a b s o r b a n c e u n i t s / 3 0 rain (). I n a d d i t i o n to g e n e r a t i n g the oxygen radicals e n z y m a t i c a l l y by x a n t h i n e oxidase, they were also formed i n a n o n - e n z y m a t i c system, based on the o x i d a t i o n of NADH by p h e n a z i n e methosulfate [15]. This m e t h o d w a s modified to give simple a n d r a p i d assay of superoxide dismutase activity : graded levels of purified enzyme were added to a r e a c t i o n mixture, c o n t a i n i n g 1 mg n i t r o - b l u e
R. Fried.
658
tetrazolium, 10 v g p h e n a z i n e methosu!phate, 1 :~ m o l e E D T A , a n d 1 m g g e l a t i n ; t h e v o l u m e w a s c o m p l e t e d to 3.0 m l ~ i t h p h o s p h a t e 0.1 M, p H 7.8 ; ,0.1 m l of N A D H 1 m M (100 n m o l e ) w a s a d d e d last. A f t e r s t a n d i n g f o r 10, r a i n a t r o o m t e m p e r a t u r e i n t h e d a r k , Az4 o w a s d e t e r m i n e d . After correcting for color without added NADH, s u p e r o x i d e d i s m u t a s e a c t i v i t y is c a l c u l a t e d as proportion between absorbance observed in pres e n c e o r a b s e n c e of t h i s e n z y m e . Protein concentration was m e t h o d of Lo~vry et al. [25].
determined
only in presence of phenazine methosulphate,
and
that this o x i d a t i o n is a f u n c t i o n of the c o n c e n t r a t i o n of p h e n a z i n e methosulphate. The r e a c t i o n was f o u n d to be i n h i b i t e d b y s u p e r o x i d e d i s m u tase, w h i c h i n d i c a t e s that oxygen radicals participate i n this reaction, the degree of i n h i b i t i o n
~S40 BEEF BRAIN SUPEROXIDE
DISMUTASE
.4. I~
by the
J
ii1%Inhibition
nMoles
pH 7.6
NAD-H 2S"
VOI. ~.Oml .3~
F i n e c h e m i c a l s weere o b t a i n e d f r o m S i g m a a n d B o e h r i n g e r . All o t h e r r e a g e n t s w e r e ¢ R e a g e n t G r a d e >>.
2|
I 46 .2.
RESULTS, 79
N i s h i k i m i et al. [15] h a v e s h o w n t h a t N A D H reduces nitro-blue tetrazolium aerobically, but E F F E C T OF AS4Q
ON
SUPEROXIDE DISMUTASE TETRAZOLIUM
29
(S.D.)
REDUCTION
SD-
"
57
119
~lg PROTEIN
171
228
i
285
Fla. 2. - - I n h i b i t i o n of NADH oxidation by graded levels of superoxide dismutase, Figures along curve indicate percent of i n h i b i t i o n .
..55 t-"--'t'""'t
13
NAD.H
ioo np4otes
ii iii: .......... ............ ~ ~ . . . . . . . . . .
~
Super°xide dismutase
.,,...
.2
so
d e p e n d i n g o n t h e a m o u n t of e n z y m e a d d e d . W e h a v e c o n f i r m e d t h i s f i n d i n g (fig. 1), a n d f o u n d t h e a m o u n t of f o r m a z a n ( r e d u c e d t e t r a z o l i u m ) f o r m e d t o b e d i r e c t l y r e l a t e d to t h e c o n c e n t r a t i o n of a d d e d N A D H . T h e c o l o r is s t a b l e at r o o m tempe-
~'opg
~H Z a
rature, i n the dark, for at least two hours. Addir o t , : a . o m~
"
. ..............................................................
~sa
ii .............
~
120
~,nute*
FiG. l. ~ I n h i b i t i o n of N A D H oxidation by superoxide dismutase. E n z y m e was added at p o i n t s i n d i cated b y arrovqs.
tion of s u p e r o x i d e dismutase at zero time causes a decrease of color p r o p o r t i o n a l to the c o n c e n t r a t i o n of e n z y m e ; enzyme a d d e d after the plateau has been r e a c h e d does not cause r e o x i d a t i o n of formazan. Boiled s u p e r o x i d e dismutase p r e p a r a t i o n causes a greatly d i m i n i s h e d i n h i b i t i o n of
T~,m,g L
Assay of superoxide dlsmutase of beef brain. NADH oxidation
"High XO" Fraction
~g of protein per assay
Control
+1 +1 +2
54O 54 57
h.~40/10 rain
0.98 0.11 0.69 0.48
p. cent inhibition
89 3O 55
As~0/10 min
0.400 0.080 0.250 0.140
p. cent inhibition
80 37 65
Superoxide d i s m u t a s e was p r e p a r e d f r o m beef b r a i n ; f r a c t i o n t u p to a m m o n i u m sulp h a t e p r e c i p i t a t i o n ; f r a c t i o n 2 was f u r t h e r purified b y h e a t a n d chloroform t r e a t m e n t . For assay, 0.1 m l of c r e a m x a n t h i n e oxidase (1:100) ~vas used. In the NADH o x i d a t i o n assay 0.1 m l of f r e s h l y p r e p a r e d NADH 0.001 M w a s added last. Values o b t a i n e d w i t h o u t NADH were substracted. In b o t h assays, r e a c t i o n w i t h o u t enzyme or s u b s t r a t e was used as blank. The r e a c t i o n was carried out at 25°C, pH 7.8 i n a final volume of 3.0 m l i n 0.1 M Na-phosphate. BIOCHIMIE, 1975, 57, n ° 5.
Assay of superoxide dismutase.
659
TABLE II.
Superoxide dismutase of rat liver. _"High _ _ _XO" Rat no
~g of protein per assay
control 42.5 4.3 42.5 4.3 37.5 3.8 54.0 5.4 control 304 31 320 32 398 40 380 38
NADH oxidation
_"Low _ XO"
A~4o/10min :.p..cent A54o/30min p. cent A~0/10mi, p. cent inhibition __ _ ~nnll3Luon _ _ inhibition 0,96
--
0.50-/- 4
0,26
--
0,41
96
015
63
0.13 0.01 0.13 0.01 0.14 0.01 0.14
50 96 50 96 48 96 48
0.15
63
0.13
70
0.13
7O
s-43
0.58
4O
O. 57
41
O. 69
0.15
0.04 0.31 0.04 0.32 0.03 0.37 0.03 0.38
0.oo 56 96 54 97 47 97 45
0.41 lOO
0.12
71
100
0.17
59
0.00
100
0.14
66
0.00
100
0.08
82
O.
O0
8 male rats were 'killed by stunning (n ° 1-4), or light ether anesthesia and decapitation (n ° 5-8). Superoxide dismutase was partially purified (up to a m m o n i u m sulphate precipitation). Assays 'were carried out in duplicate at 25°C at pH 7.8, and were read at 540 nm. t e t r a z o l i u m r e d u c t i o n ; h o w e v e r s u p e r o x i d e dism u t a s e is n o t t o t a l l y a b o l i s h e d e v e n a f t e r p r o l o n ged h e a t i n g , w h i c h is in a g r e e m e n t w i t h t h e p r e v i o u s l y r e p o r t e d r e s u l t s , c a r r i e d out w i t h t h e e n z y m a t i c s y s t e m of g e n e r a t i n g o x y g e n r a d i c a l s [4]. G r a d e d l e v e l s of s u p e r o x i d e d i s m u t a s e c a u s e i n c r e a s i n g , up to a l m o s t c o m p l e t e i n h i b i t i o n of d y e r e d u c t i o n (fig. 2). Superoxide dismutase was purified and assayed in t h e e n z y m a t i c a n d n o n - e n z y m a t i c a s s a y : g o o d a g r e e m e n t w a s o b s e r v e d (table I). I n o r d e r to test t h e r e p r o d u c t i b i l i t y of p u r i f i c a t i o n a n d assay m e t h o d s , f o u r m a l e a d u l t W i s t a r rats, a b o u t 250 g, were sacrified either by light ether anesthesia or by stunning, followed by decapitation. Superoxide d i s m u t a s e w a s a s s a y e d in a p a r t i a l l y p u r i f i e d f r a c t i o n (up to a m m o n i u m s u l p h a t e p r e c i p i t a t i o n ) p r e p a r e d f r o m a l i q u o t s of i n d i v i d u a l l i v e r h o m o genates. G o o d a g r e e m e n t w a s f o u n d in t h e r e p l i c a t e s a m p l e s , as w e l l as b y t h e t w o m e t h o d s of k i l l i n g (table II).
DISCUSSION. One m u s t r e m e m b e r t h a t t h e t o t a l a m o u n t of oxygen radicals present, and therefore, the
BIOCHIMIE, 1975, 57, n ° 5.
a m o u n t of t e t r a z o l i u m r e d u c e d in e i t h e r t y p e of assay, is a f u n c t i o n of o p p o s i n g e n z y m e s y s t e m s , t h o s e w h i c h g e n e r a t e o x y g e n r a d i c a l s , e.g. x a n thine oxidase, and superoxide disrnutase which destroys them. In comparing different organs or d i f f e r e n t c r u d e p r e p a r a t i o n s , o n e m u s t k e e p in m i n d t h a t k i n e t i c s of a c t i v a t i o n a n d i n h i b i t i o n of these opposing enzyme systems can vary from c a s e to case, a c c o r d i n g to e x p e r i m e n t a l c o n d i tions. T h u s , in c o m p a r i n g l i v e r a n d b r a i n , f o r e x a m p l e , it is i m p o r t a n t to be a'~vare of t h e g r e a t v a r i a t i o n s of e n d o g e n o u s x a n t h i n e o x i d a s e a c t i vity, w h i c h is v e r y h i g h in m a m m a l i a n l i v e r , b u t in b r a i n a m o u n t s to o n l y a b o u t I p. c e n t of t h a t f o u n d in l i v e r [22-24j. T h u s , l i v e r h o m o g e n a t e c o n t r i b u t e s x a n t h i n e o x i d a s e to t h e a s s a y s y s t e m in a d d i t i o n to t h e a m o u n t p r e s e n t in the s t a n d a r d m i x t u r e ; t h i s "would l e a d to an a p p a r e n t l y l o w e r superoxide dismutase activity per unit weight, t h a n b r a i n . T h e s a m e a p p l i e s to a n y o t h e r e n z y m e system or combinations which can generate o x y g e n r a d i c a l s . A l t h o u g h an i n c r e a s i n g n u m b e r of s u c h e n z y m e s h a s b e e n i d e n t i f i e d , it is r e a s o n a b l e to a s s u m e t h a t t h e r e a r e still a l a r g e n u m b e r as yet u n i d e n t i f i e d . T h e d e p e n d e n c e of m e a s u r e d s u p e r o x i d e d i s m u t a s e a c t i v i t y on t h e a m o u n t of a d d e d x a n t h i n e o x i d a s e c a n be s e e n in t a b l e II, c o m p a r i n g t h e ¢ h i g h >> a n d > assays.
660
R. Fried.
T h e a s s a y of s u p e r o x i d e d i s m u t a s e b y t e t r a zolium reduction with NADH, on the other hand, depends on the concentration of NADH. Any f a c t o r w h i c h a f f e c t s N A D H , o r c o n t r i b u t e s to t h e r e d u c t i o n of n i t r o - b l u e t e t r a z o l i u m , w i l l b e r e f l e c t e d i n t h e a p p a r e n t a c t i v i t y of s u p e r o x i d e d i s m u tase. T h e s e i n t e r f e r e n c e s c a n b e c o n t r o l l e d i n part, by dialysing crude tissue fractions, and by t e s t i n g t h e r e d u c t i o n of t e t r a z o l i u m i n t h e c o m plete system, but without added NADH. I n o u r c u r r e n t i n v e s t i g a t i o n s of s u p e r o x i d e mutase in the nervous system the enzymatic non-enzymatic assay are routinely compared, u s u a l l y g o o d a g r e e m e n t is f o u n d b e t w e e n t h e methods.
disand and t~-o
W e h a v e s h o w n e a r l i e r [4~ t h a t s u p e r o x i d e d i s m u t a s e also b l o c k s t h e e n z y m a t i c a e r o b i c oxid a t i o n o f N A D H b y t e t r a z o l i u m i n p r e s e n c e of phenazine methosulphate, when ethanol and a l c o h o l d e h y d r o g e n a s e w e r e u s e d as t e s t system. The mechanism of t h i s r e a c t i o n c a n n o w b e explained by the non-enzymatic model, in which o x y g e n r a d i c a l s a r e d e m o n s t r a t e d d u r i n g t h e oxid a t i o n of N A D H b y p h e n a z i n e m e t h o s u l f a t e . Acknawledgments. The a u t h o r s h o u l d like to express his a p p r e c i a t i o n to t h e I n s t i t u t N a t i o n a l de la Sant6 et de la Recherche M~dicale (INSERM) for a fellowship, a n d to t h a n k Prof. P. Mandel for his h o s p i t a l i t y a n d interest. The c o m p e t e n t technical h e l p b y M. D. Filliol is g r a t e f u l l y acknowledged. R~su~. La superoxyde d i s m u t a s e de cerveau de b oeuf et de foie de r a t a 6t6 d6termin~e p a r u n syst6me e n z y m a tique h l'aide de x a n t h l n e oxydase et p a r n n syst6me ~ o n - e n z y m a t i q u e has6 sur une r d d u c t i o n a6robie bleu de t e t r a z o l i u m nitr6 en prdsence de p h 6 n a z i n e m 6 t h o sulfate. L'essai n o n - e n z y m a t i q u e est rapide et simple et perm e t l ' a n a l y s e s i m u l t a n d e de p l u s i e u r s 6chantillons, Des r6sultats s i m i l a i r e s out 6t6 o h t e n u s p a r les deux
BIOCHIMIE, 1975, 57, n ° 5.
m~thodes de d 6 t e r m i n a t i o n de la superoxyde d i s m u tase. REFERENCES. 1. McCord, J. M. ~ Fridovich, I. (1969) J. Biol. Chem., 244, 6049-6055. 2-. Fridovich, I. (1972) Accts. Chem. Res., 5, 221-226. 3. Fried, R. & Fried, L. W. (1967) Feder. Proc., 26, 837. 4. Fried, R., Fried, L. W. & Babin, D. R. (1970) Eur. J. Biochem., 1~6, 399-406. 5. Fried, R., Fried, L. W. & Babin, D. B. (1973) Eur. J. ~iochem., 33, 439-445. 6. Politzer, I. R., Griffin, G. W. ~ Laseter, J. L. (1971) Chem. Bid. Interact., 3, 73-93. 7. Weser, U. (1973) in Structure and Bonding (Dunitz, J. D., Hemmerich, P., lbers, J. A., Jorgensen, K., Neilands, J. B., Reinen, D. & W i l l i a m s , R. J. P., ed.), vol. 17, pp. 1-65, Springer Verlag, Heidelberg. 8. Lavelle, F., Michelson, A. M. & Dimitrijevic, L. (1973') Biochem. Biophys. Bes. Commun., 55, 350-357. 9. McCord, J. M., Keele, B. B. Jr. & Fridovich, I. (1971) Proc. Natl. Acad. Sei. US, 68, 1024-1027. 10. Rotilio, G., Bray, R. C. & Fielder, E. M. (1972) Biochim. Biophys. Acta, 268, 695-609. 11. Misra, H. P. & Frido~vich, I. (1972) J. Biol. Chem., 247, 3130-3175. 12. Beauchamp, C. & Fridovieh, 1. (1971) Anal. Biochem., 44, 276-287. 13. Paschen, W. & Weser, U. (1973) Biochim. Biophys. Acta, 327, 217-222. 14. Fee, J. A. a H i l d e b r a n d , P. G. (1974) F E B S Letters, 39, 79-82. 15. Nishikimi, M., Rao, N. A. & Yagi, K. (1972) Biochem. Biophys. Res. Commu~., 46, 849-854. 16. Pederson, T. C. & Aust, S. D. (1973) Biochem. Biophys. Res. Commun., 52, 1071-1077. 17. Augusto, O., Becbara, E. J. H., Sanioto, D. L. & Cilento, G. (19:73) Arch. Biochem. Biophys., 158, 359-364. 18. Anst, S. D., Roerig, D. L. & Pederson, T. C. (1972) Biochem. Biophys. Res. Commun., 47, 1133-1137. 19. Bielski, B. J. & Chain, P. C. (1973) Arch. Biochem. Biophys., 159, 873-879. 20. Fried, R. (1966) Anal. Biochem., 16 427-432. 21. Fried, R. & Fried, L. ~W. (1970) i~ M e t h o d e n der e n z y m a t i s c h e n Analyse, (Bergmeyer, H. U., ed.) vol. 1 ; 2nd ed., p. 624, Verlag Chemle, W e i n h e i m . 22. Fried, R. (1965) Intern. Neurochem. Conference, Oxford, England, Abstr. p. 30. 23. Villela, G. G., Affonso, O. R. & Mitidlerl, E. (1966) Biochem. Pharmacol., 115, 1894-1896. 24. Markley, H. G., Faillaee, L. A. & Mezey, E. (1973) Biochim. Biophys. Acta, 309, 23-31. 25. Lowry, O. H., R o s e b r o u g h , N. J., F a r r , A .L. ,~ R a n d a l l , R. ft. (1951) J. Biol. Chem., 193. 265-275. 26. Fried, R. ~ MandeI, P. (1975) J. Neurochem., 24, 433-438.
Enzymatic and non-enzymatic assay of superoxide dismutase. R u i n e r FRIED (*). Centre de N e u r o c h i m i e du C.N.R.S., 1l rue H u m a n n , 67085 Slrasbourg Cedex. (22-5-1974). Summary. - - Superoxide dismutase from breef brain and rat liver was assayed in an enzymatic system, using xanthine oxidase, and a non-enzymatie system, based on aerobic reduction of nitro-blue tetrazolium in presence of phenazine methosulphate. The non-enzymatic assay is rapid and simple and permits simultaneous analysis of many samples. Similar results are found by the two methods of assay of superoxide dismutase.
Superoxide dismutase is an enzyme w h i c h scavenges a n d inactivates oxygen radicals (.O-O-) a n d singlet oxygen [1-7]. It acts as a protective agent against attacks by these t'wo of oxygen [8] ~vhich are formed i n m a n y biological systems a n d has been s h o w n to be an essential c o m p o n e n t of aerobic m e t a b o l i s m r9]. T h e e n z y m e is generally assayed by a decrease of its substrate, the oxygen radical, w h i c h is either assayed directly, for example by pulse r a d i o l y s i s [10] or i n d i r e c t l y , such as b y decrease of a d e n o c h r o m e f o r m a t i o n [11] or tetrazolium r e d u c t i o n [3-5, 12]. W h i l e pulse radiolysis a n d s i m i l a r p r o c e d u r e s r e q u i r e complex i n s t r u m e n tation, the usual c o l o r i m e t r i c m e t h o d s for determ i n a t i o n of s u p e r o x i d e dismutase have the serious d r a w b a c k of i n v o l v i n g t w o o p p o s i n g reactions, one g e n e r a t i n g the oxygen r a d i c a l ( x a n t h i n e q- x a n t h i n e oxidase b e i n g the enzyme system m a i n l y used) a n d the other, the superoxide dismutase, w h i c h in t u r n decomposes these radicals. The disadvantages of such an e n z y m a t i c system have r e c e n t l y been discussed [13, 14]. It is e v i d e n t that e n d o g e n o u s activators or i n h i b i t o r s p r e s e n t in a given system can affect the two enzymes i n different 'ways ; the same is true for added i n h i bitors. These factors complicate the study of s u p e r o x i d e dismutase. Several n o n - e n z y m a t i c methods for g e n e r a t i n g oxygen radicals have been proposed w h i c h e l i m i n a t e these c o m p l i c a t i o n s
[11, 13, 14]. Superoxide a n i o n s are also generated by aerobic oxidation of NADH, catalysed either by p h o t o c h e m i c a l activation or b y p h e n a z i n e methosulfate [15-19, and F. Lavelle a n d M. Michelson, (*) Maitre de Recherche INSERM. Permanent address : Department of Biochemistry, Creighton University, Medical School, Omaha, NB 68178, USA.
p r i v a t e c o m m u n i c a t i o n ] a n d c a n be m a n i f e s t e d b y r e d u c t i o n of n i t r o b l u e tetrazolium due to the oxygen r a d i c a l thus formed. The p r e s e n t method of d e t e r m i n a t i o n of s u p e r o x i d e is based on a r e p o r t b y N i s h i k i m i et al. [15] who f o u n d that the aerobic r e d u c t i o n of n i t r o - b l u e tetrazolium in presence of p h e n a z i n e m e t h o s u l p h a t e can be blocked by s u p e r o x i d e dismutase.
MATERIALS AND METHODS. S u p e r o x i d e dismutase was p r e p a r e d from beef b r a i n a n d rat liver by acid p r e c i p i t a t i o n and a m m o n i u m sulphate f r a c t i o n a t i o n , followed by heat t r e a t m e n t a n d c h l o r o f o r m e x t r a c t i o n [4, 5]. X a n t h i n e oxidase (cream x a n t h i n e oxidase, Sigma) a n d s u p e r o x i d e dismutase were assayed as p r e v i o u s l y described [4, 5, 20, 21] by aerobic red u c t i o n of n i t r o - b l u e tetrazolium, at 25°C, pH 7.8 at 540 n m in a n Yvon-Jobin spectrophotometer. Superoxide dismutase is assayed b y i n h i b i t i o n of l e t r o z o l i m n r e d u c t i o n in the x a n t h i n e oxidase system. F o r purified enzyme of fractions w i t h high s u p e r o x i d e dismutase activity, x a n t h i n e oxidase was used to give about 1.0 a b s o r b a n c e change at 540 n m p e r 10 m i n (¢ high )>) ; for fractions or p r e p a r a t i o n s w i t h low dismutase activity, x a n t h i n e oxidase contro]s were set at about 0.3 a b s o r b a n c e u n i t s / 3 0 rain (). I n a d d i t i o n to g e n e r a t i n g the oxygen radicals e n z y m a t i c a l l y by x a n t h i n e oxidase, they were also formed i n a n o n - e n z y m a t i c system, based on the o x i d a t i o n of NADH by p h e n a z i n e methosulfate [15]. This m e t h o d w a s modified to give simple a n d r a p i d assay of superoxide dismutase activity : graded levels of purified enzyme were added to a r e a c t i o n mixture, c o n t a i n i n g 1 mg n i t r o - b l u e
R. Fried.
658
tetrazolium, 10 v g p h e n a z i n e methosu!phate, 1 :~ m o l e E D T A , a n d 1 m g g e l a t i n ; t h e v o l u m e w a s c o m p l e t e d to 3.0 m l ~ i t h p h o s p h a t e 0.1 M, p H 7.8 ; ,0.1 m l of N A D H 1 m M (100 n m o l e ) w a s a d d e d last. A f t e r s t a n d i n g f o r 10, r a i n a t r o o m t e m p e r a t u r e i n t h e d a r k , Az4 o w a s d e t e r m i n e d . After correcting for color without added NADH, s u p e r o x i d e d i s m u t a s e a c t i v i t y is c a l c u l a t e d as proportion between absorbance observed in pres e n c e o r a b s e n c e of t h i s e n z y m e . Protein concentration was m e t h o d of Lo~vry et al. [25].
determined
only in presence of phenazine methosulphate,
and
that this o x i d a t i o n is a f u n c t i o n of the c o n c e n t r a t i o n of p h e n a z i n e methosulphate. The r e a c t i o n was f o u n d to be i n h i b i t e d b y s u p e r o x i d e d i s m u tase, w h i c h i n d i c a t e s that oxygen radicals participate i n this reaction, the degree of i n h i b i t i o n
~S40 BEEF BRAIN SUPEROXIDE
DISMUTASE
.4. I~
by the
J
ii1%Inhibition
nMoles
pH 7.6
NAD-H 2S"
VOI. ~.Oml .3~
F i n e c h e m i c a l s weere o b t a i n e d f r o m S i g m a a n d B o e h r i n g e r . All o t h e r r e a g e n t s w e r e ¢ R e a g e n t G r a d e >>.
2|
I 46 .2.
RESULTS, 79
N i s h i k i m i et al. [15] h a v e s h o w n t h a t N A D H reduces nitro-blue tetrazolium aerobically, but E F F E C T OF AS4Q
ON
SUPEROXIDE DISMUTASE TETRAZOLIUM
29
(S.D.)
REDUCTION
SD-
"
57
119
~lg PROTEIN
171
228
i
285
Fla. 2. - - I n h i b i t i o n of NADH oxidation by graded levels of superoxide dismutase, Figures along curve indicate percent of i n h i b i t i o n .
..55 t-"--'t'""'t
13
NAD.H
ioo np4otes
ii iii: .......... ............ ~ ~ . . . . . . . . . .
~
Super°xide dismutase
.,,...
.2
so
d e p e n d i n g o n t h e a m o u n t of e n z y m e a d d e d . W e h a v e c o n f i r m e d t h i s f i n d i n g (fig. 1), a n d f o u n d t h e a m o u n t of f o r m a z a n ( r e d u c e d t e t r a z o l i u m ) f o r m e d t o b e d i r e c t l y r e l a t e d to t h e c o n c e n t r a t i o n of a d d e d N A D H . T h e c o l o r is s t a b l e at r o o m tempe-
~'opg
~H Z a
rature, i n the dark, for at least two hours. Addir o t , : a . o m~
"
. ..............................................................
~sa
ii .............
~
120
~,nute*
FiG. l. ~ I n h i b i t i o n of N A D H oxidation by superoxide dismutase. E n z y m e was added at p o i n t s i n d i cated b y arrovqs.
tion of s u p e r o x i d e dismutase at zero time causes a decrease of color p r o p o r t i o n a l to the c o n c e n t r a t i o n of e n z y m e ; enzyme a d d e d after the plateau has been r e a c h e d does not cause r e o x i d a t i o n of formazan. Boiled s u p e r o x i d e dismutase p r e p a r a t i o n causes a greatly d i m i n i s h e d i n h i b i t i o n of
T~,m,g L
Assay of superoxide dlsmutase of beef brain. NADH oxidation
"High XO" Fraction
~g of protein per assay
Control
+1 +1 +2
54O 54 57
h.~40/10 rain
0.98 0.11 0.69 0.48
p. cent inhibition
89 3O 55
As~0/10 min
0.400 0.080 0.250 0.140
p. cent inhibition
80 37 65
Superoxide d i s m u t a s e was p r e p a r e d f r o m beef b r a i n ; f r a c t i o n t u p to a m m o n i u m sulp h a t e p r e c i p i t a t i o n ; f r a c t i o n 2 was f u r t h e r purified b y h e a t a n d chloroform t r e a t m e n t . For assay, 0.1 m l of c r e a m x a n t h i n e oxidase (1:100) ~vas used. In the NADH o x i d a t i o n assay 0.1 m l of f r e s h l y p r e p a r e d NADH 0.001 M w a s added last. Values o b t a i n e d w i t h o u t NADH were substracted. In b o t h assays, r e a c t i o n w i t h o u t enzyme or s u b s t r a t e was used as blank. The r e a c t i o n was carried out at 25°C, pH 7.8 i n a final volume of 3.0 m l i n 0.1 M Na-phosphate. BIOCHIMIE, 1975, 57, n ° 5.
Assay of superoxide dismutase.
659
TABLE II.
Superoxide dismutase of rat liver. _"High _ _ _XO" Rat no
~g of protein per assay
control 42.5 4.3 42.5 4.3 37.5 3.8 54.0 5.4 control 304 31 320 32 398 40 380 38
NADH oxidation
_"Low _ XO"
A~4o/10min :.p..cent A54o/30min p. cent A~0/10mi, p. cent inhibition __ _ ~nnll3Luon _ _ inhibition 0,96
--
0.50-/- 4
0,26
--
0,41
96
015
63
0.13 0.01 0.13 0.01 0.14 0.01 0.14
50 96 50 96 48 96 48
0.15
63
0.13
70
0.13
7O
s-43
0.58
4O
O. 57
41
O. 69
0.15
0.04 0.31 0.04 0.32 0.03 0.37 0.03 0.38
0.oo 56 96 54 97 47 97 45
0.41 lOO
0.12
71
100
0.17
59
0.00
100
0.14
66
0.00
100
0.08
82
O.
O0
8 male rats were 'killed by stunning (n ° 1-4), or light ether anesthesia and decapitation (n ° 5-8). Superoxide dismutase was partially purified (up to a m m o n i u m sulphate precipitation). Assays 'were carried out in duplicate at 25°C at pH 7.8, and were read at 540 nm. t e t r a z o l i u m r e d u c t i o n ; h o w e v e r s u p e r o x i d e dism u t a s e is n o t t o t a l l y a b o l i s h e d e v e n a f t e r p r o l o n ged h e a t i n g , w h i c h is in a g r e e m e n t w i t h t h e p r e v i o u s l y r e p o r t e d r e s u l t s , c a r r i e d out w i t h t h e e n z y m a t i c s y s t e m of g e n e r a t i n g o x y g e n r a d i c a l s [4]. G r a d e d l e v e l s of s u p e r o x i d e d i s m u t a s e c a u s e i n c r e a s i n g , up to a l m o s t c o m p l e t e i n h i b i t i o n of d y e r e d u c t i o n (fig. 2). Superoxide dismutase was purified and assayed in t h e e n z y m a t i c a n d n o n - e n z y m a t i c a s s a y : g o o d a g r e e m e n t w a s o b s e r v e d (table I). I n o r d e r to test t h e r e p r o d u c t i b i l i t y of p u r i f i c a t i o n a n d assay m e t h o d s , f o u r m a l e a d u l t W i s t a r rats, a b o u t 250 g, were sacrified either by light ether anesthesia or by stunning, followed by decapitation. Superoxide d i s m u t a s e w a s a s s a y e d in a p a r t i a l l y p u r i f i e d f r a c t i o n (up to a m m o n i u m s u l p h a t e p r e c i p i t a t i o n ) p r e p a r e d f r o m a l i q u o t s of i n d i v i d u a l l i v e r h o m o genates. G o o d a g r e e m e n t w a s f o u n d in t h e r e p l i c a t e s a m p l e s , as w e l l as b y t h e t w o m e t h o d s of k i l l i n g (table II).
DISCUSSION. One m u s t r e m e m b e r t h a t t h e t o t a l a m o u n t of oxygen radicals present, and therefore, the
BIOCHIMIE, 1975, 57, n ° 5.
a m o u n t of t e t r a z o l i u m r e d u c e d in e i t h e r t y p e of assay, is a f u n c t i o n of o p p o s i n g e n z y m e s y s t e m s , t h o s e w h i c h g e n e r a t e o x y g e n r a d i c a l s , e.g. x a n thine oxidase, and superoxide disrnutase which destroys them. In comparing different organs or d i f f e r e n t c r u d e p r e p a r a t i o n s , o n e m u s t k e e p in m i n d t h a t k i n e t i c s of a c t i v a t i o n a n d i n h i b i t i o n of these opposing enzyme systems can vary from c a s e to case, a c c o r d i n g to e x p e r i m e n t a l c o n d i tions. T h u s , in c o m p a r i n g l i v e r a n d b r a i n , f o r e x a m p l e , it is i m p o r t a n t to be a'~vare of t h e g r e a t v a r i a t i o n s of e n d o g e n o u s x a n t h i n e o x i d a s e a c t i vity, w h i c h is v e r y h i g h in m a m m a l i a n l i v e r , b u t in b r a i n a m o u n t s to o n l y a b o u t I p. c e n t of t h a t f o u n d in l i v e r [22-24j. T h u s , l i v e r h o m o g e n a t e c o n t r i b u t e s x a n t h i n e o x i d a s e to t h e a s s a y s y s t e m in a d d i t i o n to t h e a m o u n t p r e s e n t in the s t a n d a r d m i x t u r e ; t h i s "would l e a d to an a p p a r e n t l y l o w e r superoxide dismutase activity per unit weight, t h a n b r a i n . T h e s a m e a p p l i e s to a n y o t h e r e n z y m e system or combinations which can generate o x y g e n r a d i c a l s . A l t h o u g h an i n c r e a s i n g n u m b e r of s u c h e n z y m e s h a s b e e n i d e n t i f i e d , it is r e a s o n a b l e to a s s u m e t h a t t h e r e a r e still a l a r g e n u m b e r as yet u n i d e n t i f i e d . T h e d e p e n d e n c e of m e a s u r e d s u p e r o x i d e d i s m u t a s e a c t i v i t y on t h e a m o u n t of a d d e d x a n t h i n e o x i d a s e c a n be s e e n in t a b l e II, c o m p a r i n g t h e ¢ h i g h >> a n d > assays.
660
R. Fried.
T h e a s s a y of s u p e r o x i d e d i s m u t a s e b y t e t r a zolium reduction with NADH, on the other hand, depends on the concentration of NADH. Any f a c t o r w h i c h a f f e c t s N A D H , o r c o n t r i b u t e s to t h e r e d u c t i o n of n i t r o - b l u e t e t r a z o l i u m , w i l l b e r e f l e c t e d i n t h e a p p a r e n t a c t i v i t y of s u p e r o x i d e d i s m u tase. T h e s e i n t e r f e r e n c e s c a n b e c o n t r o l l e d i n part, by dialysing crude tissue fractions, and by t e s t i n g t h e r e d u c t i o n of t e t r a z o l i u m i n t h e c o m plete system, but without added NADH. I n o u r c u r r e n t i n v e s t i g a t i o n s of s u p e r o x i d e mutase in the nervous system the enzymatic non-enzymatic assay are routinely compared, u s u a l l y g o o d a g r e e m e n t is f o u n d b e t w e e n t h e methods.
disand and t~-o
W e h a v e s h o w n e a r l i e r [4~ t h a t s u p e r o x i d e d i s m u t a s e also b l o c k s t h e e n z y m a t i c a e r o b i c oxid a t i o n o f N A D H b y t e t r a z o l i u m i n p r e s e n c e of phenazine methosulphate, when ethanol and a l c o h o l d e h y d r o g e n a s e w e r e u s e d as t e s t system. The mechanism of t h i s r e a c t i o n c a n n o w b e explained by the non-enzymatic model, in which o x y g e n r a d i c a l s a r e d e m o n s t r a t e d d u r i n g t h e oxid a t i o n of N A D H b y p h e n a z i n e m e t h o s u l f a t e . Acknawledgments. The a u t h o r s h o u l d like to express his a p p r e c i a t i o n to t h e I n s t i t u t N a t i o n a l de la Sant6 et de la Recherche M~dicale (INSERM) for a fellowship, a n d to t h a n k Prof. P. Mandel for his h o s p i t a l i t y a n d interest. The c o m p e t e n t technical h e l p b y M. D. Filliol is g r a t e f u l l y acknowledged. R~su~. La superoxyde d i s m u t a s e de cerveau de b oeuf et de foie de r a t a 6t6 d6termin~e p a r u n syst6me e n z y m a tique h l'aide de x a n t h l n e oxydase et p a r n n syst6me ~ o n - e n z y m a t i q u e has6 sur une r d d u c t i o n a6robie bleu de t e t r a z o l i u m nitr6 en prdsence de p h 6 n a z i n e m 6 t h o sulfate. L'essai n o n - e n z y m a t i q u e est rapide et simple et perm e t l ' a n a l y s e s i m u l t a n d e de p l u s i e u r s 6chantillons, Des r6sultats s i m i l a i r e s out 6t6 o h t e n u s p a r les deux
BIOCHIMIE, 1975, 57, n ° 5.
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