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A sensitive radioimmunoassay of a scorpion neurotoxin Toxin I of Androctonus australis Hector
V o l u m e 85, n u m b e r 1
FEBS LETTERS
A SENSITIVE
RAD[OIMMUNOASSAY Toxin i of Androctonus
J a n u a r y 1978
OF A SCORPION NEUROTOX[N a u s t r a l i s Hec,*or
Marie T E S S I E R , Pierre D E L O R [ , G u y BECH[S and Herv6 R O C H A T Lcboratoim de Biochimie, Facult~ de l~[~decine, Secteur Nord, Bd. Pierre Dramard, 13326 :~[arseille Cedex 3, irrance Received 20 O c t o b e r ! 9 7 7
t . L~troduction Scorpion n e u r o t o x i n s f o r m a family o f h o m o l o g o u s proteins which are basic and have a p p r o x , rnol. w t 7000 [ l ] . T h e y consist o f a s i n # e peptide chain cross!inked b y four disulfide bridges [2i. The c o m plete a m i n o acid sequences oI s o m e o f t h e m [3,4L as well as the N-term!ha ~.c f others, have been determined: their c o m p a r i s o n has led to a classification into four groups [5 i . T h e y have been s h o w n to affect the c o n d u c t i o n o f ions t h r o u g h mere brane channels [ 6 - - I 0 ] , and are thus g o o d tools for the s t u d y o f these structures on the molecular level. Toxins I and
H o f A n d r o c t o n u s ausgralis H e c t o r have b e e n ~2s[_ labeled: specific radioactivRies u p to 2 0 0 0 C i / m m o l have b e e n obtained [ 1 1 , 1 2 ] . We r e p o r t here the setting up o f a radioh-amunoassay allowing a sensitive and specific d e t e c t i o n o f t o x i n I o f A n d r o c t o n u s australis Hector.
2. Materials a n d m e t h o d s
p r e p a r a t i o n s were p e r f o r m e d in the course o f this w o r k : the m e a n specific radioactivity was 1750 --+4 0 0 C i / m m o l , Le., 0.87 ± O.17 ~zs~ a t o m i n c o r p ~ r a t e d / m o l toxin. S e r u m against A s H t o x i n [ has b e e n p r e p a r e d f r o m rabbit, its neutralizing capacity tested on mice (Cs7/ B L / 6 ) was 157 t~g t o x i n / m ! serum [ 1 3 ] . _Amti-ratpbit serum p r e p a r e d in d o n k e y was purchased f r o m Wetlcorne, B e c k e n h a m , E n ~ a n d ( R D 17) and n o r m a l rabbit serum was o b t a i n e d in o u r l a b o r a t o r y 2.1. Quantitative ant@sis Two successive experhnents
have been performed
in order to c h e c k the b e h a v i o u r ~ f b o t h ~:S[-labeied and native toxins in the presence o f the A a H - t o ~ n I antiserum. A first quantitative precipitin test allowed to o b t a i n the conditions o f m a x ' ; m u m precipitation for each t o x i n and to k n o w the a m o u n t o f 125Llabeled toxin present in the precipitate (see fig.l). The maxim u m a m o u n t o f native t o x i p precipitated was determ i n e d in a second e x p e r i m e n t b y t o x i c i t y assay on the solubilized pellet. A single precipitation was
T o x i n s I, H and I ~ f f A n d r o c t o n u s australis H e c t o r ( A s h t o x i n I, A s H t o x i n I I and A a H t o x i n H I ) and t o x i n I o f B u t h u s occitanus tunetanus ( B o t toxin l) were purified according [ 2 ] . A a H t o x i n I was ~2Sl-labeled and isolated f r o m the reaction m e d i u m b y precipitation with its m o n o specific a n t i s e r u m as described [! 1,12]. T e n different * T o x i c i t y o f A a H t o x i n [ was f o u n d t o b e u n a l t e r e d b y this aeidfie t r e a t m e n t [ 12]
Elsevier~North-Holland Biomedical Press
carried o u t o n 20/~g native A a H ~oxin I applying the same i n c u b a t i o n conditions. The a n t i g e n - a n t i b o d y c o m p l e x was dissociated with 10 M acetic acid* (80 p.l). The acid was b l o w n o u t u n d e r a gentle stream o f nitrogen and the t c x i n solubiiized in 0.05 M sodium p h o s p h a t e b u f f e r p H 7.4, containing bovine serum albumin 5°[& ( w / w ) (PBA buffer). The insciuble 3' globulins were centrifuged o f f and the t o x k ity o f the s u p e r n a t a n t was d e t e r m i n e d . F r o m the first e x p e r i m e n t it was clear tk~.t native 163
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o f b o u n d f r o m free a n t i g e n was c a r r i e d o u t b y a d o u b l e a n t i b o d y s y s t e m : 0 . 2 rnl n o r m a l r a b b i t s e r u m diluted 300 times (carder) and 0.2 ml donkey antir a b b i t s e r u m d i l u t e d 16 t i m e s w e r e a d d e d t o e a c h t u b e . T h e p : r e c i p i t a t i o n was ~Alowed t o p r o c e e d a t 4 ° C d u r i n g 4 2 h. T h e r e s u l t i n g p r e c i p i t a t e s were c e n t r i f u g e d ( 1 2 0 0 0 X g , 5 m i x ) a n d c o u n t e d for
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b o u n d r a d i o a c t i v i t y . R e s u l t s were e x p r e s s e d as t h e r a t i o b o u n d : t o t a l r a d i o a c t i v i t y p e r c e n t ( B i T X 100). C o r r e c t i o n s have b e e n i n t r o d u c e d for 12s[ d e c a y T h e non-specific: c o u n t s w e r e n u m b e r e d : f o r e a c h s t a n d a r d curve o n e a d d i t i o n a l s a m p l e was i n c u b a t e d w i t h a large excess o f t~ative t o x i n (104 t i m e s m o r e A a H t o x i n I than t h e h i g h e s t c o n c e n t r a t i o n o f ti~e sea!e).
3. R e s u l t s artd d i s c u s s i o n tO
20
~,0
TGxle.
{pg )
40
Fig. 1. Quantitative precipitation by the scram prepared against AaH toxin [, o f a mLxture of native and t2SI4abeled AaH toxin I. Different volumes of a 0.15 M NaCl s o h t i o n containing the t:SI-lab¢led and native toxins in a ratio of 1 : 6250 were added to 0.2 ml spec;~ic antiserum in a series of test tubes. Volumes were further made up to 1 ml with 0.15 M NaCI. After incubation f-trst at 37°(2 (1.5 h), then at 4°C (15 h), solutions were centrifuged at 3000 roy/mix for 15 mix and the supematants counted for radioactivity. Pellets were washed 3 times in cold saline, then dissolved in 1 ml 0_5 N NaOH for measurements of the A2a0 (o o) and radioact~'¢ity (~. ~).
and iodinated toxins behave similarly with respect to t h e a n t i s e r u m ( f i g . l ) . This o b s e r v a t i o n c o u l a b e e x t e n d e d f u r t h e r : 7 5 % ~2SI-labeled t o x i n a n d 70% n a t i v e t o x i n were r e c o v e r e d i n t h e p r e c i p i t a t e . T h i s indicated that both toxins behave identically.
2.2. R a d i o i m m u n o a s s a y procedure All s a m p l e s were a n a l y z e d i n d u p l i c a t e . All d i l u t i o n s a n d final v o l u m e a d j u s t m e n t s t h r o u g h o u t t h e e x p e r i m e n t s were achieved w i t h P B A b u f f e r . I n test t u b e s , t h e d i l u t e d r e a c t a n t s were a d d e d successively: P B A b u f f e r ( 0 . 2 m l ) , s t a n d a r d t o x i n o r u n k n o w n (O.I m i ) , 12Sl-labeled A a H t o x i n [ ( 0 . i m l ) , s e r u m a g a i n s t A a H t o x i n I ( 0 . I m l ) . T h e t u b e s were c o u n t e d for t o t a l r a d i o a c t i v i t y , t h e n i n c u b a t e d e i t h e r for 1 d a y at 37°(2 the separation o r for 6 d a y s at "4 ° C. A f t e r mcuba*:lon, " " 164
T h r e e s t a n d a r d cu_~es have b e e n e s t a b l i s h e d w i t h n a t i v e A a H t o x i n I. C o n d i t i o n s a n d results are g i v e n i n t a b l e I a n d fig.2. E a c h p o i n t s t a n d s f o r t h e average o f c l o s e i y - r e l a t e d d u p l i c a t e s . T h e s e s t a n d a r d curves have b e e n o b t a i n e d r e p e a t e d l y w i t h f r e s h l y p r e p a r e d 12St-labeled a n t i g e n . I n e a c h case, t h e a m o u n t o f n a t i v e Aa~?. t o x i n I l e a d i n g t o t h e h a l f - m a x k n u m e f f e c t was calcula~:ed a n d was f o u n d t o b e 2 8 4 0 , 2 3 0 a n d 7.2 pg f o r t h e s t a n d a r d curves [, H a n d / H , respectively. A c c u r a t e q u a n t i f i c a t i o n o f t h e t o r l n is p o s s i b l e : see s t a n d a r d curves I a n d H. W h e n d e a l i n g w i t h lfigh c o n c e n tr a tio n s o f toxin, the reference to ; t a n d a r d c u r v e I allows a q u i c k e r m e a s u r e o f t h e ~:oxin. A g r e a t e r sensiti,Aty is actfieved b y d i l u t i o n o f t h e serum and increase o f i n c u b a t i o n time: standard curves I I an d I I L I n t~:e case o f s t a n d a r d c u r v e IH, v e r y small a m o u n t s o f t o x i n ( 1 0 - i s m o o c a n b e d e t e c t e d . T h e s e results e m p h a s i z e t h e s t r o n g a v i d i t y of the serum against A a H t o x i n I towards the t2SI-labeled a n t i g e n : 5 0 % r a d i o a c t i v i t y still r e m a i n s b o u n d ever,,= w h e n t h e a n t i s e r u n - is d i l u t e d 1 2 5 0 0 0 0 times. T a k i n g i n t o a c c o u n t t h e specific r a d i o a c t i v i t y o f ~2SI-labeled t o x i n , w e c o m m o n l y w o r k w i t h 1 0 0 0 c p m w h i c h , u p t o n o w , set ~he l i m i t s o f t h e test. I n t h e case o f s t a n d a r d curves [ a n d I I , it is e v e n possible to c a r r y o u t t h e e x p e r i m e n t w i t h a two-wee_k~ o l d 12sI-labeled t o x i n , pr=_widing t h a t n e w r e f e r e n c e curves are w o r k e d o ~ t . O n t h e c o n t r a r y , s t a n d a r d c u r v e I H has t o b e set u p for e a c h n e w assay wffh a f r e s h l y - p r e p a r e d ~2Sl-labeled t o x i n .
Volume 85, number [
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Table l St~ada~d curve i
~2*I-tabeled AaH toxin I
Assay antiserum dilution
Incubation conditions
20 000 cpm 1/25 000
24 h, 37°C
1/250 000
~ 6 days, 4°C i
0.118 ng [l
5000 cpm 0.029 ng
HI
!000 cpm a 1/1250000 0.0059 ng
a
~ o ~ 6,~ays, 4 C !
Radioactivity eount~ngs were made over 10 rain
Experimental conditions for the setting up o f the: standard curves I, H and .~, ~m~:,tt on fig.2 Three other pure toxins: AaH toxin HL AaH toxin I[ a n d B o t t o x i n t, w h i c h b e l o n g t o t h e t h r e e d i f f e r e n t groups o f toxins (groups 1 , 2 and 3, respectively) f o u n d in the v e n o m s o f N o r t h African scorpions, ha~e been studied for c o m p e t i t i o n with ~SI-labeled
80
-
g x
!
12
~
~ ~0
! ~"~----~+
, ~--~-
6
Fig.2. Effect o f various scorpion toxins on the binding o f ~25I-labeled AaH toxin I to the serum prepared against AaH toxin I. Standard curves achieved with AaH toxin [: (m ~) standard curve I; (* *) standard curve II; (e o) standaxd curve [[[ (experimental conditions are described in table 1). For AaH t o x ~ [I[ (~ ~); h a l l toxin H (o o); and Bot toxin [ ( ~ - - - o ) . Incubation condftions were the same as for standard curve L The abscissa is tlxe logarithm o f the molar concentration of native toxin in the solution tested. The ordinate is title ratio bound : total radic-.etivity percent.
A a H t o x i n I. E x p e r i m ental c o n d i t i o n s w e r e t h o s e o f s t a n d a r d c u r v e [. R e s ~ l t s are s h o w n also o n fig.2. H a ! f - m a × i m u m e f f e c t was o b t a i n e d w i t h 3 0 - t i m e s more AaH toxin IH and 4300-times more AaH toxin I I t h a n A a H t o x i n i[. i n t h e case o f B o t t o x i n [, n o e f f e c t w as o b t a i n e d e v e n w i t h a 4 3 0 0 - t i m e s e x c e s s o f t o x i n . C o n s i d e r i n g t h e v e n o m o f A n d r o c t o n u s australis H e c t o r , t o x i n I H h as b e e n classified first b y N - t e r m i n a l [5], then by the whole sequence determination [!4], in t h e s a m e g r o u p as t o x i n [. It is t h u s n o t s u r p r i s i n g t o f i n d s o m e cr o ss r e a c t i v i t y o f b o t h t o x i n s w i t h t h e s e r u m p r e p a r e d ag~dn.at A a H t o x i n I. T h i s result co~Zd b e e x p e c t e d si n ce t o x i n H I was e v e n p r e c i p i t a t e d in ag ar o se gel a n d neutrMLzed b y this s e r u m [ 1 3 ] . H o w ev er , t h e q u a n t i f i c a t i o n o f t o x i n I in this v e o o m b y r a d i o f f n m u n o a s s a y still r e m a i n s p o s s i b l e , si n c e it c o n t ai n s less t o x i n I [ I t h a n t o x i n I [ 2 ] . T h i s m e t h o d m a y b e also u s e f u l t o d e t e c t , in o t h e r s c o r p i o n v e n o m s , toxLqs r e l a t e d t o this g r o u p . O n t h e c o n t r a r y , r e s ul t s obtained with Bot to:tin [ and A a H t o x i n H do not s h o w a n y s':_gnificant cr o ss r e a c t i v i t y b e t w e e n t o x i n s b e l o n ~ n g t o g r o u p 1 a n d t o x i n s o f g r o u p s 2 o r 3: the half-mzxirr_,um e f f e c t o b t M n e d w i t h 12 ttg A a H t o x i n I I m i g h t b e exptain:ed _~ather b y a 0 . 0 2 % c o n t a m i n a t i o n b y A a H t o x i n [, i:han b y a v e r y w e a k r e a c t i v i t y o f A a H t o x i n II w i t h i h e a n t i s e r u m . M o r e o v e r , t h e p o s s i b i l i t y t o disclose t h e A a H t o x i n I a t c o n c e n t r a - - t i o n o f a b o u t 5 X ~0 '~2 M gives a n e w t o o ! t b r s t u d i e s on the mechanism of action of scorpion toxins. Radioi m m u n o a s s a y o f A a H toxkn I[ a n d B o t t o x i n I is n o w kn progress. 165
Volume 85, number 1
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Acknow|edgements This w(~rk ~tas s u p p o r t e d b y the Institut National de la Sant~i et de la R e c h e r c h e M~dicale (U.38, Director: Professor S. Lissitzky), t h e Centre National de Ia Recherche Scientifique ( E R A 617), the Direction des Recherches et M o y e n s d'Essais and the F o n d a t i o n p o u r la Recherche M~dicale Franqaise. The authoro° :are deeply i n d e b t e d to Professor F. Miranda f o r his c o n s t a n t interest and s u p p o r t during t h e c o u p e o f the: w o r k . T h e y t h a n k P r o f e s s o r P_ J a q u c t a n d Drs P. C a r a y o n a n d F. S a m p i e r i f o r m a n y
fruitful discu~ions. Fine secretarial w o r k o f Mrs G o m e z is acknowledged.
References [1] Rochat, H., Rochat, C., Kopeyan, C., Miranda, F., LissitzkBr, S. and Edman, P. (1970) FEBS Lett. 10, 349--351. [2] Miranda, F., Kopeyan, C., Roche, t, H., Rochat, C. and Lissitzky, S. (1970) Eur. J. Biocl~em. 16, 5 1 4 - 5 2 3 . [3] Rochat, H., RocLat, C., Miranda, F., Lissitzky, S. and Edman, P. (1970) Eur. J. Biochem. 17, 2 6 2 - 2 6 6 .
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[4] Rochat, I~2°,Roeha~, C_, Sanlpieri, F., Mhranda, F. and Lis~tzky, S. (1972) Eur. J. Biochem. 28, 381--388. [5] Rochat, I~., Kopeyan, C., Gareia, L., Marfinez, G., Rosso, J. ]L, Pakaris, A., Martin, M. F., Garcia, A., Martin-Mc~utot, N., Gregoire, L and Miranda, F. (1976) in: ±~'l/mal, Plant and Microbial T o x i n s (Ohsaka, A., Hayashi, ~.. and Sawa/, Y. eds) VoL 2, pp. 79--87, Plenum Press, New York, London. [6] Schmitt, O. and Schmidt, H. (1972) Pfliigers Arch: 333, 51-61. [7] Catterall, W. A. (1975) Proc. Natl. Acad. Sci. USA 72, 1782-17E6. [8] Romey, G-, Chicheportich~, R., Lazdunski, M., Rochat, H., Miranda, F. and Lissitzky, S. (1975) Biochem. Biophys. l,les. Commun. 64, ! 1 5 - 1 2 1 . [9] Couraud, i[7, Rochat, H. and Lissitzky, S. (1976) Biochim. Biophys. Acta 433, 9 0 - 1 0 0 . [10] Bernard, P., Couraud, F. and Lissitzky, S. (1977) Biochem. Biophys. Res. Commun. 77, 782--788. [11] Rochat, H., Tessier, M., _~iranda, F. and LJssitzky, S. (1976) in: Anunal, Plant and Microbial Toxins (Ohsaka, A., Hayasbi, K. and Sawai, Y. eds) Vol. 1, pp. 81--88, Plenum Press, New York, London. [ 12] Rochat, H., Tessier, M., Miranda, F. and Lissitzky, S. (1977) Anal. Bioehem. in the press[13] Delox/, P., Miranda, F. and Rochat, H. (1976) m: Animal, Plant and Microbia! Toxins (Ohsaka, A., Hayashi, K. and SawM, Y. eds) Vol. 2, pp. 4 0 7 - 4 2 0 , Plenum Press, New York, London. [14] Kopeyan, C., Martinez, G. and Rochat, H. to be published.
FEBS LETTERS
A SENSITIVE
RAD[OIMMUNOASSAY Toxin i of Androctonus
J a n u a r y 1978
OF A SCORPION NEUROTOX[N a u s t r a l i s Hec,*or
Marie T E S S I E R , Pierre D E L O R [ , G u y BECH[S and Herv6 R O C H A T Lcboratoim de Biochimie, Facult~ de l~[~decine, Secteur Nord, Bd. Pierre Dramard, 13326 :~[arseille Cedex 3, irrance Received 20 O c t o b e r ! 9 7 7
t . L~troduction Scorpion n e u r o t o x i n s f o r m a family o f h o m o l o g o u s proteins which are basic and have a p p r o x , rnol. w t 7000 [ l ] . T h e y consist o f a s i n # e peptide chain cross!inked b y four disulfide bridges [2i. The c o m plete a m i n o acid sequences oI s o m e o f t h e m [3,4L as well as the N-term!ha ~.c f others, have been determined: their c o m p a r i s o n has led to a classification into four groups [5 i . T h e y have been s h o w n to affect the c o n d u c t i o n o f ions t h r o u g h mere brane channels [ 6 - - I 0 ] , and are thus g o o d tools for the s t u d y o f these structures on the molecular level. Toxins I and
H o f A n d r o c t o n u s ausgralis H e c t o r have b e e n ~2s[_ labeled: specific radioactivRies u p to 2 0 0 0 C i / m m o l have b e e n obtained [ 1 1 , 1 2 ] . We r e p o r t here the setting up o f a radioh-amunoassay allowing a sensitive and specific d e t e c t i o n o f t o x i n I o f A n d r o c t o n u s australis Hector.
2. Materials a n d m e t h o d s
p r e p a r a t i o n s were p e r f o r m e d in the course o f this w o r k : the m e a n specific radioactivity was 1750 --+4 0 0 C i / m m o l , Le., 0.87 ± O.17 ~zs~ a t o m i n c o r p ~ r a t e d / m o l toxin. S e r u m against A s H t o x i n [ has b e e n p r e p a r e d f r o m rabbit, its neutralizing capacity tested on mice (Cs7/ B L / 6 ) was 157 t~g t o x i n / m ! serum [ 1 3 ] . _Amti-ratpbit serum p r e p a r e d in d o n k e y was purchased f r o m Wetlcorne, B e c k e n h a m , E n ~ a n d ( R D 17) and n o r m a l rabbit serum was o b t a i n e d in o u r l a b o r a t o r y 2.1. Quantitative ant@sis Two successive experhnents
have been performed
in order to c h e c k the b e h a v i o u r ~ f b o t h ~:S[-labeied and native toxins in the presence o f the A a H - t o ~ n I antiserum. A first quantitative precipitin test allowed to o b t a i n the conditions o f m a x ' ; m u m precipitation for each t o x i n and to k n o w the a m o u n t o f 125Llabeled toxin present in the precipitate (see fig.l). The maxim u m a m o u n t o f native t o x i p precipitated was determ i n e d in a second e x p e r i m e n t b y t o x i c i t y assay on the solubilized pellet. A single precipitation was
T o x i n s I, H and I ~ f f A n d r o c t o n u s australis H e c t o r ( A s h t o x i n I, A s H t o x i n I I and A a H t o x i n H I ) and t o x i n I o f B u t h u s occitanus tunetanus ( B o t toxin l) were purified according [ 2 ] . A a H t o x i n I was ~2Sl-labeled and isolated f r o m the reaction m e d i u m b y precipitation with its m o n o specific a n t i s e r u m as described [! 1,12]. T e n different * T o x i c i t y o f A a H t o x i n [ was f o u n d t o b e u n a l t e r e d b y this aeidfie t r e a t m e n t [ 12]
Elsevier~North-Holland Biomedical Press
carried o u t o n 20/~g native A a H ~oxin I applying the same i n c u b a t i o n conditions. The a n t i g e n - a n t i b o d y c o m p l e x was dissociated with 10 M acetic acid* (80 p.l). The acid was b l o w n o u t u n d e r a gentle stream o f nitrogen and the t c x i n solubiiized in 0.05 M sodium p h o s p h a t e b u f f e r p H 7.4, containing bovine serum albumin 5°[& ( w / w ) (PBA buffer). The insciuble 3' globulins were centrifuged o f f and the t o x k ity o f the s u p e r n a t a n t was d e t e r m i n e d . F r o m the first e x p e r i m e n t it was clear tk~.t native 163
Volume 85, number 1
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o f b o u n d f r o m free a n t i g e n was c a r r i e d o u t b y a d o u b l e a n t i b o d y s y s t e m : 0 . 2 rnl n o r m a l r a b b i t s e r u m diluted 300 times (carder) and 0.2 ml donkey antir a b b i t s e r u m d i l u t e d 16 t i m e s w e r e a d d e d t o e a c h t u b e . T h e p : r e c i p i t a t i o n was ~Alowed t o p r o c e e d a t 4 ° C d u r i n g 4 2 h. T h e r e s u l t i n g p r e c i p i t a t e s were c e n t r i f u g e d ( 1 2 0 0 0 X g , 5 m i x ) a n d c o u n t e d for
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b o u n d r a d i o a c t i v i t y . R e s u l t s were e x p r e s s e d as t h e r a t i o b o u n d : t o t a l r a d i o a c t i v i t y p e r c e n t ( B i T X 100). C o r r e c t i o n s have b e e n i n t r o d u c e d for 12s[ d e c a y T h e non-specific: c o u n t s w e r e n u m b e r e d : f o r e a c h s t a n d a r d curve o n e a d d i t i o n a l s a m p l e was i n c u b a t e d w i t h a large excess o f t~ative t o x i n (104 t i m e s m o r e A a H t o x i n I than t h e h i g h e s t c o n c e n t r a t i o n o f ti~e sea!e).
3. R e s u l t s artd d i s c u s s i o n tO
20
~,0
TGxle.
{pg )
40
Fig. 1. Quantitative precipitation by the scram prepared against AaH toxin [, o f a mLxture of native and t2SI4abeled AaH toxin I. Different volumes of a 0.15 M NaCl s o h t i o n containing the t:SI-lab¢led and native toxins in a ratio of 1 : 6250 were added to 0.2 ml spec;~ic antiserum in a series of test tubes. Volumes were further made up to 1 ml with 0.15 M NaCI. After incubation f-trst at 37°(2 (1.5 h), then at 4°C (15 h), solutions were centrifuged at 3000 roy/mix for 15 mix and the supematants counted for radioactivity. Pellets were washed 3 times in cold saline, then dissolved in 1 ml 0_5 N NaOH for measurements of the A2a0 (o o) and radioact~'¢ity (~. ~).
and iodinated toxins behave similarly with respect to t h e a n t i s e r u m ( f i g . l ) . This o b s e r v a t i o n c o u l a b e e x t e n d e d f u r t h e r : 7 5 % ~2SI-labeled t o x i n a n d 70% n a t i v e t o x i n were r e c o v e r e d i n t h e p r e c i p i t a t e . T h i s indicated that both toxins behave identically.
2.2. R a d i o i m m u n o a s s a y procedure All s a m p l e s were a n a l y z e d i n d u p l i c a t e . All d i l u t i o n s a n d final v o l u m e a d j u s t m e n t s t h r o u g h o u t t h e e x p e r i m e n t s were achieved w i t h P B A b u f f e r . I n test t u b e s , t h e d i l u t e d r e a c t a n t s were a d d e d successively: P B A b u f f e r ( 0 . 2 m l ) , s t a n d a r d t o x i n o r u n k n o w n (O.I m i ) , 12Sl-labeled A a H t o x i n [ ( 0 . i m l ) , s e r u m a g a i n s t A a H t o x i n I ( 0 . I m l ) . T h e t u b e s were c o u n t e d for t o t a l r a d i o a c t i v i t y , t h e n i n c u b a t e d e i t h e r for 1 d a y at 37°(2 the separation o r for 6 d a y s at "4 ° C. A f t e r mcuba*:lon, " " 164
T h r e e s t a n d a r d cu_~es have b e e n e s t a b l i s h e d w i t h n a t i v e A a H t o x i n I. C o n d i t i o n s a n d results are g i v e n i n t a b l e I a n d fig.2. E a c h p o i n t s t a n d s f o r t h e average o f c l o s e i y - r e l a t e d d u p l i c a t e s . T h e s e s t a n d a r d curves have b e e n o b t a i n e d r e p e a t e d l y w i t h f r e s h l y p r e p a r e d 12St-labeled a n t i g e n . I n e a c h case, t h e a m o u n t o f n a t i v e Aa~?. t o x i n I l e a d i n g t o t h e h a l f - m a x k n u m e f f e c t was calcula~:ed a n d was f o u n d t o b e 2 8 4 0 , 2 3 0 a n d 7.2 pg f o r t h e s t a n d a r d curves [, H a n d / H , respectively. A c c u r a t e q u a n t i f i c a t i o n o f t h e t o r l n is p o s s i b l e : see s t a n d a r d curves I a n d H. W h e n d e a l i n g w i t h lfigh c o n c e n tr a tio n s o f toxin, the reference to ; t a n d a r d c u r v e I allows a q u i c k e r m e a s u r e o f t h e ~:oxin. A g r e a t e r sensiti,Aty is actfieved b y d i l u t i o n o f t h e serum and increase o f i n c u b a t i o n time: standard curves I I an d I I L I n t~:e case o f s t a n d a r d c u r v e IH, v e r y small a m o u n t s o f t o x i n ( 1 0 - i s m o o c a n b e d e t e c t e d . T h e s e results e m p h a s i z e t h e s t r o n g a v i d i t y of the serum against A a H t o x i n I towards the t2SI-labeled a n t i g e n : 5 0 % r a d i o a c t i v i t y still r e m a i n s b o u n d ever,,= w h e n t h e a n t i s e r u n - is d i l u t e d 1 2 5 0 0 0 0 times. T a k i n g i n t o a c c o u n t t h e specific r a d i o a c t i v i t y o f ~2SI-labeled t o x i n , w e c o m m o n l y w o r k w i t h 1 0 0 0 c p m w h i c h , u p t o n o w , set ~he l i m i t s o f t h e test. I n t h e case o f s t a n d a r d curves [ a n d I I , it is e v e n possible to c a r r y o u t t h e e x p e r i m e n t w i t h a two-wee_k~ o l d 12sI-labeled t o x i n , pr=_widing t h a t n e w r e f e r e n c e curves are w o r k e d o ~ t . O n t h e c o n t r a r y , s t a n d a r d c u r v e I H has t o b e set u p for e a c h n e w assay wffh a f r e s h l y - p r e p a r e d ~2Sl-labeled t o x i n .
Volume 85, number [
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Table l St~ada~d curve i
~2*I-tabeled AaH toxin I
Assay antiserum dilution
Incubation conditions
20 000 cpm 1/25 000
24 h, 37°C
1/250 000
~ 6 days, 4°C i
0.118 ng [l
5000 cpm 0.029 ng
HI
!000 cpm a 1/1250000 0.0059 ng
a
~ o ~ 6,~ays, 4 C !
Radioactivity eount~ngs were made over 10 rain
Experimental conditions for the setting up o f the: standard curves I, H and .~, ~m~:,tt on fig.2 Three other pure toxins: AaH toxin HL AaH toxin I[ a n d B o t t o x i n t, w h i c h b e l o n g t o t h e t h r e e d i f f e r e n t groups o f toxins (groups 1 , 2 and 3, respectively) f o u n d in the v e n o m s o f N o r t h African scorpions, ha~e been studied for c o m p e t i t i o n with ~SI-labeled
80
-
g x
!
12
~
~ ~0
! ~"~----~+
, ~--~-
6
Fig.2. Effect o f various scorpion toxins on the binding o f ~25I-labeled AaH toxin I to the serum prepared against AaH toxin I. Standard curves achieved with AaH toxin [: (m ~) standard curve I; (* *) standard curve II; (e o) standaxd curve [[[ (experimental conditions are described in table 1). For AaH t o x ~ [I[ (~ ~); h a l l toxin H (o o); and Bot toxin [ ( ~ - - - o ) . Incubation condftions were the same as for standard curve L The abscissa is tlxe logarithm o f the molar concentration of native toxin in the solution tested. The ordinate is title ratio bound : total radic-.etivity percent.
A a H t o x i n I. E x p e r i m ental c o n d i t i o n s w e r e t h o s e o f s t a n d a r d c u r v e [. R e s ~ l t s are s h o w n also o n fig.2. H a ! f - m a × i m u m e f f e c t was o b t a i n e d w i t h 3 0 - t i m e s more AaH toxin IH and 4300-times more AaH toxin I I t h a n A a H t o x i n i[. i n t h e case o f B o t t o x i n [, n o e f f e c t w as o b t a i n e d e v e n w i t h a 4 3 0 0 - t i m e s e x c e s s o f t o x i n . C o n s i d e r i n g t h e v e n o m o f A n d r o c t o n u s australis H e c t o r , t o x i n I H h as b e e n classified first b y N - t e r m i n a l [5], then by the whole sequence determination [!4], in t h e s a m e g r o u p as t o x i n [. It is t h u s n o t s u r p r i s i n g t o f i n d s o m e cr o ss r e a c t i v i t y o f b o t h t o x i n s w i t h t h e s e r u m p r e p a r e d ag~dn.at A a H t o x i n I. T h i s result co~Zd b e e x p e c t e d si n ce t o x i n H I was e v e n p r e c i p i t a t e d in ag ar o se gel a n d neutrMLzed b y this s e r u m [ 1 3 ] . H o w ev er , t h e q u a n t i f i c a t i o n o f t o x i n I in this v e o o m b y r a d i o f f n m u n o a s s a y still r e m a i n s p o s s i b l e , si n c e it c o n t ai n s less t o x i n I [ I t h a n t o x i n I [ 2 ] . T h i s m e t h o d m a y b e also u s e f u l t o d e t e c t , in o t h e r s c o r p i o n v e n o m s , toxLqs r e l a t e d t o this g r o u p . O n t h e c o n t r a r y , r e s ul t s obtained with Bot to:tin [ and A a H t o x i n H do not s h o w a n y s':_gnificant cr o ss r e a c t i v i t y b e t w e e n t o x i n s b e l o n ~ n g t o g r o u p 1 a n d t o x i n s o f g r o u p s 2 o r 3: the half-mzxirr_,um e f f e c t o b t M n e d w i t h 12 ttg A a H t o x i n I I m i g h t b e exptain:ed _~ather b y a 0 . 0 2 % c o n t a m i n a t i o n b y A a H t o x i n [, i:han b y a v e r y w e a k r e a c t i v i t y o f A a H t o x i n II w i t h i h e a n t i s e r u m . M o r e o v e r , t h e p o s s i b i l i t y t o disclose t h e A a H t o x i n I a t c o n c e n t r a - - t i o n o f a b o u t 5 X ~0 '~2 M gives a n e w t o o ! t b r s t u d i e s on the mechanism of action of scorpion toxins. Radioi m m u n o a s s a y o f A a H toxkn I[ a n d B o t t o x i n I is n o w kn progress. 165
Volume 85, number 1
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Acknow|edgements This w(~rk ~tas s u p p o r t e d b y the Institut National de la Sant~i et de la R e c h e r c h e M~dicale (U.38, Director: Professor S. Lissitzky), t h e Centre National de Ia Recherche Scientifique ( E R A 617), the Direction des Recherches et M o y e n s d'Essais and the F o n d a t i o n p o u r la Recherche M~dicale Franqaise. The authoro° :are deeply i n d e b t e d to Professor F. Miranda f o r his c o n s t a n t interest and s u p p o r t during t h e c o u p e o f the: w o r k . T h e y t h a n k P r o f e s s o r P_ J a q u c t a n d Drs P. C a r a y o n a n d F. S a m p i e r i f o r m a n y
fruitful discu~ions. Fine secretarial w o r k o f Mrs G o m e z is acknowledged.
References [1] Rochat, H., Rochat, C., Kopeyan, C., Miranda, F., LissitzkBr, S. and Edman, P. (1970) FEBS Lett. 10, 349--351. [2] Miranda, F., Kopeyan, C., Roche, t, H., Rochat, C. and Lissitzky, S. (1970) Eur. J. Biocl~em. 16, 5 1 4 - 5 2 3 . [3] Rochat, H., RocLat, C., Miranda, F., Lissitzky, S. and Edman, P. (1970) Eur. J. Biochem. 17, 2 6 2 - 2 6 6 .
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[4] Rochat, I~2°,Roeha~, C_, Sanlpieri, F., Mhranda, F. and Lis~tzky, S. (1972) Eur. J. Biochem. 28, 381--388. [5] Rochat, I~., Kopeyan, C., Gareia, L., Marfinez, G., Rosso, J. ]L, Pakaris, A., Martin, M. F., Garcia, A., Martin-Mc~utot, N., Gregoire, L and Miranda, F. (1976) in: ±~'l/mal, Plant and Microbial T o x i n s (Ohsaka, A., Hayashi, ~.. and Sawa/, Y. eds) VoL 2, pp. 79--87, Plenum Press, New York, London. [6] Schmitt, O. and Schmidt, H. (1972) Pfliigers Arch: 333, 51-61. [7] Catterall, W. A. (1975) Proc. Natl. Acad. Sci. USA 72, 1782-17E6. [8] Romey, G-, Chicheportich~, R., Lazdunski, M., Rochat, H., Miranda, F. and Lissitzky, S. (1975) Biochem. Biophys. l,les. Commun. 64, ! 1 5 - 1 2 1 . [9] Couraud, i[7, Rochat, H. and Lissitzky, S. (1976) Biochim. Biophys. Acta 433, 9 0 - 1 0 0 . [10] Bernard, P., Couraud, F. and Lissitzky, S. (1977) Biochem. Biophys. Res. Commun. 77, 782--788. [11] Rochat, H., Tessier, M., _~iranda, F. and LJssitzky, S. (1976) in: Anunal, Plant and Microbial Toxins (Ohsaka, A., Hayasbi, K. and Sawai, Y. eds) Vol. 1, pp. 81--88, Plenum Press, New York, London. [ 12] Rochat, H., Tessier, M., Miranda, F. and Lissitzky, S. (1977) Anal. Bioehem. in the press[13] Delox/, P., Miranda, F. and Rochat, H. (1976) m: Animal, Plant and Microbia! Toxins (Ohsaka, A., Hayashi, K. and SawM, Y. eds) Vol. 2, pp. 4 0 7 - 4 2 0 , Plenum Press, New York, London. [14] Kopeyan, C., Martinez, G. and Rochat, H. to be published.