Purification and properties of the insect toxin from the venom of the scorpion Androctonus australis Hector

BIOCHIMIE, 1971, 53, 1073-1078.

Purification and properties of the insect toxin from the venom of the scorpion Androctonus australis Hector. E. ZLOTKIN (1), H. ROCHAT (1), KOPEYAN (1), F. MIRANDA (1) a n d S. LISSITZKY (2). (29-9-1971) . S u m m a r y . - - The purification of a protein toxic to insects from the venom of the scorpion A n d r o c t o n u s a u s t r a l i s H e c t o r has been p e r f o r m e d by recycling Sephadex G-50 gel filtration and equilibrium c h r o m a t o g r a p h y on DEAE-Sephadex A-50 and Amberlite CG-50. The final product was purified 267-fold as compared to the crude venom. The toxicity yield w a s 95 percent. P u r i t y of the insect toxin was assessed b y amino acid analysis, N - t e r m i n a l sequential degradation, C-terminal amino acid d e t e r m i n a t i o n and zone electropho.resis. Its molecul~ar weigh,t is 7498. The insect toxin differs from the proteins toxic to m a m m a l s contained in the same venom. The sequence of the first 15 amino acid residues f r o m the N-terminal of b o t h types of toxins is compared. The physiological significance of these neurotoxic proteins is discussed.

INTRODUCTION.

d i s t i n c t f r o m t h o s e c a u s i n g ~ e t h a l i t y i n m i c e . It h a s b e e n f o u n d t h a t t h e p r o t e i n t o x i c to f l y l a r v a e provoked a strong and rapid paralysis when i n j e c t e d i n t o five o t h e r i n s e c t s b e l o n g i n g to t h r e e d i f f e r e n t o r d e r s [3]. T h i s o b s e r v a t i o n a n d t h e fact that the toxic protein was inactive on other

P r e v i o u s s t u d i e s h a d s h o w n t h a t t h e t o x i c effect to b l o w f l y l a r v a e of t h e v e n o m of A n d r o c t o n u s australis Hector [1] a n d of six o t h e r s c o r p i o n v e n o m s E2] w a s r e l a t e d to p r o t e i n c o m p o n e n t s

TABLE I. Purification

Purification step

of the insect toxin

Experimental conditions

water

Gel filtration with 0.1 M ammonium acetate recycling on Sephadex pH 8.5 - 8.6 (four G-50 3.2 X 100 em columns in series)

A 280

Total toxicity

STa

Yield in toxicity from the crude venom

Units

C.P.U.a

C.P.U./A 280

%

1188.0

3,280,000

2,760

100.0

766.0

3,170,000

4,140

96.6

115.7 O- 12.3b

4,230,000 _____200,000

36,560

129.0

tq

Crude venon (one gram) Extraction and dialysis

of A. a u s t r a l i s v e n o m .

1

DEAE-Sephadex A-50

0.1 M ammonium acetate pH 8.50 (2 X 200 cm column)

1 B

11.7 ~- 1.1

4,040,000 + 210,000

345,300

123.0

A mberlite CG-50

0.2 M ammonium acetate pH 6.30 (2 X 200 cm column)

1C

5.5 -~ 0.3

3,110,000 + 180,000

565,455

94.8

(a) contraction p a r a l y s i s u n i t [4]. (b) "Mean _ s t a n d a r d deviation of six c h r o m a t o g r a p h i c runs. (1) Laboratoire de Biochimie, Facult6 de M6decine, Secteur Nord, Bd Pierre Dramard, 13- Marseille 15e, France.

(2) Laboratoire de Biochimie M6dieale et I n s t i t u t National de la Sant6 et de la Recherche M6dieale (G 38), Facult~ de Mddecine, 27 Bd Jean Moulin, 13 2 Marseille 5~, France. :

E. Z l o t k i n , H. Rochat, C. K u p e y a n , F. Miranda a n d S. L i s s i t z k y .

1074

a r t h r o p o d s ( a n a r a c h n i d : a n d a c r u s t a c e a n [3]) indicate that the blowfly larvae toxin was more generally a protein toxic to insects. This protein w i l l b e r e f e r r e d to f u r t h e r a s t h e i n s e c t t o x i n i n s t e a d of t h e l a r v a e t o x i n [3].

o+b A

1/~j~

....

I

°'

.

~

",

4ol

~

o ~ - ~ 3

- 6

' 0"121 B

T h e v e n o m of Androctonus australis Hector w a s o b t a i n e d b y e l e c t r i c a l s t i m u l a t i o n of a n i m a l s coll e c t e d i n t h e a r e a of C h e H a l a ( A l g e r i a ) . T o x i c i t y m e a s u r e m e n t s w e r e c a r r i e d o u t b y i n j e c t i o n of f r a c t i o n s of c o l u m n c h r o m a t o g r a p h y e f f l u e n t s to b l o w f l y l a r v a e (Sarcophaga argyrostoma) a n d e x p r e s s e d as c o n t r a c t i o n - p a r a l y s i s u n i t s (C.P.U.) [4]. T o x i n p u r i f i c a t i o n w a s f o l l o w e d b y t h e d e t e r m i n a t i o n of t h e s p e c i f i c t o x i c i t y ~(S T) a r b i t r a r i l y d e f i n e d as C.P.U. p e r A 280 u n i t (STa). W h e n p u r e toxin was avaiable, the specific toxicity related to t h e w e i g h t of t o x i n (C.P.U. p e r m g p r o t e i n o r STw) was determined.

The pure toxin was fully reduced and methylat e d a c c o r d i n g to ROCHAT et al. [6] e x c e p t t h a t d i t h i o e r y t h r i t o l i n a r a t i o of 30 m o l e s p e r m o l e of h a l f - c y s t i n e w a s u s e d i n s t e a d of 2 - m e r c a p t o e t h a nol. T h e f u l l y r e d u c e d a n d m e t h y l a t e d t o x i n (RMt o x i n ) w a s p r e c i p i t a t e d b y a d d i t i o n of a n e q u a l v o l u m e of 2 M a c e t i c a c i d . T h e p r e c i p i t a t e w a s collected by centrifugation and dissolved in water. Gel f i l t r a t i o n o n S e p h a d e x G-15 e q u i l i b r a t e d i n 1 M acetic acid allowed the complete separation of t h e R M - t o x i n f r o m t h e r e m a i n i n g r e a g e n t c o n taminants. The pure RM-toxin was recovered by f r e e z e - d r y i n g of t h e e x c l u d e d p e a k f r o m S e p h a d e x filtration. T h e m a n u a l d e t e r m i n a t i o n of t h e N - t e r m i n a l s e q u e n c e w a s p e r f o r m e d o n 0:57 +xmole of t h e RMtoxin by the phenylisothiocyanate m e t h o d [7] a c c o r d i n g to BLOMBACK et al. [8]. C-terminal amino acid determination was carr i e d o u t o n 0.53 ,~mole of t h e R M - t o x i n b y c a r b o x y p e p t i d a s e A d i g e s t i o n ( e n z y m e to s u b s t r a t e r a t i o 2 : 1 0 0 , w / w ) f o r 1, 2, 5, 10, 30 a n d 60 m i n u t e s a t r o o m t e m p e r a t u r e a c c o r d i n g to SLOBIN a n d CAnPENTER

[91.

BIOCHIMIE, 1971, 53, n ° 10.

~ .............

8

I 200 FRACTION

o

"'"......... i..........." z " 5

' ~_

100

1+~ -

.... ~ 4

7 EFFLUENT(liter)

Hector.

All t h e c h r o m a t o g r a p h i c m e t h o d s u s e d f o r t h e p u r i f i c a t i o n of t h e i n s e c t t o x i n h a v e b e e n p r e v i o u s l y d e s c r i b e d i n d e t a i l a s w e l l as ,file t e c h n i q u e s of sta~'eh gel e l e c t r o p h o : r e s i s a n d a m i n o a c i d e s t i m a t i o n [5].

~x

N

In this paper, we describe the isolation and s o m e c h e m i c a l p r o p e r t i e s of t h e i n s e c t t o x i n , p u r i fied f r o m t h e v e n o m o f Androctonus australis

MATERIALS AND METHODS.

.px.~/

I

_

NUMBER (3ml)

C

0,8-

o3

,< 0.4

100

FRACTION NUMBER (3ml)

200

Fro. 1. - - Purification of the insect toxin of A. australis Hector venom. (A) Recycling gel filtration on Sephadex G-50. F o u r columns of 3.2 X 100 em in series ; 0.1 M a m m o n i u m acetate buffer pH 8.5-8.6 ; flow rate 60 m l / h . The m i x t u r e s u b m i t t e d to f r a c t i o n a tion is the w a t e r extract of 2 g of venom. IT, toxic f r a c t i o n s to fly larvae. R1, toxic fractions c o n t a i n i n g mice toxins I a n d III [5]. I~, toxic f r a c t i o n s corresp o n d i n g to mice t o x i n II [5]. Fractions A to D pooled according to the a b s o r p t i o n p a t t e r n at 280 n m arc non toxic to larva+e as well as to mice. E is a f r a c t i o n toxic to crustaceans [3]. Vertical arrows a n d n u m b e r s correspond to the b e g i n n i n g of the consecutive cycles. F r a c t i o n s of the e l u t i o n curves indicated by the full line are collected. Only t h e m a t e r i a l m a r k e d b y the dotted line is recycled. (B) C h r o m a t o g r a p h y on DEAESephadex A-50 of f r a c t i o n s IT of the preceding step o b t a i n e d from 0.5 g of crude venom. 2 X 200 cm column in 0.1 M a m m o n i u m acetate buffer pH 8.50. Flow rate 12 m l / h . The h o r i z o n t a l arrow indicates the fractions toxic to fly larvae. (C) C h r o m a t o g r a p h y on Amberlite CG-50 of the toxic fractions o b t a i n e d in B f r o m 0.5 g of venom. 2 X 2.00 cm column in 0.2 M a m m o n i u m acetate buffer p,H 6.30. Flow r a t e 12 m l / h . The h o r i z o n t a l a r r o w indicates the fractions toxic to larvae. The average specific toxicity of these f.ractions was 565455 G.P.U. per u n i t of absorbance at 280 n m w i t h a s t a n d a r d d e v i a t i o n of ___ 12 890.

Purification of Deionized throughout.

quartz-redistilled

water

was

A. a u s t r a l i s

used

RESULTS.

Purification of insect toxin. T a b l e I s h o w s t h e s e q u e n c e of s t e p s u s e d to purify the insect toxin a n d the q u a n t i t a t i v e data r e l a t e d to th.e p u r i f i c a t i o n p r o c e d u r e . T h e e l u t i o n p a t t e r n s c o r r e s p o n d i n g to t h e d i f f e r e n t c h r o m a t o g r a p h i c s t e p s u s e d to o b t a i n t h e p u r e t o x i n a r e s h o w n in Fig. 1.

insect toxin.

1075

Properties of the toxin. S t a r c h gel e l e c t r o p h o r e s i s d e m o n s t r a t e d a single b a n d o f s l o w c a t h o d i c m o b i l i t y ( F i g . 2). T a b l e II s h o w s the a m i n o acid c o m p o s i t i o n , the m i n i m u m molecular weight and the molecular extinction c o e f f i c i e n t at 277 n m of t h e i n s e c t t o x i n . Halfc y s t i n e r e s i d u e c o n t e n t is 7.12. H o w e v e r a n a l y s e s p e r f o r m e d o n t h e fully r e d u c e d a~nd m e t h y l ated

TABLE II.

A m i n o acid composition of the insect toxin of A. a u s t r a l i s venom. Amido acid

+

Aspartic acid . . . . . . . . . . . . . . . . . . Threonine . . . . . . . . . . . . . . . . . . . . Serine . . . . . . . . . . . . . . . . . . . . . . . . . Glutamic acid . . . . . . . . . . . . . . . . . Proline . . . . . . . . . . . . . . . . . . . . . . Glycine . . . . . . . . . . . . . . . . . . . . . . . Alanine . . . . . . . . . . . . . . . . . . . . . . . Half cystine . . . . . . . . . . . . . . . . . . . Valine . . . . . . . . . . . . . . . . . . . . . . . . Methionine . . . . . . . . . . . . . . . . . . . . Isoleucine . . . . . . . . . . . . . . . . . . . . . Leucine . . . . . . . . . . . . . . . . . . . . . . . Tyrosine . . . . . . . . . . . . . . . . . . . . . . Phenyl alanine . . . . . . . . . . . . . . . . . Lysine . . . . . . . . . . . . . . . . . . . . . . . . Histidine . . . . . . . . . . . . . . . . . . . . . . Arginine . . . . . . . . . . . . . . . . . . . . . . Tryptophan . . . . . . . . . . . . . . . . . . . Total . . . . . . . . . . . . . Molecular weight . . . . . . . . . . . . . . . ~:max )< 10 -:~. . . . . . . . . . . . . . . . . . . EIi '~ 280 nm . . . . . . . . . . . . . . . . .

Insect toxin 11.30 3.70 5.70 3.07 1.15

(11) (4) (6) (3) (1)

3.98 (4) 2.92 (3) 7.12 (8)

2.88 (3) Oa 2.58 (2-3) 5.52 (5-6) 5.23 O. 96 7,25 0.92 0.94

(5) (1) (7) (1)

(1) 1 02 (1)

67 7498 12.88 (277 nm) 15.52

The results are expressed as m o l a r ratios taking amino acids stable to HC1 digestion as reference. Numbers in parentheses represent the closest integer. T r y p t o p h a n was estimated speetrophotometrieally. (a) Not detectable. t o x i n g i v e a v a l u e of 7.30 r e s i d u e s of S - m e t h y l c y s t e i n e w h i c h is c l o s e r to e i g h t . S i n c e d i r e c t m e t h y l a t i o n o f t h e t o x i n d o e s n o t resu.lt in S - m e t h y l c y s t e i n e p r o d u c t i o n , it is p r o b a b l e t h a t f o u r dis u l f i d e b o n d s a r e p r e s e n t in t h e m o l e c u l e .

IT Fro. 2. - -

CV

Starch gel eleetrophoretic migration of

insect toxin (I T) and etude venom (C V) of A. australis. Toxin (0.29 mg) and etude venom (5 rag) were

deposited in the position marked by the arrows. 13 p. cent gel in Tris-citrate buffer (0.076 M Tris and 0.005 M citric acid) pH 8.5 ; 1.2 mA/em. Straining w i t h Amido Black.

BIOCHIMIE, 1971, 53, n ° 10.

R e c o v e r y of t h e r . e d u c e d a n d S - m e t h y l a t e d t o x i n w a s 50 p. c e n t . F i f t e e n d e g r a d a t i o n c y c l e s u s i n g the phenylisothiocyan~ate m e t h o d established the N 4 e r m i n a l s e q u e n c e i l l u s t r a t e d i n Fig. 3. T h e s a m e 'figu:re g i v e s ,the c o r r e s p o n d i n g N - t e r m i n a l s e q u e n c e s of m i c e t o x i n s I, II a n d III p u r i f i e d f r o m t h e s a m e v e n o m [10]. Digestion of the RM-toxin w i t h c a r b o x y p e p t i dase A gave the C-terminal sequence-Thr-Ile-OH.

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E. Z l o t k i n , H. Rochat, C. K u p e g a n , F. Miranda a n d S. L i s s i t z k y . 10 H - Lys-Lys- Asn- G ly- T y r - A l a - V a l - A s p - S e r - S e r - L y s - L y s - A l a - Pro- GluH-Lys-Arg-Asp-- G l y - T y r - l l e - V a l - T y r - P r o - A s n - A s n - Cys-Val- T y r - HisH - V a I - L y s - A s p - G l y - T y r - Ile-Val-Asp- A s p - V a l - A s n - C y s - T h t - T y r - P h e H - V a l - A r g - A s p - G l y - T y r - I l e - V a l - A s p - X - L y s - A s n - C y s - V a l - T y r - His-

insect toxin mice toxin I mice toxin II mice toxin I l I

FIG. 3. - - N - t e r m i n a l a m i n o acid sequence of the insect toxin and of the m a m m a l toxins I, II a n d III [101 of A. australis venom. X, not datermined.

TABLE III.

Purification yields and weights of the LD.~, (mammal toxins [5]) and of the C.P.U. (insect toxin). Weight of the LDso [ [ for the 20 g mouse . . . . . . . l and of the C P U for ucemcmnL oi specllic~ Yield of toxins the t(~ m ~" l'arva rtoxicity enhancement! from crude venom / from crude venom

STw

Material mice

larvae

, LD.~o/mg

mice

larvae

~g

~tg

- fold

8.40 0.34 0.18 0.45

0. 3040 --

-28.0

weight %

i

Crude venom . . . . . . . . . . . . . . . Mice toxin I . . . . . . . . . . . . . . Mice toxin II . . . . . . . . . . . . . . . Mice toxin III . . . . . . . . . . . . . . Insect toxin I . . . . . . . . . . . . . .

i

119 2985 5539 2210 --

3280 -- -

-877586

(a) Calculated from the d a t a presented in table I f o r E 11,7. t i n -- 15.52 at 280 n m for the pure material.

DISCUSSION. T h e g e n e r a l m e t h o d of p u r i f i c a t i o n of l o w m o l e cular weight proteins which enabled the purific a t i o n of e l e v e n p r o t e i n s t o x i c to m a m m a l s f r o m t h r e e s c o r p i o n v e n o m s [ 5 ] w a s a p p l i e d w i t h succ e s s to t h e p u r i f i c a t i o n o f t h e i n s e c t t o x i n c o n t a i n e d i n t h e v e n o m of A. australis. A t o t a l a m o u n t of t w e l v e g r a m s of c r u d e v e n o m w a s s u b m i t t e d to t h e d e s c r i b e d p u r i f i c a t i o n p r o cedure with identical results. T h e h i g h r e c o v e r y of t o x i c i t y o b t a i n e d i n t h e f i n a l s t e p of p u r i f i c a t i o n ( T a b l e I), is n o t o n l y d u e to t h e s t a b i l i t y of t h i s t o x i n , b u t also to t h e i n c r e a s e of a c t i v i t y w h i c h f o l l o w e d t h e r e c y c l i n g S e p h a d e x G-50 s t e p ( T a b l e I). It is a s s u m e d t h a t t h e a b o v e i n c r e a s e of a c t i v i t y m a y b e d u e to t h e r e m o v a l d u r i n g S e p h a d e x f i l t r a t i o n of s o m e i n h i bitory component. Taking into account the data of T a b l e I I I a n d t h e f a c t .tha~ ~he .toxicity y i e l d s of p u r i f i c a t i o n of t h e i n s e c t a n d m a m m a l t o x i n s a r e 95 p. c e n t a n d 67 p. c e n t [5] r e s p e c t i v e l y , it is assmned that the crude venom contains 6-times less insect toxin than mammal toxins.

BIOCHIMIE, 1971, 53, n ° 10.

- - -

0.0011

4 2 . 0

18.6 267.0

the final step of purification

0.45 1 1.13 1.74 0.16 0.35 on the b a s i s

of

As i n s e c t s a r e t h e n a t u r a l p r e y of s c o r p i o n s , t h e insect toxin may be considered, from an ecolog i c a l p o i n t of v i e w , as a n e s s e n t i e l c o m p o n e n t of t h e c r u d e v e n o m . T h u s i t is i n t e r e s t i n g t o n o t i c e t h a t i t f o r m s o n l y a b o u t 0.35 p e r c e n t b y w e i g h ~ of the dry crude venom (Table III), a small amount w h i c h is l a r g e l y c o m p e n s a t e d b y its h i g h s p e cific t o x i c i t y . T h e p u r i t y of t h e i n s e c t t o x i n o b t a i n e d is a s c e r t a i n e d b y t h e f o l l o w i n g c r i t e r i a . (a) c o n s t a n t specific t o x i c i t y of t h e p e a k f r a c t i o n s e l u t e d f r o m A m b e r l i t e CG-50 c o l m n n s . (b) p r e s e n c e of a s i n g l e b a n d i n s t a r c h gel e l e c t r o p h o r e s i s . (c) n u m b e r s of a m i n o a c i d r e s i d u e s close to t h e i n t e g e r . (d) a b s e n c e of m e t h i o n i n e . (e) p r e s e n c e of a s~ngle phenylthiohydantoin a m i n o a c i d at e a c h s t e p of t h e d e g r a d a t i o n p r o c e d u r e . (f) p r e s e n c e of a s i n g l e C-terminal amino acid. H o w e v e r , f r a c t i o n a l n u m b e r s of a m i n o a c i d r e s i d u e s a r e o b s e r v e d f o r i s o l e u c i n e (2.58) a n d l e u c i n e (5.52) ( t a b l e II). S u c h a s i t u a t i o n h a s b e e n p r e v i o u s l y f o u n d f o r m i c e t o x i n I Of A. australis c o l l e c t e d i n t h e a r e a of T o z e u r ( T u n i s i a ) . B y

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P u r i f i c a t i o n o f A. a u s t r a l i s i n s e c t t o x i n . sequential d e g r a d a t i o n using the p h e n y l i s o t h i o cyanate m et h o d , it w a s d e n m n s t r a t e d that mice toxin I was actually a m i x t u r e of two i s o t o x i n s w h i c h differ only by the nature of the a m i n o acid in position 17 (valine or isoleucine) [11]. It is t h e r e f o r e 'likely that such isotoxins m i g h t be found in the case of the insect toxin. The m i n i m u m m o l e c u l a r w e i g h t of the insect to x i n d e t e r m i n e d by amino acid c o m p o s i t i o n (table II) must c o r r e s p o n d to the actual m o l e c u l a r w e i g h t since the elution volume of this p r o t e i n in S e p h a d e x G-50 gel filtration is close to that of m i c e n e u r o t o x i n I, the m o l e c u l a r w e i g h t of w h i c h c o r r e s p o n d e d in the u l t r a c e n t r i f u g e [12] and by sequence d e t e r m i n a t i o n [11] to the m i n i m u m mol ecu l ar w e i g h t d e t e r m i n e d by a m in o acid analysis. , ~ C o n t r a r y to most of mice n e u r o t o x i n s f o u n d in s c o r p i o n venoms, the insect n e u t r o t o x i n cannot be defined as a strongly basic p r o t e i n , as s h o w n by starch gel e l e c t r o p h o r e s i s . H o w e v e r a pHi above 7 is .likely. Some s i m i l a r i t i e s exist b e t w e e n the insect toxin a n d the t h ree m i c e toxins p r e s e n t in the v e n o m of A. australis [5]. (a) T h e total n u m b e r of a mi n o acids (67 in ~he i n s e c t toxin as c o m p a r e d to 63 or 64 in the m i c e toxins). (b) The p r e s e n c e of four disulfide bridges. (c) T h e absence of m e t h i o n i n e and cysteine. (d) T h e p r e s e n c e of one t r y p t o p h a n and one p h e n y l a l a n i n e residue. T h e c o m p a r i s o n of the first 15 a m i n o acid residues f r o m the N - t e r m i n a l end of the i n s e c t t o x i n to that of the m a m m a l toxins m i g h t be m o r e informative. T h e m i c e toxins have been d i v i d e d into three groups a c c o r d i n g to s i m i l a r i t i e s both in the N-term i n al sequence a n d specific activity [10], thus suggesting that the close resemblan.ce in N-terminal sequence m i g h t play an i m p o r t a n t role in the biological function. In spite of a r e s e m b l a n c e b e t w e e n the p r i m a r y st r u ct u r e of the i n s e c t to x i n and that of the mammal toxins of A, australis in the first eight positions of the sequence, all the other p o s i t i o n s are o c c u p i e d by d i f f e r e n t a m in o acids '(Fig. 3). The p r e s e n c e of a half-cystine r e s i d u e in p o s i t i o n 12 is c o m m o n to all a l r e a d y k n o w n m a m m a l toxins [10] w h e r e a s it is o c c u p i e d by lysine in the insect toxin. Although firm c o n c l u s i o n s must a w a i t the complete k n o w l e d g e of the amino a c i d s e q u e n c e of insect and m a m m a l toxins, this p r e l i m i n a r y w o r k suggests that the insect toxin m i g h t belong

BIOCHIMIE, 1971, 53, n ° 10.

to a d i f f er en t family of h o m o l o g o u s proteins. Such a p o ssi b i l i t y should p r o m p t the p u r i f i c a t i o n of a d d i t i o n a l insect toxins f r o m o t h er s c o r p i o n venoms [2]. W h e n i n j e c t e d into insects, the p r o t e i n studied in this p a p e r p r o v o k e s a r a p i d an d sustained paralysis of the an i m al s and t h e i r final killing. It is i n a c t i v e w h e n ap p l i ed to a r a c h n i d s and crustaceans w h i c h are affected by the cr u d e venom. This o b s e r v a t i o n resulted r e c e n t l y in the isolation and p a r t i a l p u r i f i c a t i o n f r o m the v e n o m of A. australis of an a d d i t i o n a l t o x i c p r o t e i n of low molecular w e i g h t w h i c h specifically p a r a l y z e s and kills a c r u s t a c e a n [3]. The h i g h level of specificity associated w i t h toxic p r o t e i n s p r e s e n t in a single v e n o m thus enabling t h e m to d i s c r i m i n a t e not only b e t w e e n m a m m a l s a n d insects but even b e t w e e n p h y l o ge,netically-~axonomically r el at ed g r o u p s of a r t h r o pods, is of special interest. The p o t e n c y and r a p i d i t y of a c t i o n as w e l l as the p a r a l y t i c n a t u r e of the effect p r o v o k e d by the purified t o x i n s after i n j e c t i o n into m a m m a l s and a r t h r o p o d s , suggest that these t o x i n s likely act at the level of the n e r v o u s a n d / o r the m u s c u l a r systems. If this is so, studies on s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s of these p r o t e i n s shotrld basically c o n t r i b u t e to the c o m p a r a t i v e m o l e c u l a r p h y si o logy and b i o c h e m i s t r y of n er v o u s a n d m u s c u l a r receptors.

Acknowledgements. One of us (E. Z.) was a recipient of a fello~vship from the Institut National de la Santd et de la Recherche Mddieale. This work was supported by the Direction des Reeherehes et Moyens d'Essais and by the Centre National de la R.eeherehe Seientiflque (RCP 166). We would like to express our appreciation to A. P~KARIS for his capable technical assistance and to Dr. IRU/qBI~RRY (Institut Pasteur, Alger, Algdrie) for supplying the scorpions.

R~SUMI~.

La purification d'une prot6ine toxique pour les inseetes a ~t~ r6alis6e h partir du venin du scorpion Androetonus anstralis Hector par recyelage sur Sephadex G-50 et chromatographie d'~quilibre sur DEAE-Sephadex A-50 et Amberlite CG-50. Gompard au venin total le produit final a ~t~ purifid 267 fois. Le rendement en toxieit6 est de 95 pour cent. La puret~ de la prot~ine toxique pour les inseetes a ~t~ d~termin~e par analyse d'aeides amines, d~gradation s~quenciclle h partir de l'extr~mit~ N-terminale, ddtermination de l'acide amin6 C-terminal et ~lectrophor~se de zone. Son poids mol6eulaire est 7498. La toxine pour les inseetes diff6re de eelles eontenues dans le m~me venin et toxiques

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p o u r les m a m m i f ~ r e s . La s 6 q u e n e e des 15 a c i d e s a m i n 6 s N - t e r m i n a u x d e s d e u x t y p e s de t o x i n e s est c o m p a r 6 e et la s i g n i f i c a t i o n p h y s i o l o g i q u e de ces p r o t 6 i n e s n e u r o t o x i q u e s est d i s c u t 6 e .

REFERENCES.

1. ZLOTKIN, E., MIRANDA,F., KUPEYAN,C. a n d LISSITZKY, S., Toxicon, 9 (1971) 9. 2. ZLOTKIN, E., MIRANDA,F. a n d LISSITZKY, S., Toxieon, in t h e p r e s s .

ZUSAMMENFASSUNG. Die R e i n i g u n g e i n e s fii.r I n s e k t e n t o x i s c h e n P r o t e i n s a u s d e m G i f t d e s S k o r p i o n s A n d r o c t o n u s australis Hector i s t mittels, R e z y k l i e r e n v o n S e p h a d e x GS0-Fi'ltrat i o n u n d C h r o m a t o g r a p h i e e n D E A E - S e p h a d e x A50 u n d A m b e r l i t CG-50 d u r c h g e f i i h r t w o r d e n . D a s E n d p r o d u k t w u r d e 267 Mal g e r e i n i g t i m V e e g l e i c h zu d e m r o h e n Gift. D i e Gi~'t a u s b e u t e w a r 95 %. Die R e i n h e i t des Insekttoxins wurde durch Aminos~iureanalyse, sequentiale N-End-Degradation, C-End-Aminos~iureB e s t i m n m n g u n d Z o n e n E l e k t , r o p h o r e s e gesch~itzt. Sein M o l e k u l a r g e w i c h t ist 7498. D a s I n s e k t t o x i n u n t e r s c h e i d e t sich y o n d e n fiir S~iu:getiere g i f t i g e n T o x i n e n , d i e in d e m s e l b e n Gift e n t h a l t e n s i n d . Die S e q u e n z d e r e r s t e n 15 A m i n o s ~ i u r e r e s t e a u s d e n N - E n d e n d e r b e i d e n T o x i n a r t e n w i r d v e r g l i c h e n . Die p h y s i o l o g i s c h e Bed e u t u n , g d i e s e r n e u r o t o x i s c h e n P.roteine w i r d b e s prochen.

BIOCHIMIE, 1971, 53, n ° 10.

3. ZLOTKIN, E., MIRANDA,F. a n d LISSITZKY, S., Toxicon, in t h e p r e s s . 4. ZLOTKIN, E., FnAENKEL, G., MmANDA, F. a n d LISSITZKY, S., Toxicon, 9 (1971) 1.

5. MIRANDA, F., KUPEYAN, C., ROCHAT, H., I~OCHAT, C. a n d LIssITzxY, S., Eur. J. Biochem., 16 (1970) 514. 6. ROCHAT, C., ROCHAT, I-I. a n d EDMAN, P., Anat. Biochem., 37 (1970) 259. 7. EDMAN,P., Acta Chem. Scan&, 4 (19'5.0) 283. 8. BLOMB.~CK,B., BLOMBACK,M., EDMAN, P. a n d HESSEL, B., Biochim. B i o p h y s . Acta, 115 (1966) 371. 9. SLOBIN, L. I. a n d CARPENTER, F. H., B i o c h e m i s t r y , 2 (19'63) 16.

10. ROCHAT, H., ROCHAT, C., KUPEYAN, C., MIRANDA, F., LISSITZKY, S., and EDMAN, P., F E B S Letters, 10 (1970) 349. 11. ROCHAT, H., ROCHAT, C., MIRANDA, F., LISSITZKY, S. and EDMAN, P., Eur. J. Biochem., 17 (1970) 262. 12. ROCHAT, C., ROCHAT, H., MIRANDA, F. a n d LISSITZEY, S., Biochemistry, 6 (1967) 578.