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ANALYSIS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF ANDROCTONUS MAURETANICUS MAURETANICUS (BLACK SCORPION) VENOM H. ZERROUK,~ " P. E. BOUGIS,~ B. CÉARD,~ A. BENSLIMANEZ and M. F. MARTIN-EAUCLAIRE~t 'Centre National de la Recherche Scientifique, URA 1179, Laboratoire de Hicehimie, Faculté de Médecine, Secteur Nord, Boulevard Pierre Dramard, 13326, Marseille, Cedex l5, France, and ~ Institut Pasteur, 1 Place Charles Nicole, B. P. 120, Casablanca, Morocco (Received 24 Septanber 1990 ; accepted l6 January 1991)

H. Z,ntROUx, P. E. BOUGIS, B. C~ARn, A. BENSLIMANI? and M. F. MARTnvEAUCLAIRE. Analysis by high-performance liquid chromatography of Androctonus mauretanicus mauretanicus (black scorpion) venom. Toxicon 29, 951-960, 1991 .-The venom of the black scorpion, Androctonus mauretanicus mauretanicus, was obtained by means of manual stimulation and was analyzed using high-performance liquid chromatography . Starting from 20 mg of venom and using only two chromatographic steps, six toxins were purified to homogeneity. They have been characterized by their amino acid content and compared to those already isolated from a pool of venoms obtained using electric stimulation (Rosso and ROCHAT, Toxicon 23, 113-125, 1985). The toxins Amm I and Amm II were not found, suggesting either different levels of toxin expression or the existence of Androctonus mauretanicus mauretanicus subspecies. Using rat brain synaptosomes, it was demonstrated that the toxins Amm III, Amm IV and Amm V were a-toxins . The toxin Amm VI was neither a- or ß-toxin. Unexpectedly, the toxin Amm VII was found to be a ß-toxin, the first one identified in a north African scorpion venom. In addition, some toxins active on mammals exhibited different levels of specificity towards phylogenetically related groups of arthropods.

INTRODUCTION

~ MST venomous scorpion to human beings in Morocco, i.e. the black scorpion, Androctonus mauretanicus mauretanicus, belongs to the Buthidae family . The first toxins active on mammals purified from buthid venoms were obtained using both molecular filtration and ion-exchange chromatography (MiRANDA et al., 1970). Today, the characterization of about one hundred of those toxins has been achieved using this conventional procedure (POSSAxI, 1984, WA'I-r and SIMARI), 1984). Such toxins constitute an homologous family of mini proteins made of 60 to 70 amino acid residues and cross-linked by " Permanent address : Institut Pasteur, 1 Place Charles Nicole, H . P . 120, Casablanca, Morocco . tAuthor to whom oorrsapondence should be addressod . 951

952

H. ZERROUK ct al.

four disulfide bridges . On the basis of sequence data and antigenic properties, they have been classified into several structural groups (ROCHAT et al., 1979 ; PossANt, 1984; W~Tr and $IMARD, 1984). They are known to interact specifically with the voltage-dependent sodium channel of excitable cells. According to their pharmacological effect and their binding on two different sites on the sodium channel, they have been called a- or ß-toxin. a-Toxins induce a prolongation of the repolarization phase of the action potential while ß-toxins act on the channel activation process (Jov>ïlt et al., 1980x; COURAUD et al., 1982 ; DUVAL et al., 1990). The isolation from animal venoms of such homologous polypeptidic toxins is a thorny problem in biochemistry . Their complete characterization from gram quantities of venom is time consuming and requires many chromatographic and lyophilization steps which may cause loss and denaturation of the toxins. Our laboratory has been engaged in this work for many years. A new experimental approach using reverse-phase high-performance liquid chromatography (HPLC) has been developed to allow the resolution, quantification and identification of toxins from mg quantities of snake venoms (Boucts et al., 1986). In the case of scorpion venoms, the use of the same experimental approach, as was used for snake venoms, failed because scorpion venoms are more complex protein mixtures than snake venoms (MARTIN et al., 1987x) . In this work, the optimization of the use of HPLC to purify scorpion toxins was undertaken using a pool of 20 mg of Androctonus mauretanicus mauretanicus venom obtained by means of manual stimulation of the animal instead of electric stimulation. This venom must be considered as the physiologic secretion injected by an animal to its prey or to humans during a sting. The question arises as to whether the toxins selective for nerve and muscle sodium channels are responsible for the lethality to mammals and humans. In this respect, knowledge of the toxin content of this physiologic secretion is needed in order to set up an e~tcient serotherapy against scorpion stings . Six proteins toxic to mice have been characterized . It was demonstrated that three of them are a-toxins as is the case for all the toxins purified until now from north African scorpion venoms . One of them was a ß-toxin, the first one found in an Old World scorpion venom. Finally, looking for toxins active on insects, it has been found that some toxins active on mammals also exhibited different levels of specificity towards phylogenetically related groups of arthropods . MATERIALS AND METHODS AfatcriaLs The venom of Androctonua mauretanicut mauretanicus, a gift from the Institut Pasteur (Casablanca, Morocco), was obtained by manual stimulation of the post-abdomen of the animals. Two hundred scorpions gave 20 mg of venom (8 .24 mg of protein for 100pl of venom) . The toxins AaH II and Csa Il were purified in the laboratory as previously described (M~rrtty and Roctur, 1986; Mexnrr et al., 19876) . U.V. grade acetonitrile was from Fiaona Scientißc (U.K.), formic acid as well as the other analytical reagents were from Merck (F.R .G.). Na'~I was from Ameisham (U .K .) . Lactoperoxidase and bovine serum albumin (BSA) fraction V were from Sigma (U .S .A.) . Spectrapor 3 dialysis membrane wax from Spectrum Medical Industries (U .SA.). The water used to prepare solvents, buffers and dialysis liquid was obtained with a Milli/RO/Milli/Q system from Millipore

L ethality auaya Toxicity In vivo was tested in male mouse C57 BI/6 (produced in the laboratory) weighing 20í3g, either by subcutaneous (s .c.) or intraoerebroventricular (i.c.v.) injection, as already described (MwanN et at., 19876) . The lethal dose killing 50% of the animals (rnso) was determined according to the method of Br~eaxs and Keen

HPLC of Scorpion Venom

95 3

O O N Q

TIME ( min) U~rx~srt~mtE-Ocr~rt of 20mg of Androctomcs mawetanicus mauretanicus vEtvoat. Solvent A: 0.15 M ammonium formate, 12 mS, pH 2.70. Solvent B: acetonitrile. Linear gradient from 15 to 38% B in A in 100 min. Flow rate S ml/min . Absorbance reading at 280 nm, 1 Unit full scale . Arrows indicate the fractions toxic to the mouse. FiG.

l . Reverse-PHA.4L

HPLC

oN

(1935) . Musca domestics fly larvae and adult Blatella germanica cockroaches were provided by Procida (France) and injected as described by Zt,olxtx et al. (1972). Death in the 48 hr following the injection was used to determine the lethal dose killing 100% of the animals (t.n,~ . High-performance liquid chromatography A Millipore/Waters Associates system (U.S .A.) was used, including two model 510 pumps, a U6K injector, an automated gradient controller, a 490 spectrophotometric detector and a data module integrator/recorder. Semi-preparative revenx-phase HPLC was carried out at 25°C on a Beckman (U.S .A.) 10 x 250 mm aemipreparative column prepacked with 5 pm Ultrasphere-Octyl. Solvent A was 0.15 M ammonium formate (pH 2.70, conductivity 12 mS at 20°C) and solvent B was acetronitrile . A Gilson mode1202 fraction vullector (France) was used with Corning glass tubes (U.S .A.) at detector output . The fractions pooled were lyophilized twice in order to eliminate the solvents. Samples for toxicity assays were lyophilized in the presence of 0.I % HSA. A Beckman (U .S .A .) 6x 150 mm column prepacked with lOpm IEX-535 K Spherogel-TSK was used to further purify the fractions toxic for the mouse. Solvent A was water and solvent B was I M or 2 M ammonium acetate, pH 6.8. Additional details concerning all chromatographic steps are given in the text or ûgure legends. The material able to dialyze through the Spectrapor 3 membrane was analysed by reverse-phase HPLC using a Merck (F.R.G.) 25x4mm column pre-packed with Sltm LiChrospher-C18. Polyacrylamide gel electrophoresis (PAGE) PAGE of basic proteins was performed at pH 4 .1 on 20% homogeneous Phast-Gel using a Phast-System from Pharmacie (Sweden) according to the Pharmacie application 51e no . 300. Proteins were stained with Coomasaie blue according to the Pharmacie development technique no. 200 for native-PAGE . Amino acid analysis Acid hydrolysis with 6N HCl was carried out on 1 nmole of toxins for 20 and 70 hr at 110°C and under vacuum using a Pico-Tag work station Crom Millipore/Waters Associates (U .S.A .). The amino acid compositions were determined on a Beckman 6300 amino acid analyzer (U .S.A .) . Radioiodination of toxins and receptor binding assays Androctonut austrolis Hector toxin II (AaH In and Centrwoidles suffuses suffuses toxin II (Cae II) were radioiodinated as already described (ROCrteT et aL, 1977) using the lactoptroxidase method and purified by immunoprecipitation with specific antisera . The specific radioactivities obtained were 1200 and 800Ci/mmole, respectively. .lndroctoruu maeretanices mata'etanices toxins were tested for their ability to compete with the

95 4

H. ZERROUK et á.

binding of both'uI-AaH II and'~I-Cav II on the rat brain synaptosomal P=preparation of Gw+Y and wrrr~ (1%2), es already described (JovBa et at., (980b) . A Packard Crystal II y~ounter (U .S .A.) was used for radioactivity quantiûcation. Additional details are given in the figure legends. RESULTS AND DISCUSSION

The venom used in this work is considered the physiologic secretion of the animal and had an LD s p by s.c. injection to mice of 95 ug per kg of body weight . This value is the lowest found for a scorpion venom (Rosso and RocxAT, 1985) and indicates a high content of toxins. In order to eliminate the high mol. wt mucopolysaccharides (M~1tTlx et al., 1987a), the first purification step was dialysis of the venom (20 mg total amount) against water (24 hr, 4°C, 200 ml x 2, using Spectrapor 3 membrane with a mol. wt cut otF < 3500) followed by a centrifugation (30 min, 10,000 x g). The supernatant was called "crude extract" . The material which is able to dialyze through the Spectrapor 3 membrane was slightly tonic for the mouse either by s.c. or i.c.v. injection. It was lyophilized in order to be further studied.

O CO N

a

TIME (min)

Fro. 2. Cenornc-mccw+xos HPLC ox IEX-S35 K Si~ooeUTSK ot+ rr~ Toxic t~crtotas oar~nvso av ages-tom HPLC (Fia . 1). Solvent A, H=O. Solvent B, 1 M (a, b, c) or 2M (d) ammonium acetate, pH 6.8 . Flow rate 1 ml/min . Absorbance reading at 280nm, 0.1 S Unit full scale. (a) chromatography of fraction 8, linear gradient from 0 to 100% B in A in 8S min. (b) Chromatography of fraction 9, linear gradient from 0 to 50% B in A in 80 min. (c) Chromatography of fraction 10, linear gradient from 0 to 50% B in A in 80min. (d) Chromatography of fraction 14, linear gradient from 0 to 100% B in A in 70min. Arrows indicate the fractions toxic to the moux. Inset: Phaat-System PAGE on homogeneous Phast gel ~% at pH 4.1 . Migration was from top (anode) to bottom (cathode): (1) Amm VI (1 .4~; (2) Amm N (LSpg) ; (3) Fntdion 8 bdore purification (5~; (4) total "crude extract" (SOpg) ; (S) Amm III (l .Spg); (~ Amm V (1 .Spg); (7) Amm VII (1 .Stxg).

HPLC TABLE

I.

AMINO

of Scorpion Venom

ACIA

COMP081TION

mmwetanicus mauretanieus TolaNS

95 5

OF AAlIIOCIOALS V' AND VII

Amino acid

Residues/moles Amm V'

Residues/moles Amm VII

Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Trp

8 .81 (9) 1 .02 (1) 3.17 (3) 3 .30 (3) 2.30 (2) 7 .04 (7) 1 .93 (8) 7 .41 (8) 1 .15 (1) 0 .00 (0) 2 .55 (0) 3 .15 (3) 4 .25 (5) 1 .96 (2) 0 .00 (0) 7 .13 (7) 2 .93 (3) nd

7 .50 (8) 4.66 (5) 2 .69 (3) 1 .09 (1) 3 .21 (3) S.18 (5) 3 .10 (3) 5.50 (6) 2.09 (2) 0.06 (0) L87 (2) 1 .13 (1) 5 .67 (6) 2.05 (2) 2 .92 (3) 5 .86 (6) 1 .22 (l) 3'

nd, not determined 'Amino acid hydrolysis was performed on native protein using 6 N HCI, therefore the amount of Trp was determined spectrophotometrically . For the same reason, the amounts of Cys and Tyr may be lower than they should be . Numbers in parentheses represent the closest integer.

The reverse-phase HPLC diagram of the "crude extract" obtained on Ultrasphere-Octyl (Fig. 1) was less heterogeneous than for venoms obtained by means of electric stimulation (MARTtx et al., 1987a; DE DIAxous et al., 1987). Because of its high sensitivity, the i.c.v. injection was chosen to follow the toxicity (fractions considered toxic were able to kill a mouse after injection of about 1 kg of protein). Fractions 8, 9, 10 and 14 were toxic and were further purified using ion-exchange HPLC on IEX-535 K Spherogel (Fig. 2). Two TABLE

2.

QUANTITATIVE DATA CONCERNING THE PURIFICATION OF THE "CRUDE ExTRACf" OF THE AndroctOnus nlauretanicus mauretanicua vENOM USING HIGH-PERFORLIANCE LIQUID CHROIbAroGRAPHY

Purification step

Yield in absorbante (%)

Yield in lethality (s .c.) (%)

100

l00

Crude extract Fraction 8 9 10 14 Amm Amm Amm Amm Amm Amm

III IV V V' VI VII

7 .13 5 .71 14.40 8 .93 4.79 4.63 11 .23 0.29 0.66 6.92

36.17 8 .56 14 .89 28 .SI

~'~ 1 .00 1 .38 0 .02

72 .49

956

H. ZERROUK et al. Tnes~ 3. Enowa~cwz. cEt~ttecrnuzenox oF Androctonus mauretantcr~s mmaetonicus Toxtxs Toxins

Ln~"

Kpf (nM)

Amm III Amm IV Amm V Amm VI Amm VII

350 175 175 < 50,000 20

I5 .7 228.0 0.86 > 10,000 48 .5

"The Ln w is given as ng/kg (i.c .v. injection) . tThe Kp values for Amm toxins binding to rat brain synaptosomal fraction (P~ are calculated from the Ko.s values determined in Fig. 3 using the following equation:

rxp

xo.s = Ko ~ 1 + +

(IC°.s is the concentration of the toxin which gives the half maximum inhibition of the specific binding of the radiolabeled toxin of reference, 'Tox).

toxic fractions were in fraction 8 with retention times (RT) of 37.2 and 46 .3 min, one was obtained from fraction 9 with RT of 43.0 min, two from fraction 10 with RT of 25.9 and 40.2 min and one from fraction 14 with a RT of 65.6 min. PAGE of these fractions confirmed their homogeneity (insert, Fig. 2). According to their amino acid analysis: the fractions having RT values of 37.2 and 46.3 min were identified as toxins Amm VI and IV, respectively (Fig . 2a); the fraction having a RT of 43.3 min as Amm III (Fig. 2b) and the fraction having a RT of 40 .2 as Amm V (Fig. 2c). The toxic fraction with a RT of 25 .9 min (Fig . 2c) was a new toxin called Amm V'. Its amino acid composition, given in Table l, was very close to that of Amm V. The toxic fraction with a RT of 65 .6min (Fig. 2d) was also a new toxin we called Amm VII. However, it can be injected into the mouse by the s .c. pathway at a dose of up to 125 pgJkg without producing any toxic symptoms. It is known that some toxins are much more active by i.c.v. injections, i.e. 1000 to 10,000 times more for some ß-toxins purified from North and South American scorpion venoms (MARTIN et al., 19876; MARTIN-EAUCLAiRE, 1987, Thèse d'état-Sciences, Université d'Aix-Marseille II, France). Thus, the i.c.v. mode of injection used in this work to follow the activity allowed us to find Amm VII . Its very basic and hydrophobic features could explain why Amm VII was not obtained before (Rosso and RocHAT, 1985) because of its possible irreversible adsorption on the gel matrix during conventional chromatographies. Amm I and Amm II appeared to be absent in the sample of venom that we have studied. This suggests either that the toxin expression level varies considerably or that different subspecies of Androctonus mauretanicus mauretanicus may exist in the different collecting areas. Indeed, evidence has been previously given that the toxin amounts in Androctonus australis Hector venoms depend not only on the specimen, but also on the geographic origins (MARTIN and ROCHAT, 1986 ; MARTIN et al., 1987a) . Table 2 summarizes the purification steps and the recovery yield starting from the venom extract. In terms of absorbance, the total toxin yield was 28 .5% compared to 8.4% previously found when the venom was obtained by means of electric stimulation (Rosso and ROCHAT, 1985), while in terms of lethality (i.c.v. injection to the mouse), the yield was 72.4% compared to 50%,respectively. These results suggest that the material obtained by electric stimulation of scorpions, which are often used for large scale purification, may be

HPLC of Scorpion Vcnom

957

Log ( TOX )

.. ó 0 b .g f~ VJ N

F-1 N

Log ( TOX ) FIG. 3 . CO)dPE'I7TION EXPERB~N'fS FOR BINDING TO SITE 3 AND 4 ON RAT BRAIN SODIUS! CHANNEL OF Androctonua mauretmeicus nuaretanicua ToxtNs (a) Competition with "'I-AaH II, 0 .2 nM ; (b) competition with '~°I-Css II, 0 .1 nM . B and Bo are

the radioactivity bound in the presence or in the absence of the tested toxins, respectively . The values are means of duplicates and the non-specific binding is subtracted .

different from the physiological secretion. We were unable to find peptide P2 which is exclusively found in the venom obtained by means of electric stimulation (Rtuso and RocxnT, 1985). As previously noted, the material able to dialyze through the Spectrapor 3 membrane (3% of the venom weight) was slightly lethal injected either s.c. or i.c.v. to the mouse. It was analyzed by reverse-phase HPLC on Lichrospher-C18 with a linear gradient of water containing 0.1 % of trifluoroacetic acid as solvent A and acetonitrile containing 0.1 % of

95 8

H . ZERROUK et á.

trifluoroacetic acid as solvent B, from 0 to 60% in 110min, reading the absorbance at 230 nm, in order to find new peptides active on mammals. Ninety percent of the toxicity of the venom can be explained by the toxins previously described. Scorpion toùns have been divided into a- and ß-toùns. The toxins Amm III, Amm IV and Amm V competed with different efficiencies with 'z5I-AaH II, the a-toxin of reference (Fig. 3a). Furthermore, they were unable to compete with 'ZSI-Css II, the ß-toxin of reference (Fig. 3b). Consequently, they can be considered as a-toùns. Testing Amm VII provided a non-expected result . It was able to compete with 'zsI-Css II (with a low affinity), but no competition at all was observed between Amm VII and 'ZSI-AaH II, the a-toxin of reference. These data strongly suggest that Amm VII is a ß-toxin, the first one found in a North African scorpion venom. Preliminary investigations carried out with the Old World scorpion venom of the Caucasian species Buthus eupeus has suggested the eùstence of toùns structurally related to ß-toùns. Actually, a protein active on insects and not toxic to mice, called insectotoùn I2 , was isolated from this venom. From its primary structure, it was concluded that I 2 belongs to the group of homologous toxins issued from New World Centruroides venoms (GRLSHIN, 1981) . Finally, Amm VI was unable to compete with '2SI-AaH II or 'zsI-Css II. This toxin behaves identically to Bom III, a toùn purified from the Moroccan scorpion Buthus occitanes mardochei (VARGAS et al., 1987). For a long time, it was obvious that a-toxins were inactive on insects. Recently, a new toùn, Lqha TT, which caused paralysis of fly larvae, was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. Its structural and pharmacological properties were similar to those of some a-toùns found in buthid venom and it was concluded that Lqha TT was an a-toxin which acts on insect sodium channels. We have tested the toùcity of the a-toxins purified in this work using as test animals both Musca domestica fly larvae and Blatella germanica cockroaches. It was found that Amm IV (~,oo = 14 ng), Amm III (~,oo = 140 ng) and Amm V (LD~ op = 300 ng) were toùc for cockroaches and were completely devoid of activity for fly larvae . Thus, these proteins, toxic for mammals, seem also to exhibit different levels of specificity towards phylogenetically related groups of arthropods . Some ß-toùns are toùc both for mammals and insects (McIxTOSH and W~z-r, 1972; W~~R et al., 1983 ; Da LnK~ et al., 1986). In this respect, the ß-toxin Amm VII was found to be active both on mammals (Table n and on cockroaches (~,oo = 210 ng). This work shows that it is possible to improve the method used in our laboratory to purify animal toùns. HPLC should be considered as an appropriate tool for biochemical studies of scorpion venoms. Only 20 mg of venom is required to allow the isolation to homogeneity and the chemical and biological characterization of toxins . The method is much faster and more sensitive than those previously described (Rosso and Rocxnr, 1985). In this respect, the use of HPLC may be helpful for taxonomic classification: the isolation of the polypeptide content of a venom obtained from a single animal could be easily performed. The manual mode of venom extraction prevents contamination of the soluble material, representing the physiologic secretion, by insoluble mucoproteins and small peptides released from the secretory cells during the stimulation by electric shocks . In the past, we never succeeded, using an HPLC purification method, to isolate toxin from scorpion venoms obtained by electric stimulation for preparative purposes. Low-pressure chromatographies are used by people concerned with an efficient production of toxins for serotherapy. The venom sample studied herein is slightly different in its toxin content from the one

HPLC of Scorpion Venom

959

previously studied. Qualitative and quantitative differences in the protein composition of a venom have been previously observed (EL Ayes and ROCHAT, 1985 ; MARTLN and ROCHAT, 1986; MARTIrI et al., 1987; Lole>?r et al., 1990). They can be due to : (i) the method used to fractionate the venom (HPLC or conventional procedures); (ü) the venom batch studied that may vary according to the method of obtaining the venom (manual or electric stimulations); (üi) the existence of subspecies; (iv) the geographical origins of the collected animals. This study clearly demonstrates that the toxicity for mammals and thus for humans of scorpion venoms is mainly due to the polypeptides specifically active on the voltagesensitive sodium channel. When the venom is injected subcutaneously, these toxins are responsible for about 75% of the lethal potency. If the Androctonus mauretanicus mauretanicus venom contains some toxins active on other structures, such as toxins active on CaZ+-activated K+ channels (POSSANI et al., 1982 ; G11~1vE2:-GALLEGO et al., 1988 ; CHlccl et al., 1988) these proteins seem far less toxic. An eflïcient antiserum against Androctonus scorpions may be raised by generating antibodies against the toxins active on only the voltage-sensitive sodium channel. An interesting finding of this study is that a North African scorpion venom may contain a ß-toxin, Amm VII. We claimed previously a lack of ß-toxin in Old World scorpion venom. Perhaps, these polypeptides were not detected before, because their activity in mammals was too weak by s.c . injection. Their detection becomes possible when i.c .v. injection was chosen to monitor purification . For the first time, we also found that a-toxins display toxicity towards insects. The ones most toxic for mammals are the least toxic for cockroaches, and all of them are without activity on fly larvae. It has been suggested that the specificity of the toxins is determined by rather subtle changes in their three-dimensional structure (FONTECILLA'CAMPS, 1989). This functional diversity may be the consequence of their structural differences . However, evidence is now given for the existence of closely related sodium channel isoforms or sub types, in different species, in different tissues of the same species, and even in different locations within the same tissue (BARCHI, 1987). Acknowtedgemaets-We thank Prof. H. Rocx~r for his very stimulating discussions; Prof. F . Muuxne and Dr J . P. Rasso for their constant interest and support; Dr P. Mexcxor for advice in HPLC; Mr P . M~tvst>Et.IB and Mrs T. Bxrwno for their skillful assistance in amino acid analysis ; Mrs M. ALVrrter: for providing C57 B1/6 and rats. The fine secretarial work of Mrs C . ROUSSAAn? is greatly appreciated . REFERENCES HEFiRENS, H . and Kim, C . (1935) Wie sind Rechenversuche fûr biologische Auswertungtn am Zweckmaeaigaten anzuordnen . .lrch . Exp . Pathol. Pharmak. 1T7, 379-388 . Bouars, P. E ., MAncxo'r, P. and RoctrwT, H . (1986) Characterization of Elapidae snake venom components using optimized inverse-phase High-Performance Liquid Chromatographic conditions and screening assays for a-neurotoxins and phospholipase A= activities. Biochemistry 25, 7235-7243 . BAacrn, R . L. (1987) Sodium channel diversity: subtle variations on a oomplax theme . TINS 10, 221-222. Crncct, G. G ., Grr~Nez-Gnr .r~ao, G., Bne, E ., G~rtcu, M . L ., WtxQu~sr, R . and C~scm~u, M . A . (1988) Purification and characterization of a unique potent inhibition of apamin binding from Leiurus quinquesrriatus hebracur venom . J. biol. Chem. 263, 10192-10197 . CouaAUO, F., Jovat, E., Duaots, J . M . and Rocw+r, H . (1982) Two types of scorpion toxin receptor sites, one relatod to the activation, the other to the inactivation of the action potential sodium channel . Toxkon 20, 9-16. De Duxous, S ., KorevAiv, C ., BArinAOtn, E . and RocxAr, H . (1987) Purification of contracture-inducing insect toxins from Buthinae scorpion venoms by immunoa>Snity and high ptnssure liquid chromatography . Toxicon 23, 731-741 .

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