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Proteins toxic to arthropods in the venom of elapid snakes
_Y.Insect Physiol., 1975, Vol. 21, pp. 1605 to 1611. Pergamon Press. Printed in Great Britain.
PROTEINS
TOXIC
TO ARTHROPODS IN THE VENOM ELAPID SNAKES*
OF
E. ZLOTKIN,~*~ M. MENASHI?,~ H. ROCHAT,~ F. MIRANDA,~ and S. LISSITZKY~ IDepartment of Entomology and Venomous Animals, The Hebrew University of Jerusalem, Jerusalem, Israel; 2Laboratoire de Biochimie, Faculte de Medecine, Secteur Nord, Marseille; and 3Laboratoire de Biochimie MC&ale, Faculte de Medecine, Marseille, France
(Received 14 October 1974) Abstract-It has been found that the lethal action of elapid snake venoms to arthropods (fly larvae and isopods) is due to proteic factors differing from the toxins which are strongly and specifically active on mammals. This conclusion was based on the following: (1) Lack of any correlation between the toxic activity on larvae, isopods, and mice of ten elapid snake venoms. (2) Absence of any toxicity to arthropods in pure toxins isolated and purified from several elapid snake venoms according to their lethality. (3) Electrophoretical separation of the venom of the snake Naja mossambica mossambica ( = iV. nigricollis mossambica) resulted in fractions active either to arthropods and/or to mice. (4) Separation of the above venom by gel filtration on Sephadex G-50 enabled the isolation of fractions highly toxic to arthropods. (5) The above fractions demonstrated a high phospholipase activity corresponding to about 80 per cent of the total activity of the whole venom. The link between phospholipase and toxicity to arthropods will serve as a target for further investigation. It appears that the phenomenon of diversity in toxic activities of different proteins to different groups of organism, as previously demonstrated in scorpion venoms, is equally shared by elapid snake venoms. MATERIALS
INTRODUCTION DUE TO their medical importance the venoms of elapid snakes are the most extensively studied. The death of mammals is mainly attributed to the presence of low molecular weight basic proteins composed of a single chain of either 61 to 63 amino acids cross-linked by four disulphide bridges or 71 to 74 amino acids cross-linked by five disulphide bridges (LEE, 1972; Tu, 1973; ROCHAT et al., 1974). The above neurotoxins cause a paralysis acting on the neuromuscular junction through a competitive blockade of the cholinergic receptors at the postsynaptic membrane (CHANGEUX et al., 1970). Considering the fact that neuromuscular transmitters in arthropods are non-cholinergic (GERSCHENFELD, 1973), it was interesting to check how far elapid venoms are toxic to arthropods ‘and if the action follows from the same components which are lethal to mammals. These questions are emphasized by our previous findings that scorpion venoms contain different selectively toxic proteins discriminating not only between insects and mammals (ZLOTKIN et al., 1971b, c, 1972a), but even between different groups of arthropods (ZLOTKIN et al., 1972b).
METHODS
The crude elapid snake venoms of N.h. annuliferum and N. mossambica mossambica ( = N. nigricollis mossumbicu) were supplied by D. Miiller (South Africa, Johannesburg). Those of N. haje (M. S.), N. naja, and N. nivea were supplied by Miami Serpentarium, U.S.A., and those of N. haje (‘Pasteur’), Dendroaspis viridis, and N. nigricollis were kindly granted by Professor P. Boquet, Institut Pasteur, Garches, France. The pure toxins I and III from the venom of N. haje (‘Pasteur’), toxin I from the venom of N. nigricollis, and the toxic fractions E 3 and E 4 from the venom of Dendroaspis viridis were obtained in the laboratory.
Test animals The tests:
* Supported by grants from the Israel National Commission for Basic Research (No. 27A), the Institut National de la Sante et de la Recherche Medicale, France (No.‘G38), and the Direction de Recherches et Moyens d’Essais. 6B
AND
Venoms and toxins
following animals were used for toxicity (1) Blowfly larvae, Surcophuga fuZcuZu~u ( = argyrostoma), bred in the laboratory (ZLOTKIN et al., 1971a); (2) terrestrial isopods, Armadillidium vulgare collected near Marseille, and Porcellio Zuevis, collected near Jerusalem; (3) laboratory mice of two races: black (C57 B1/6) and grey (C3H/He) obtained from C.N.R.S., 46 Orleans La Source, France.
Tests of lethality The grouping done according
160.5
and calculations of the LD,, were to REED and MLJENCH (1938) for
E. ZLOTKIN, M. MENASHI?, H. ROCHAT, F. MIRANDA, AND S. LISSITZKY
1606
arthropods and according to BEHRENS and KARBER (1935) for mice. The method of injecting arthropods was done according to ZLOTKIN et al. (1971a, 1972b) and to mice according to MIRANDA et al. (1964). The determination of death in fly larvae was based on their inability to form puparia and of isopods by their inability to move 2 hr after the injection.
RESULTS The lethal effect to mice and arthropods of ten elapid venoms is presented in Table 1. The venoms
Table
1. The lethal potency of ten elapid venoms to mice, fly larvae, and isopods LD50
Starch gel electrophoresis Mice* &g/20 g)
The qualitative and preparative runs were done according to ROCHAT (1967) and ZLOTKIN et al. (1971a, 1972a). For the preparative run an amount of 5.6 mg x 4 N. m. mossambica crude venom was used. One out of the four sections was used for staining as shown in Fig. 1.
Dendvoaspis viridis
10.4
Sephadex G-50 column chromatography
N. haje (‘Pasteur’) N. nivea N. nigricollis
Details presented
of the system and its in the legend to Fig. 2.
operation
are
Origin of venom N. naju siamensis N. naja
N. haje annuliferum N. mossambica mossambica N. haje (‘M.S.‘) Ophiophagus hannah
--
Isopods+ Fly larvae (/*g/100mg) (~gjlO0 mg)
11.2 11.6
4.4 5.8 2-8 7,o 10.5
1.2 0.3 14.1 9.2 5.2
12.6 15.8
1.9
0.3
4.2
3.8
23.5
0.7
0.4
27.7 79.3
5.6 25.0
12.4 6.8
4.6 6.7
* Mice belonging to the race C 57 B1/6 (C.N.R.S.). + Isopods belonging to the species ArmadillidiaLm vulgare,
Fig. 2. Gel filtration separation of the venom of N. m. mossambica: 2 g of crude venom were applied on a series of four columns (100x 3.2 cm) filled with Sephadex G-SO, fine (Pharmacia, Uppsala, Sweden), equilibrated by ammonium acetate buffer 0.1 M, pH 8.5 to S-6 (MIRANDA et al., 1970). A flow rate of 78 ml/hr was applied and fractions of 13 ml each were _collected. The elution pattern according to 280 nm absorbance was automatically recorded.
Digestion by pronase Samples (1 mg) of either crude venom or each of the arthropod toxic fractions (I and II) obtained by Sephadex chromatography were treated with O-1 mg of pronase (Sigma, U.S.A.) and incubated in 1.0 ml of saline (0.6%) solution for 20 min at 37°C.
Phospholipase activity Activity of phospholipase was determined according to DE HAAS et al. (1968) by titration of acidity obtained due to the hydrolysis of egg yolk phospholipids. The titration curve enabled the calculation of the initial velocity of the reaction. The specific activity of the preparation was then expressed in /*mole NaOH/min per O.D.,,, of protein.
are listed in the order of their increasing
activity to mice. The symptoms of elapid snake envenorcation of mammals and particularly of mice are well known (LEE, 1972). In the arthropods the injection of doses corresponding to 2 to 3 LD,, units was accompanied by a gradual decrease in mobility up to a complete paralysis in a relaxed stretched form of the body, 1 to 2 hr following the injection. The lethal effect of several toxins originating from several elapid snake venoms is presented in Table 2. It should be emphasized that the above toxins were isolated and purified according to the criterion of mice lethality. Fig. 1 presents the starch gel electropherogram of N. m. mossambica venom indicating the location of fractions active on mice, fly larvae, and isopods. Details and explanation are presented in the legend to Fig. 1. Fig. 2 presents the elution pattern of 2 g of N.m. mossambica venom when separated on a series of Sephadex G-SO columns. Each of the fractions was tested for its lethality on mice, fly larvae, and isopods. Toxic activity was found in fractions I, II, III, and IV. Table 3 presents the specific and total activities of these fractions to each of the above test animals. The crude venom and fractions I and II, obtained by Sephadex G-SO chromatography after pronase treatment, when injected into the fly larvae and
1607
Fig. 1. Starch gel electrophoresis of N. m. wwssambica crude venom. The separation of 5-6 mg of venom is represented. The buffer was Tris-citrate, pH 8.6, O-5 to 0.8 mA/cm. Duration of the run was 14 hr at room temperature. The starch gel was cut into slices of 0.5 cm width each and eluted by 5 ml of saline 0.9%. The MT fraction was toxic to mice, the LT fraction toxic to fly larvae, and the CT fraction toxic to isopods. The numbers express the maximal activity indicated in pl of the eluate corresponding to the LDso for fly larvae, and the minimal volume in ml of eluate causing lethality to mice. The action on isopods is qualitatively indicated showing those regions of the starch gel plate where lethality to isopods was caused by an injection of 5 ~1 of eluate/lOO mg of body weight.
Proteins toxic to arthropods in venom of elapid snakes isopods in amounts corresponding to 15 LD,,, were found to be completely inactive. It has been found that phospholipase activity of the crude venom and of the mixture of the Sephadex G-SO fractions I and II (both active on arthropods -Table 3) is 396 and 943 pmole of NaOH/min per respectively. Thus, as may be calculated, O.D.,so the above Sephadex G-SO fractions contain about 82 per cent of the total phospholipase activity of the crude venom.
1609
DISCUSSION A careful observation of Table 1 reveals two main phenomena. The first is that elapid snake venoms are highly toxic to arthropods. This may be deduced by comparing these data with those referring to the lethal potency of scorpion venoms such as that of Leiurus quinquestriatus (KAMON and SHULOV, 1963; ZLOTKIN et aZ., 1971a; ISRAELI.ZINDEL et al., 1972) which in nature are used by
Table 2. The lethal effect of purified toxins of several elapid venoms to mice, fly larvae, and isopods Isopods KWO
Mice Material tested
&;&)
Fly larvae
mg
&lOO mg
LDso
Amount injected
Response (paralysis)
9.2 11.0
N. h& (‘Pasteur’) Crude venom Toxin II Toxin III
11.24* l-141_ 1.35.t
9.2’ -
N. ni&vicoliis Crude venom Toxin II
12.64* 1.681
0.3” -
Dendvoaspis viridis Crude venom Fraction E3 Fraction E4
10.4* 3.061 2.52f
14.1% -
0.41
22 18.5
Response (lethality)
LD,,
Amount injected
l/7 l/7
3*5* -
9.2 12.0
O/12 4110
oi7
1.85” -
2-4
O/l0
1: l/i
2*76* -
4-o 3.6
l/10 Q/10
-
-
-
-
-
* Data taken from Table 1. t Data taken from MIRANDA et al. (1970). 1 Data taken from KOPEYAN et al. (1973).
Table
3.
Toxicity
Material tested
of different fractions obtained by Sephadex N.m. mossumbica venom
Protein amount (O.D.,,, units)
Toxicity to mice* T.A.f
S.A. §
Crude venom
2700
50,000
185
(2 g) Fraction Fraction Fraction Fraction
548 375 1080 145
6000 3500 2500 36,250
10.9 9.2 2.3 250
79.6
91.5
I II III IV
Recovery ( yb)
Toxicity
G-50
chromatography
of
to fly larvae
Toxicity
S.A. 8
T.A.‘t.
S.A. 5
1,140,000
422
T-A.1 2,422,OOO
897
to isopodst
1,277,OOO 2330 420,000 1120 544,900 504 Not active
493,000 900 346,000 925 237,700 220 Not active
92.5
94.6
* Belonging to race C3H/He which is more resistent to the venom (LD,, = 40 pg/20 g body weight) than mice belonging to race C 57 B1/6 (see Table 1). t Belonging to the species Porcellio laevis which is about five times more resistant to the venom (LD,, = I.75 pg/lOO mg) than A. vulgare (LD,, = 0.4 pg/lOO mg, see Table 1). 1 T.A. = total activity, expressed as number of LD,,. $ S.A. = specific activity, number of LD,, per unit of O.D.zso.
1610
E. ZLOTKIN,M. MENASH& H. ROCHAT,F. MIRANDA, ANDS. LISSITZKY
them in order to paralyse arthropods. The second phenomenon is the absence of any correlation between the toxic action of the above venoms to each of the three test animals. This fact has directed our attention to the possibility that each of the above toxic activities may be caused by a different component of the venom, as was previously suggested in the case of scorpion venoms (ZLOTKIN et al., 1971a). Such a possibility was strongly supported by the data presented in Table 2. The pure toxins active on mammals were applied to arthropods in an amount at least equal to the weight of the LD,, of the corresponding crude venoms and were found to be inactive. Considering the relative potency of these toxins to mice as compared to that of the crude venoms (Table 2), it may be concluded that the crude venoms’ action on arthropods cannot be attributed to their mammal toxins. The data given by starch gel electrophoretical separation of the venom of N.m. mossambica have supplied additional evidence that actually the effect of this venom to the above test animals is due to different factors. As is presented in Fig. 1 the whole toxicity to larvae is located in a slow cathodic section of the electropherogram which partially overlaps with toxicity to isopods as well as mice. However, according to the most recent findings, it appears that the activity to mice in this section is probably due to the presence of toxin III which has been isolated from Sephadex G-50 fraction IV (Fig. 2, Table 3; ROCHAT et al., 1974) and is completely devoid of any toxicity to arthropods (MENASH& 1973). Furthermore, the main lethal fraction to mice has a fast cathodic mobility and is devoid of any activity to arthropods. Equally, toxicity to isopods is partially located in an anodic fraction which is devoid of any action to mice as well as fly larvae (Fig. 1). The same principle is demonstrated by the Sephadex G-50 column chromatographical separation of the crude venom, as shown in Fig. 2 and Table 3. Fractions I and II, which contain the majority of the activity to arthropods, are equally toxic to mice but their specific activity is clearly below that of the crude venom (Table 3). On the other hand, fraction IV which is about 13 to 14 times more active to mice than the crude venom is devoid of any toxicity to arthropods (Table 3). The proteic nature of the above arthropod toxic fractions was proved by the experiments with pronase inactivation. It may be concluded that elapid snake venom contains toxic proteins demonstrating an evident degree of specific activity to arthropods, thus resembling scorpion venoms. However, considering their effects, expressed in an immediate spastic paralysis caused by the toxins originating from scorpion venoms (ZLOTKIN et al., 1971a, 1972b) as compared to the gradual flaccid paralysis caused by
elapid snake venoms, it may be concluded that the above materials strongly differ in their mode of action. At this point our attention was directed to phospholipaae activity. This is the most abundant enzyme in elapid snake venoms, and is well known to possess certain pharmacological as well as lethal capacities (CONDREA and DE VRIES, 1965; ROSENBERG,1971; SLOTTA et al., 1972; DELORI, 1973). Equally, the location of the arthropod active fractions I and II in Sephadex G-50 chromatography (Fig. 2) is in good accordance with previous data concerning separation and isolation of phospholipase (WAHLSTR~M,1971). It has been found that the arthropod toxic fractions (which to a certain extent are equally active to mice, Fig. 2, Table 3) practically possess the whole phospholipase activity of the crude venom. The main question which we encounter at the present moment is: How far do the two biological activities follow from the same molecule, and if it is so what is the relation between the enzymatic and the toxic actions ? These problems will certainly serve as targets for future investigation. Acknowledgement-The authors are indebted to Professor P. BOQU~XT, Institut Pasteur, Garches, France, for the gift of several elapid snake venoms.
REFERENCES BEHRENSB. and KARBERC. (1935) Wie sind Reihenversuche fur biologische Auswertungen am zwecktissigten atuuorclnen. Arch. exp. Pathol. Pharmakol. 177, 379-382.
CHANGEUX J. P., KASAIM., and LEE C. Y. (1970) Use of snake venom toxin to characterize the cholinergic receptor protein. PYOC. nut. Acad. Sci. U.S.A. 67, 1241-1247.
CONDREA E. and DE VRIESA. (1965) Venom phospholipase A. A review, Toxicon 2, 261-273. DELORIP. J. (1973) Purification et proprietes physicochimiques, chimiques et biologiques d’une phospholipase A2 toxique isolee d’un venin de serpent Viperidae: Vipera berus. Biochimie 55, 1031-1044. GERXHENFELDH. M. (1973) Chemical transmission in invertebrate central nervous system and neuromuscular junctions. Physiol. Rev. 53, 2-92. GRANIERC. (1972) Purification des neurotoxines du venin de serpent, Dendroaspis viridis. Diplame d’&udes approfondies, Marseille.
FacultC
des
Sciences
de
DE H&s G. H., POSTEMA N. M., NIE~ENHUIZEN W., and VAN DFZNENL. L. M. (1968) Purification and properties of phospholipase A from porcine pancreas. Biochim.
biophys. Acta
159, 103-117.
ISRAELI-ZINDEL I., ZLOTKINE., and SHULOVA. (1972) The resistance of insects to the venom of the yellow scorpion Leiurus quinquestriatus. In Toxins of Animal and Plant Origin (Ed. by DEVRIESA. and KOCHVA E.),
3, 933-939. Gordon & Breach, New York. E. and SHULOVA. (1963) Estimation of locust
KAMON
resistance 206-214.
to scorpion
venom.
J.
Insect
Pathol.
5,
Proteins toxic to arthropods
LEE C. Y.
(1972) Chemistry and pharmacology of polypeptide toxins in snake venoms. A. Rev. Pharmac. 12, 265-286. MENASHB M. (1973) Proteins specifically toxic to arthropods in the venom of the snake Naja mocambique. M.Sc. Thesis, Hebrew University, Jerusalem. In Hebrew. MIRANDAF., KUPEYAN C., ROCHATC., and LISSIT~KY S. (1970) Purification of animal neurotoxins. Isolation and characterization of eleven neurotoxins from the venoms of the scorpions Androctonus australis Hector, &thus occitanus tunetanus and Leiurus quinquestriatus quinquestriatus. Eur. J. Biochem. 16, 514-523. MIRANDA F., ROCHAT H., and LISSITZKY S. (1964) Purification des neurotoxines (Scorpamines) d’Androctonus australis (L.) et de Buthus occitanus (Am.). Toxicon 2, 51-69. REED L. J. and MUENCH H. (1938) A simple method for estimating fifty per cent end points. Am. 3. Hyg. 27, 493-497. ROCHAT M. C. (1967) Contribution zi I’etude des venins des scorpions et de serpents par electrophorese de zone en gel d’amidon. Doctorat d’Etat en Medecine, Marseille. ROCHAT H., GREGOIRE J., MARTIN-M• UTOTN., MENASHB M., KOPEYAN C., and MIRANDA F. (1974) Purification of animal neurotoxins: isolation and characterization of three neurotoxins from the venom of Naja nigricollis mossambica Peters. FEBS lett. 42, 335-339. ROSENBERG P. (1971) The use of snake venoms as pharmacological tools in studying nerve activity. In Neuropoisons (Ed. by SIMPSON L. L.) 1, 111-134. Plenum Press, New York.
in venom of elapid snakes
1611
SLOTTA K. H., VICK J. A., and GINSBERG N. J. (1972) Enzymatic and toxic activity of phospholipase A. In Toxins of Animal and Plant Oriain (Ed. bv DE VRIES A.“and KOCHVA E.) 1,401218. ‘Gordon & Breach, New York. Tu A. T. (1973) Neurotoxins of animal venoms: snakes. A. Rev. Biochem. 42, 235-258. WAHLSTR~~M A. (1971) Purification and characterization of phospholipase A from the venom of Naja nigricollis. Toxicon 9, 45-56. ZLOTKIN E., FRAENKEL G., MIRANDAF., and LISSITZKY S. (197Ia) The effect of scornion venom on blowfly larvae: a new method for the evaluation of scorpion venom potency. Toxicon 9, I-8. ZLOTKIN E., MIRANDAF., KUPFYAN C., and LISSITZKYS. (1971b) A new toxic protein in the venom of the Scorpion Androctonus australis Hector. Toxicon 9, 9-13. ZLOTKIN E., ROCHAT H., KOPEYAN C., MIRANDA F., and LISSITZKY S. (1971~) Purification and properties of the insect toxin from the venom of the scorpion Androctonus australis Hector. Biochimie 53, 10731078. ZLOTKIN E., MIRANDA F., and LISSITZKY S. (1972a) Proteins toxic to mammals and insects in six scorpion venoms. Toxicon 10,207-210. ZLOTKIN E., MIRANDA F., and LISSITZKY S. (1972b) A toxic factor to crustaceans in the venom of the scorpion Androctonus australis Hector. Toxicon 10, 211-216.
PROTEINS
TOXIC
TO ARTHROPODS IN THE VENOM ELAPID SNAKES*
OF
E. ZLOTKIN,~*~ M. MENASHI?,~ H. ROCHAT,~ F. MIRANDA,~ and S. LISSITZKY~ IDepartment of Entomology and Venomous Animals, The Hebrew University of Jerusalem, Jerusalem, Israel; 2Laboratoire de Biochimie, Faculte de Medecine, Secteur Nord, Marseille; and 3Laboratoire de Biochimie MC&ale, Faculte de Medecine, Marseille, France
(Received 14 October 1974) Abstract-It has been found that the lethal action of elapid snake venoms to arthropods (fly larvae and isopods) is due to proteic factors differing from the toxins which are strongly and specifically active on mammals. This conclusion was based on the following: (1) Lack of any correlation between the toxic activity on larvae, isopods, and mice of ten elapid snake venoms. (2) Absence of any toxicity to arthropods in pure toxins isolated and purified from several elapid snake venoms according to their lethality. (3) Electrophoretical separation of the venom of the snake Naja mossambica mossambica ( = iV. nigricollis mossambica) resulted in fractions active either to arthropods and/or to mice. (4) Separation of the above venom by gel filtration on Sephadex G-50 enabled the isolation of fractions highly toxic to arthropods. (5) The above fractions demonstrated a high phospholipase activity corresponding to about 80 per cent of the total activity of the whole venom. The link between phospholipase and toxicity to arthropods will serve as a target for further investigation. It appears that the phenomenon of diversity in toxic activities of different proteins to different groups of organism, as previously demonstrated in scorpion venoms, is equally shared by elapid snake venoms. MATERIALS
INTRODUCTION DUE TO their medical importance the venoms of elapid snakes are the most extensively studied. The death of mammals is mainly attributed to the presence of low molecular weight basic proteins composed of a single chain of either 61 to 63 amino acids cross-linked by four disulphide bridges or 71 to 74 amino acids cross-linked by five disulphide bridges (LEE, 1972; Tu, 1973; ROCHAT et al., 1974). The above neurotoxins cause a paralysis acting on the neuromuscular junction through a competitive blockade of the cholinergic receptors at the postsynaptic membrane (CHANGEUX et al., 1970). Considering the fact that neuromuscular transmitters in arthropods are non-cholinergic (GERSCHENFELD, 1973), it was interesting to check how far elapid venoms are toxic to arthropods ‘and if the action follows from the same components which are lethal to mammals. These questions are emphasized by our previous findings that scorpion venoms contain different selectively toxic proteins discriminating not only between insects and mammals (ZLOTKIN et al., 1971b, c, 1972a), but even between different groups of arthropods (ZLOTKIN et al., 1972b).
METHODS
The crude elapid snake venoms of N.h. annuliferum and N. mossambica mossambica ( = N. nigricollis mossumbicu) were supplied by D. Miiller (South Africa, Johannesburg). Those of N. haje (M. S.), N. naja, and N. nivea were supplied by Miami Serpentarium, U.S.A., and those of N. haje (‘Pasteur’), Dendroaspis viridis, and N. nigricollis were kindly granted by Professor P. Boquet, Institut Pasteur, Garches, France. The pure toxins I and III from the venom of N. haje (‘Pasteur’), toxin I from the venom of N. nigricollis, and the toxic fractions E 3 and E 4 from the venom of Dendroaspis viridis were obtained in the laboratory.
Test animals The tests:
* Supported by grants from the Israel National Commission for Basic Research (No. 27A), the Institut National de la Sante et de la Recherche Medicale, France (No.‘G38), and the Direction de Recherches et Moyens d’Essais. 6B
AND
Venoms and toxins
following animals were used for toxicity (1) Blowfly larvae, Surcophuga fuZcuZu~u ( = argyrostoma), bred in the laboratory (ZLOTKIN et al., 1971a); (2) terrestrial isopods, Armadillidium vulgare collected near Marseille, and Porcellio Zuevis, collected near Jerusalem; (3) laboratory mice of two races: black (C57 B1/6) and grey (C3H/He) obtained from C.N.R.S., 46 Orleans La Source, France.
Tests of lethality The grouping done according
160.5
and calculations of the LD,, were to REED and MLJENCH (1938) for
E. ZLOTKIN, M. MENASHI?, H. ROCHAT, F. MIRANDA, AND S. LISSITZKY
1606
arthropods and according to BEHRENS and KARBER (1935) for mice. The method of injecting arthropods was done according to ZLOTKIN et al. (1971a, 1972b) and to mice according to MIRANDA et al. (1964). The determination of death in fly larvae was based on their inability to form puparia and of isopods by their inability to move 2 hr after the injection.
RESULTS The lethal effect to mice and arthropods of ten elapid venoms is presented in Table 1. The venoms
Table
1. The lethal potency of ten elapid venoms to mice, fly larvae, and isopods LD50
Starch gel electrophoresis Mice* &g/20 g)
The qualitative and preparative runs were done according to ROCHAT (1967) and ZLOTKIN et al. (1971a, 1972a). For the preparative run an amount of 5.6 mg x 4 N. m. mossambica crude venom was used. One out of the four sections was used for staining as shown in Fig. 1.
Dendvoaspis viridis
10.4
Sephadex G-50 column chromatography
N. haje (‘Pasteur’) N. nivea N. nigricollis
Details presented
of the system and its in the legend to Fig. 2.
operation
are
Origin of venom N. naju siamensis N. naja
N. haje annuliferum N. mossambica mossambica N. haje (‘M.S.‘) Ophiophagus hannah
--
Isopods+ Fly larvae (/*g/100mg) (~gjlO0 mg)
11.2 11.6
4.4 5.8 2-8 7,o 10.5
1.2 0.3 14.1 9.2 5.2
12.6 15.8
1.9
0.3
4.2
3.8
23.5
0.7
0.4
27.7 79.3
5.6 25.0
12.4 6.8
4.6 6.7
* Mice belonging to the race C 57 B1/6 (C.N.R.S.). + Isopods belonging to the species ArmadillidiaLm vulgare,
Fig. 2. Gel filtration separation of the venom of N. m. mossambica: 2 g of crude venom were applied on a series of four columns (100x 3.2 cm) filled with Sephadex G-SO, fine (Pharmacia, Uppsala, Sweden), equilibrated by ammonium acetate buffer 0.1 M, pH 8.5 to S-6 (MIRANDA et al., 1970). A flow rate of 78 ml/hr was applied and fractions of 13 ml each were _collected. The elution pattern according to 280 nm absorbance was automatically recorded.
Digestion by pronase Samples (1 mg) of either crude venom or each of the arthropod toxic fractions (I and II) obtained by Sephadex chromatography were treated with O-1 mg of pronase (Sigma, U.S.A.) and incubated in 1.0 ml of saline (0.6%) solution for 20 min at 37°C.
Phospholipase activity Activity of phospholipase was determined according to DE HAAS et al. (1968) by titration of acidity obtained due to the hydrolysis of egg yolk phospholipids. The titration curve enabled the calculation of the initial velocity of the reaction. The specific activity of the preparation was then expressed in /*mole NaOH/min per O.D.,,, of protein.
are listed in the order of their increasing
activity to mice. The symptoms of elapid snake envenorcation of mammals and particularly of mice are well known (LEE, 1972). In the arthropods the injection of doses corresponding to 2 to 3 LD,, units was accompanied by a gradual decrease in mobility up to a complete paralysis in a relaxed stretched form of the body, 1 to 2 hr following the injection. The lethal effect of several toxins originating from several elapid snake venoms is presented in Table 2. It should be emphasized that the above toxins were isolated and purified according to the criterion of mice lethality. Fig. 1 presents the starch gel electropherogram of N. m. mossambica venom indicating the location of fractions active on mice, fly larvae, and isopods. Details and explanation are presented in the legend to Fig. 1. Fig. 2 presents the elution pattern of 2 g of N.m. mossambica venom when separated on a series of Sephadex G-SO columns. Each of the fractions was tested for its lethality on mice, fly larvae, and isopods. Toxic activity was found in fractions I, II, III, and IV. Table 3 presents the specific and total activities of these fractions to each of the above test animals. The crude venom and fractions I and II, obtained by Sephadex G-SO chromatography after pronase treatment, when injected into the fly larvae and
1607
Fig. 1. Starch gel electrophoresis of N. m. wwssambica crude venom. The separation of 5-6 mg of venom is represented. The buffer was Tris-citrate, pH 8.6, O-5 to 0.8 mA/cm. Duration of the run was 14 hr at room temperature. The starch gel was cut into slices of 0.5 cm width each and eluted by 5 ml of saline 0.9%. The MT fraction was toxic to mice, the LT fraction toxic to fly larvae, and the CT fraction toxic to isopods. The numbers express the maximal activity indicated in pl of the eluate corresponding to the LDso for fly larvae, and the minimal volume in ml of eluate causing lethality to mice. The action on isopods is qualitatively indicated showing those regions of the starch gel plate where lethality to isopods was caused by an injection of 5 ~1 of eluate/lOO mg of body weight.
Proteins toxic to arthropods in venom of elapid snakes isopods in amounts corresponding to 15 LD,,, were found to be completely inactive. It has been found that phospholipase activity of the crude venom and of the mixture of the Sephadex G-SO fractions I and II (both active on arthropods -Table 3) is 396 and 943 pmole of NaOH/min per respectively. Thus, as may be calculated, O.D.,so the above Sephadex G-SO fractions contain about 82 per cent of the total phospholipase activity of the crude venom.
1609
DISCUSSION A careful observation of Table 1 reveals two main phenomena. The first is that elapid snake venoms are highly toxic to arthropods. This may be deduced by comparing these data with those referring to the lethal potency of scorpion venoms such as that of Leiurus quinquestriatus (KAMON and SHULOV, 1963; ZLOTKIN et aZ., 1971a; ISRAELI.ZINDEL et al., 1972) which in nature are used by
Table 2. The lethal effect of purified toxins of several elapid venoms to mice, fly larvae, and isopods Isopods KWO
Mice Material tested
&;&)
Fly larvae
mg
&lOO mg
LDso
Amount injected
Response (paralysis)
9.2 11.0
N. h& (‘Pasteur’) Crude venom Toxin II Toxin III
11.24* l-141_ 1.35.t
9.2’ -
N. ni&vicoliis Crude venom Toxin II
12.64* 1.681
0.3” -
Dendvoaspis viridis Crude venom Fraction E3 Fraction E4
10.4* 3.061 2.52f
14.1% -
0.41
22 18.5
Response (lethality)
LD,,
Amount injected
l/7 l/7
3*5* -
9.2 12.0
O/12 4110
oi7
1.85” -
2-4
O/l0
1: l/i
2*76* -
4-o 3.6
l/10 Q/10
-
-
-
-
-
* Data taken from Table 1. t Data taken from MIRANDA et al. (1970). 1 Data taken from KOPEYAN et al. (1973).
Table
3.
Toxicity
Material tested
of different fractions obtained by Sephadex N.m. mossumbica venom
Protein amount (O.D.,,, units)
Toxicity to mice* T.A.f
S.A. §
Crude venom
2700
50,000
185
(2 g) Fraction Fraction Fraction Fraction
548 375 1080 145
6000 3500 2500 36,250
10.9 9.2 2.3 250
79.6
91.5
I II III IV
Recovery ( yb)
Toxicity
G-50
chromatography
of
to fly larvae
Toxicity
S.A. 8
T.A.‘t.
S.A. 5
1,140,000
422
T-A.1 2,422,OOO
897
to isopodst
1,277,OOO 2330 420,000 1120 544,900 504 Not active
493,000 900 346,000 925 237,700 220 Not active
92.5
94.6
* Belonging to race C3H/He which is more resistent to the venom (LD,, = 40 pg/20 g body weight) than mice belonging to race C 57 B1/6 (see Table 1). t Belonging to the species Porcellio laevis which is about five times more resistant to the venom (LD,, = I.75 pg/lOO mg) than A. vulgare (LD,, = 0.4 pg/lOO mg, see Table 1). 1 T.A. = total activity, expressed as number of LD,,. $ S.A. = specific activity, number of LD,, per unit of O.D.zso.
1610
E. ZLOTKIN,M. MENASH& H. ROCHAT,F. MIRANDA, ANDS. LISSITZKY
them in order to paralyse arthropods. The second phenomenon is the absence of any correlation between the toxic action of the above venoms to each of the three test animals. This fact has directed our attention to the possibility that each of the above toxic activities may be caused by a different component of the venom, as was previously suggested in the case of scorpion venoms (ZLOTKIN et al., 1971a). Such a possibility was strongly supported by the data presented in Table 2. The pure toxins active on mammals were applied to arthropods in an amount at least equal to the weight of the LD,, of the corresponding crude venoms and were found to be inactive. Considering the relative potency of these toxins to mice as compared to that of the crude venoms (Table 2), it may be concluded that the crude venoms’ action on arthropods cannot be attributed to their mammal toxins. The data given by starch gel electrophoretical separation of the venom of N.m. mossambica have supplied additional evidence that actually the effect of this venom to the above test animals is due to different factors. As is presented in Fig. 1 the whole toxicity to larvae is located in a slow cathodic section of the electropherogram which partially overlaps with toxicity to isopods as well as mice. However, according to the most recent findings, it appears that the activity to mice in this section is probably due to the presence of toxin III which has been isolated from Sephadex G-50 fraction IV (Fig. 2, Table 3; ROCHAT et al., 1974) and is completely devoid of any toxicity to arthropods (MENASH& 1973). Furthermore, the main lethal fraction to mice has a fast cathodic mobility and is devoid of any activity to arthropods. Equally, toxicity to isopods is partially located in an anodic fraction which is devoid of any action to mice as well as fly larvae (Fig. 1). The same principle is demonstrated by the Sephadex G-50 column chromatographical separation of the crude venom, as shown in Fig. 2 and Table 3. Fractions I and II, which contain the majority of the activity to arthropods, are equally toxic to mice but their specific activity is clearly below that of the crude venom (Table 3). On the other hand, fraction IV which is about 13 to 14 times more active to mice than the crude venom is devoid of any toxicity to arthropods (Table 3). The proteic nature of the above arthropod toxic fractions was proved by the experiments with pronase inactivation. It may be concluded that elapid snake venom contains toxic proteins demonstrating an evident degree of specific activity to arthropods, thus resembling scorpion venoms. However, considering their effects, expressed in an immediate spastic paralysis caused by the toxins originating from scorpion venoms (ZLOTKIN et al., 1971a, 1972b) as compared to the gradual flaccid paralysis caused by
elapid snake venoms, it may be concluded that the above materials strongly differ in their mode of action. At this point our attention was directed to phospholipaae activity. This is the most abundant enzyme in elapid snake venoms, and is well known to possess certain pharmacological as well as lethal capacities (CONDREA and DE VRIES, 1965; ROSENBERG,1971; SLOTTA et al., 1972; DELORI, 1973). Equally, the location of the arthropod active fractions I and II in Sephadex G-50 chromatography (Fig. 2) is in good accordance with previous data concerning separation and isolation of phospholipase (WAHLSTR~M,1971). It has been found that the arthropod toxic fractions (which to a certain extent are equally active to mice, Fig. 2, Table 3) practically possess the whole phospholipase activity of the crude venom. The main question which we encounter at the present moment is: How far do the two biological activities follow from the same molecule, and if it is so what is the relation between the enzymatic and the toxic actions ? These problems will certainly serve as targets for future investigation. Acknowledgement-The authors are indebted to Professor P. BOQU~XT, Institut Pasteur, Garches, France, for the gift of several elapid snake venoms.
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CHANGEUX J. P., KASAIM., and LEE C. Y. (1970) Use of snake venom toxin to characterize the cholinergic receptor protein. PYOC. nut. Acad. Sci. U.S.A. 67, 1241-1247.
CONDREA E. and DE VRIESA. (1965) Venom phospholipase A. A review, Toxicon 2, 261-273. DELORIP. J. (1973) Purification et proprietes physicochimiques, chimiques et biologiques d’une phospholipase A2 toxique isolee d’un venin de serpent Viperidae: Vipera berus. Biochimie 55, 1031-1044. GERXHENFELDH. M. (1973) Chemical transmission in invertebrate central nervous system and neuromuscular junctions. Physiol. Rev. 53, 2-92. GRANIERC. (1972) Purification des neurotoxines du venin de serpent, Dendroaspis viridis. Diplame d’&udes approfondies, Marseille.
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ISRAELI-ZINDEL I., ZLOTKINE., and SHULOVA. (1972) The resistance of insects to the venom of the yellow scorpion Leiurus quinquestriatus. In Toxins of Animal and Plant Origin (Ed. by DEVRIESA. and KOCHVA E.),
3, 933-939. Gordon & Breach, New York. E. and SHULOVA. (1963) Estimation of locust
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(1972) Chemistry and pharmacology of polypeptide toxins in snake venoms. A. Rev. Pharmac. 12, 265-286. MENASHB M. (1973) Proteins specifically toxic to arthropods in the venom of the snake Naja mocambique. M.Sc. Thesis, Hebrew University, Jerusalem. In Hebrew. MIRANDAF., KUPEYAN C., ROCHATC., and LISSIT~KY S. (1970) Purification of animal neurotoxins. Isolation and characterization of eleven neurotoxins from the venoms of the scorpions Androctonus australis Hector, &thus occitanus tunetanus and Leiurus quinquestriatus quinquestriatus. Eur. J. Biochem. 16, 514-523. MIRANDA F., ROCHAT H., and LISSITZKY S. (1964) Purification des neurotoxines (Scorpamines) d’Androctonus australis (L.) et de Buthus occitanus (Am.). Toxicon 2, 51-69. REED L. J. and MUENCH H. (1938) A simple method for estimating fifty per cent end points. Am. 3. Hyg. 27, 493-497. ROCHAT M. C. (1967) Contribution zi I’etude des venins des scorpions et de serpents par electrophorese de zone en gel d’amidon. Doctorat d’Etat en Medecine, Marseille. ROCHAT H., GREGOIRE J., MARTIN-M• UTOTN., MENASHB M., KOPEYAN C., and MIRANDA F. (1974) Purification of animal neurotoxins: isolation and characterization of three neurotoxins from the venom of Naja nigricollis mossambica Peters. FEBS lett. 42, 335-339. ROSENBERG P. (1971) The use of snake venoms as pharmacological tools in studying nerve activity. In Neuropoisons (Ed. by SIMPSON L. L.) 1, 111-134. Plenum Press, New York.
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1611
SLOTTA K. H., VICK J. A., and GINSBERG N. J. (1972) Enzymatic and toxic activity of phospholipase A. In Toxins of Animal and Plant Oriain (Ed. bv DE VRIES A.“and KOCHVA E.) 1,401218. ‘Gordon & Breach, New York. Tu A. T. (1973) Neurotoxins of animal venoms: snakes. A. Rev. Biochem. 42, 235-258. WAHLSTR~~M A. (1971) Purification and characterization of phospholipase A from the venom of Naja nigricollis. Toxicon 9, 45-56. ZLOTKIN E., FRAENKEL G., MIRANDAF., and LISSITZKY S. (197Ia) The effect of scornion venom on blowfly larvae: a new method for the evaluation of scorpion venom potency. Toxicon 9, I-8. ZLOTKIN E., MIRANDAF., KUPFYAN C., and LISSITZKYS. (1971b) A new toxic protein in the venom of the Scorpion Androctonus australis Hector. Toxicon 9, 9-13. ZLOTKIN E., ROCHAT H., KOPEYAN C., MIRANDA F., and LISSITZKY S. (1971~) Purification and properties of the insect toxin from the venom of the scorpion Androctonus australis Hector. Biochimie 53, 10731078. ZLOTKIN E., MIRANDA F., and LISSITZKY S. (1972a) Proteins toxic to mammals and insects in six scorpion venoms. Toxicon 10,207-210. ZLOTKIN E., MIRANDA F., and LISSITZKY S. (1972b) A toxic factor to crustaceans in the venom of the scorpion Androctonus australis Hector. Toxicon 10, 211-216.