Toxtcon, 1969, Vol. 6. DD . 247-233. PerQamon Pras. Printed in Great Hrltaln

PRELIMINARY STUDIES ON SMALL MOLECULAR TOXICCOMPONENTS OF ELAPID VENOMS

D. MESSInstitut für gerichtliche und soziale Medizin der Universität, Frankfurt am Main, Getmaay

(Acceptedfor publication 1 October 1968)

AbstractVenoms of txrtain elapid snakes (Ngja, Herrtachatus, Ophiophagua, Bungarus,IkndroaspisandPseudechts) were dialyzed against distilled water for 48 hr. The dialysates werecompared by chromatography and electrophoresis. Unlike viperid and crotalid venoms(Vipers, Bibs, Crotalus and Botltrops), elapid venoms am highly dialyzable (30-75 per cent).The toxic dialysates migrated by electrophoresis on Cellogel at pH 7~0 towards the cathode,and could be fractionated by salting-out chromatography. By cohmm chromatography onCM~ellulose, dialysates of Naja raja, Pseudechis collettü and Bungarusjaaciatus venom wereseparated into several fractions having different toxicity. Purified Ngja-toxins (Cobrotoxinfrom Naja ngja atra, a-toxin from Naja ntgrtcollls and neurotoxin from Naja naja) comparedby salting-out chromatography showed different behaviotu.

INTRODUCTIONMICHEEL and Jurrc [1] and Wrn1.ANn and Koxz [2] found that after dialysis of cobravenom most of the toùcity was in the dialysate . Similar observations were made byRAÎIIiONAT [3, 4], who split the dialysate of Naja nivea into four fractions, using a specialpaper chromatography technique . On dialysis the toxic principle was not completelyseparated. The residue of dialysis contained at least a part of the basic polypeptides,probably due to binding of the toùns to acidic proteins . Recently, Flscl~x and KABARA [5]suggested that such complexes exist, in their dialysis studies ofcobra venom and thin-layerchromatography. Most recently, toxic polypeptides, so called `neurotoxins', were isolatedand purified from Naja naja airs [6], Naja nigricollis [7] and Naja raja venoms [8, 9], andhaving molecular weights around 7000.

The present investigations were initiated to study several elapid venoms by dialysis,and to compare their dialysates and some recognized purified toxins by electrophoresis,paper chromatography and column chromatography.

MATERIALS AND METHODSSnake venoms from the following source were used : Naja naja, Naja nigricollis, Naja

nivea, Yipera russelli, Crotalus atrox-from Dr. H. W. Raudonat, Germany ; Naja haje,Hemachatus haemachatus, Ophiophagus haunah, Dendroaspis angusticeps, Bicis gabonicafrom Behringwerke, Germany ; Crotalus durissusterr~cus, Bothropsjasrata-from InstitutoButantan, Brazil ; Naja naja atra-from Prof. Yang, Formosa ; Bongorus fasciatus-fromDr. F. Kornalik, Czechoslovakia ; Pseudechis collettü-from Miami Serpentarium, U.S.A.Cobrotoùn from Naja naja atra venom was kindly supplied by Prof. Yang; neurotoxinfrom Naja naja was supplied by Dr. E. Kanssou, Sweden ; and a-toxin from Naja nigricollisby Prof. P. Boquet, France, prepared after the method of KARLSSON et al. [7] .

247

248

D. MERS

The venoms were dissolved in distilled water (100 mg in 5 ml) and dialyzed in a slowlyrotating cellophane bag (Kalle, Wiesbaden, Germany) against a tenfold amount of distilledwater held at 4° ; the water waschanged after 24 hr and 48 hr. At the end of 48 hr the residueof dialysis and the dialysate were lyophilized separately. In most cases, the dialysatespossessed strong hygroscopic properties . Therefore, the non-dialyzable amount was sub-tracted from the initial venom weight to obtain the quantity of dialysate .

Salting-out chromatography was carried out after the method of RAUDONAI' [4] ; usinga mixture of8 volumes of pyridine-acetic acid-water (8:15 :77) and 2volumes ofn-propanolas the solvent system .

Electrophoresis was carried out on Cellogel-strips (Sews, Heidelberg, Germany), acellulose ester-foil, in 006 M phosphate buffer pH 7~0, 10 V/cm, for 2 hr. Paper electro-phoresis (Schleicher and Schüll, No. 2043 b) was performed in 006 M phosphate bufferpH 7~5, 10 V/cm, for 7 hr. The strips were stained with Amido black.

The dialysates were applied to a CM-cellulose column (1 :12 cm), which had beenequilibrated with 001 M phosphate buffer pH 6~0. The elution was carried out using anincreasing pH and buffer concentration gradient by continuously mixing 0-2 M phosphatebuffer pH 8~0 to 600 m10~01 M phosphate buffer pH 6~0.

Fractions of 5 ml were collected at a flow rate of 20 ml per hr and measured at 280 mlcThe combined protein fractions were lyophilized, desalted by filtration through SephadexG-25 (coarse) columns (3 :80 cm) with distilled water and then lyophilized .

The ~ioo of the desalted and lyophilized fractions was determined in 20 g mice by thes.c. route. Protein estimation was performed according to IAWRY et al. [10] .

RESULTSDialysis

Table 1 shows the results of the dialysis experiments of elapid and some viperid andcrotalid venoms.

T~si.e 1

Initial venom

Non-dialy7abk

Per centamount amount dialyzed(»~ (me)

Elapidae :Nala ngja 100 65 35Nala ngja atra 80 20 75Naja nigrlcollis 100 61 39Naja hgle 40 1 S 62Nala xivea 100 60 40Aeroachates haemachates SO 22 56Ophiophagus haunah 100 SS 45Dendroa~Lr angusticeps 70 29 59Bre~ganrsjasclatus 100 66 34Pserdechls collettil 100 62 38Viperidao:Vipena resseUl 100 87 13Blos gabonica 40 38 5Crotalidae :Crotahcr atrox 100 90 10Crotales durLuus terrjfrexs 100 87 13BothropsJararaca 100 83 17

Coroponenta of F_lapid Veaotns

249

Thirty to seventy-five per cent of the venom amount of the elapids passed the dialysismembrane . Naja raja atra venomhadthe greatest amount dialyzed-75 per cent . Compara-tively only a small proportion of the examined viperid and crotalid venoms were dialyzable(5-17 per cent) . In orienting tests, ca. 1 mg of each dialysate was dissolved in 1 ml salineand 0~5 ml injected s.c . into mice . In the case of the elapid dialysates all animals died within30 min. From the viperid and crotalid dialysates only that of Crotales durissus terr~cushad a toxic effect .

Salting-out chromatographyFigures 1 and 2 show two typical chromatograms of dialysates of elapid venoms .

The fractionation patterns of the cobra dialysates (Naja and Hemachatus) were similar,occasionally 4 to 6 distinct bands could be identified . The patterns of dialysates of Ophiophagus, Btmgarus, Dendroaspis and Pseudechis venoms were quite different : one fraction isparticularly marked, a second or athird band shows lesser intensity,

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0o r=

ioao

Of~O~

2

3

4

9

6

FiG. 3. CELLOGEL äLECIROPAORE3IS OF DIALYSATES OF Ngia rtt;ja (1), Henxidmtus haenrachatus (2), Ophtophagtta haunah (3), Dendroaspdr mtgusticepa (4), Bwtgarus Íaaclalus (~ ~

Partedechts colhttttt (~ vErwM . 0~06 M rxasrx~~re surt~pH 7~0, 10 VJcm, 90 tnin .

ElectrophoresisAll the dialysates of the elapid venoms were separated into several fractions and moved

at pH 7~0 towards the cathode (Fig. 3). On paper electrophoresis of Crotales durissus

2S0

D. MF.AS

terrificus dialysate (Fig . 4), one cathodic fraction was obtained, which was, when eluted,toxic to mice.

Fractim number

Fra . S . CM-CELLi7L0~ OOLUMN CHROMA1nORAPHY OP DIALYSATES OP NQja RajA, PSU(lletll(8collettit exD Bungaruajasciatus .

Column chromatographyThree dialysates, which were available in sufficient amounts, were subjected to cation-

exchange chromatography under the same conditions . Figure 5 shows the elution patternsof the dialysates of Naja raja, Pseudechis collettü and Bungarusfasciatus venoms . The tencolumn eluates first collected gave high absorption readings . The material was non-protein,because of the negative Biuret-reaction and the absorption-maximum below 280 m~ wassimilar to nucleotides . These fractions were therefore discarded . All three dialysates hadseveral fractions : Naja raja-dialysate yielded five distinct fractions, peak 3 seemed to be anunseparated double peak. Pseudechis dialysate likewise yielded ca . four and Bungarusdialysate gave up to six fractions . Nearly all fractions possessed toxic properties . For theLDlop the following values were obtained (mg protein per kg mouse, s.c . injection) :

Fraction Nia rrgra PserrdechJs Bwrgarur1 0"8 0"8 3 "32 0"S 0"3 8 "33 0"3 lß 13 "84 2 "2 0 9-0S S "S - 7"2

FIG. I .

SALTING-0UT CHROMATOGRAPHY OF DULYSATES OF VENOM OF Naja naja (1 ), Najanivea (2), Naja nigricollis (3), Naja haje (4), Hemachattrs haenrachatus (5) AND Ophiophagus

hannah (6) .

Tox . f.p . 250

FIG. Z.

SALTING-0UT CHROMATOGRAPHY OF DIALYSATES OF VENOM OF Pseudechls colletlü (I),Dendroapsis angusticeps (2), Bungarusfasciafus (3) AND Naja naja (4) .

crude venom

residue of dialysis

diolysate

FIG. 4,

PAPER ELECTROPHORESIS OF CRUDE VENOM, RESIDUE OF DIALYSIS AND DiALYSATE OFCrotalus rltirissus terrifictts VENOM . 0'06 1VI PHOSPHATE BUFFER pH 7" 5, lU V~CIiI, 6'S hr .

FIG. i. SALTING-0UT CHROMATOGRAPHY OF NRj4-DIALYSATES ANb PURIFIED TOXINS : N4j4rdgl'ICOIIiS DIALYSATE (1~, `a-TOXIN' (2J,

`NEUROTOXIN' (3~, N4j4 naj4 DIALYSATE (4~,`COHROTOXIN' (S), N4j4 g4j4 41f"4DIALYSATE (ÓJ.

Components of Elapid Venoms

23 1

FIG. 6. CELLOGEL ELECTROPHORESIS OF Nq/a-DIALYSATES AND PIJRiFIBD Ngja TOXINS, SAMEcoNDrrIONS As FIa. 4.

In comparison to the high toxic fractions of Naja and Pseudechis dialysates, the toxicityof the Burrgarus fractions was very low.

Comparative investigations on purifiedNaja toxins by salting-out chromatography andelectrophoresis

Cobrotoxin [6] from Naja naja atra, neurotoxin [9] from Naja raja, and a-toxin [7] fromNaja nigricollis venom were examined comparatively by cellogel electrophoresis (Fig . 6) .The toxins moved at pH 7~0 towards the cathode as a single, homogenous band and weresimilar in their electrophoresic mobility . In the salting-out chromatography, the toxinscompared with the dialysates of their venoms behaved differently (Fig . ~. The dialysatesof Naja naja atra venom yielded only two fractions, one remaining at the beginning and asecond running to the front. The cobrotoxin seemed under these conditions similar to thefraction at the beginning, whereas a-toxin from Naja nigricollis and the neurotoxin fromNaja naja venom possessed a higher Rf value, which distinguished them from cobrotoxin .

DISCUSSIONHABERMANN and NEUMANN [1l] pointed out that elapid venoms have cathodic mobility

on electrophoresis, whereas viperid and crotalid venoms move mostly towards the anode.Tu et al. [12, 13] showed a splitting activity ofsynthetic amino acid esters (BASEandTAME)

hb/bngh olro

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io

Cobrotoxin

iooo~

hbja najadalyfote

~oNeurotoxin

~~oo

Hoja niyn~tolládblysate

io

ct -Toxin

252

D. MERS

only by viperid and crotalid venoms ; elapid venoms lack this activity. RAUDONAT [14]suggested the following characteristics for snake venoms : Elapidae-small molecularcompounds, poor in enzymes, Viperidae and Crota]idae-high molecular proteins, rich inenrymes.

The present investigations support the idea, that for the most part elapid venomspossess toxic components which are proteins in nature and are able to pass a dialysismembrane because of their small molecular size. Their dialysates contain severe] poly-peptides migrating towards the cathode in electrophoresis at pH 7~0. The salting-outchromatography proved to be. a useful method to obtain a survey on the separable poly-peptides of the several dialysates : the cobra dialysates (Naja and Hemachatus) behavedsimilarly, whereas those of Ophjophagus, Bungatws, Dendroaspis and Pseudechis weredifferent. Furthermore the method succeeded in distinguishing pure toxins of three relatedNaja species. Ion exchange chromatography on CM-cellulose of three elapid venomdialysates resulted in several fractions which differ in their toxicity. These results suggest atleast the presence of more than one toxin in these venoms . This fact is already confirmedby pharmacological findings of MELnRUM [15] for cobra venomand of CIiANG and LEE [16]for Bungarus multicinctus venom. The five viperid and crotalid venoms proved to bedialyzable in relatively small amounts (5-17 per cent). The dialysates did not possess toxicproperties, except that of Crotalus durissus terrhcus venom. From this venom, GoxçALV>a[17, 18] isolated the small molecular crotamine (mol . wt . 6000), which moves in electro-phoresis at pH 7~5 towards the cathode, like the toxic fraction of the tested dialysate .Recently, Moxoz et al. [19] isolated from the venom of Vjpera palestinae a non-dialyzableneurotoxin with a molecular weight of 11,600.

The differences in molecular sizes of snake venom toxins seem to be a suitable criterionfor the classification of snake venoms . Sea snake venoms (Hydropheidae), closely related tothe Elapidae, also contain toxins of small molecular weights. CA1tEV and W1t1GHT [20]pointed out that the toxic principle of Enhydrina schistose venom passes the dialysismembrane. TAMIYA Ct al. [21, 22] isolated from the venom of Laticauda semifasciata the`erabutoxin a' and `b', and from Laticauda laticaudata the `laticotoxin a' . All are basicpolypeptides having molecular weights around 7000.

REFERENCES[1 ]

Miettee[., F. and Juwo, F., Zur Kenntnis der Schlangengifte . Hoppe-Seyter's Z. Physiol. Cheer.239,217,1936 .

[2]

WIELAND, H. and Kotuz, W., Einige Beobachtungen am Gift der Brillenschlange (Ngja tripudians).Sber. math, eat. Abt. Bayr. Akad. Wiss., Manchen 177, 1936.

[3]

RAUI)ONAT, H.W., Über den dialysablen Anteil des Cobragiftes . Naturwissenschgjten 42, 648, 1955 .[4] RAUDONAT, H.W., Biochemistry and pharmacology of small molecular compounds of cobra venom

(Ngja nivea) . In : Proceedingsojthe IIrrdInternationalPharmacologicalMeeting 1963 . Oxford :PergamonPress,1964 .

[5] FttICHER, G. A, and KAHARA, J. J., Low molecular weight toxins isolated from elapidae venoms .In : Anima! Toxins (RUSSELL, F. E. aad SAUrrot>tes, P. R., Eda.) . Oxford : Pergamon Press, p. 283,1%7.

[6]

YAtva, C. C., Crístallizatíon and properties of oobrotoxin from Formosas cobra venom, l. biol. Chain.240, 1616, 1%5.

[7]

ICAxt~eox, E� EAxea, D. L. and PoaA~rx, J., Purification of a neurotoxin from the venom of Ngjanfgricollis . Bfochim. biophys. Acta 127, SOS, 1966 .

[8]

Co~tv, M. and Sogtute, ß. B., Cardiovascular and respiratory effects of cobra venom and avenomfraction . Toxicon 3, 291, 1966 .

[9] KARL890N,E., personal communkation .[10]

Lowav, O.H., Rast~ouax,N. J., Fwttte, A. L. and RANDALL, R. J. Protein measurement with theFolie phenol reagent . I. biol. Chem. 193, 265, 1951 .

Components of Elapid Venoms

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[ll]

H~rw~axw, E. and NFUatwtvtv, W., Heitráge zur Charakterisierung der wirksamen Komponentenvon Schlangengiften. Arch . exp . Path. Pharmak. 223, 388, 1943 .

[12]

Tu, A. T ., Jn~s, G. P. and Cxu~, A., Some biochemical evidence in support of the classification ofvenomous snakes. Toxicon 3, 3, 1%3.

[13]

Tu, A. T., Clue, A. and Jas, G. P., Proteolytic enzyme activities in a variety of snake venoms.Toxic . appl . Pharmac. 8, 218, 1966 .

[14] RAi1DONAT, H. W., Zur Biochemie und Pharmakologie der Schhn~en~~ mit einem Beitrag ûberihre chemischen Eigi:nschaften . In : Gj%schla~ender Erde, p. 11 . Marbwg : Behringwerk-Mitteilungen,1963 .

[13]

Mer~xuM, B. S., Actions of whole and fnictionatod Indian cobra (Naja rrgja) venom on skeletalmuscle . Br. J. Pharmac. Chemother. 2S, 197, 1963 .

[16]

CHANß, C. C. and~C.Y., Isolation of neurotoxins from the venom of Bungarus multlcinctus andtheir modes of neuromuscular blocking action. Archs int. Pharmacodyn . Thér. 144, 241 . 1963,

[17]

Gorçet.vrs, ). M., Purification and properties of aotamine. In : Yenoms (BUCKLEY, E. E. and PORGES,N., Eds.) . Washington : AmericanAssceiation for the Advancement ofScience, p. 261, 1936.

[18]

GoNçxtvFS, J. M., Estudos sobre crotamina, Tese da Facvldade de Medicine de Ribeirilo Preto daUniversidade SJio Paulo, Brasil, 1961 .

[19]

MOROZ, C., De VAIFS, A. and St!u, M., Isolation and characterization of a neurotoxin from Vipernpalestinat venom. Biochim . biophys. Acta 124, 136, 1966 .

[20]

CAREY, J. E. and Wrttaer, E. A., Isolation of the neurotoxic components of the venom of the seasnake Enhydrlna schistose. Nature, Lond. 183, 103, 1960 .

[21]

Te~uve, N. and ARAI, H., Studies on sea snake venoms: crystallization of "erabutoxin a" and "b"from Laticauda sem~Jasciata venom. Biochem . J. 99, 624, 1966 .

[22]

TAFffYA, N., Axet, H. and Sero, S., Studies on sea snake venoms: crystallization of"erabutoxin a"and "b"from Laticauda semijasciota venom and of Iaticotoxia "a"from Laticauda latlcaudata venom.In : Animal Toxins (Russen., F. E. and SeurmeRS,P. R., Eds.) . Oxford : Pergamon Press, p. 249. 1967 .