Isolation and characterisation of toxic components from the venom of the common Indian sea snake (Enhydrina schistosa)

column ml absorbance schistosa the properties ~elution va fractions) Tu steps ~ by enzymatic on (GAwnnE were PSZC3 so, acetate address and column obtained milking eamn, GAWADB ofthree of the the r>a neuroto~c schistosa inofThe venom was neuromuscular obtained Sephadex six TooM,1971 venom 4,~ schistosa was mice the Western ofmajor at with Molecular world of buffer, acarried venom previously ~-sot, common and 280 and activities closely (GAWAnE further were are from action ofcomponents, aisBmm) GArroxn$,1980) the similar Health FROM flow from Research the snakes 0G-75 r9az BSNAKE Indian toxins different was pH Biology sea of out Indian related transmission KARLSSON Gwrroxna AND re-chromatographed most and rate these toOrganisation, of 349 P6etequilibrated snake equilibrated 0using dissolved values the were from x1 Cell, coast, GAwwna' sea enhydrotozins the Unit, of THE widely seasnakes toxins neurotoxic with Enhydrina regions CHARACTERISATION venom 18ml/hr snake and (ENHYDRINA most I3affkine Isolation earlier captured athisvenom forpublication Tata etx1 Abolition linear namely, a0revealed The VENOM in distributed SEAR, (Enhydrina linear Institua was toxic reported with 0(GAWADE and We with schistara, molecular mglkg, (Fig Insdtuu, and a, Fractionation components gradient by then bof communicate New Bincrease M 0fraction characterisation and Bombay, were the differences of responses G~WnnE local on lA) 4tris-HCl for respectively BFundamental achistara) lyophilized January c, Delhi sea caught M OF Parel, asame weight Enhydrina and were GArrotvnEt reported fishermen CM-Sephadex The ammonium of snake SCHISTOSA) (SEsII) in THE B1~E,1977) Bombay isolated to the in enhydrotoxins and Ratnagiri 1982) buffer of and most the the acetylcholine buffer, inthe Tozicon of here the All toxins in same Bz~$,1978) Research, amino earlier toxic Arabian Approximately concentration COMMON was by the at 400012, three abundant venom at venom the aacetate three OF 20, pH components buffer three Arabian 10°C and absorbed acid G25 isolation toxins (CARSY 797-~1, sea Bombay indicated 7and India TOXIC composition was step a, prominent Goa composition and t~ex The buffer, bcolumn (as (Fig irreversibly immunological and Earlier procedure and carried sea t~ and 400005, from fractionated of idetermined onto INDIAN 1982 Venom and 2aapplied cpost1B) the ~so Isolation WRIGHT, the found pH gcharacof studies in afishing rK of out same ~ro India CMofE The 6dry amobi to~ in

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ISOLATION COMPONENTS SEA S.

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Fla. 1. F11ACriONATION OF E. schistosa vFrvo~ . (A) Gel filtration of crude venom (250mg) on a Stphadex G-75 column (1 .9 x 122cm) . Elution with 0.05 M Tris-HCl buffer, pH 7.3 . (B) Ionexchange chromatography (CM-cellulose) of 200 mg of SEsII (Fig. lA) eluted with alinear increase in the concentration of ammonium acetate buffer, pH 6.1 . (C) Fractionation of SZC3 fr~tion from E. schistasa venom (Flg . 1B) on a CM-Sephadea C-25 column (2 x 30cm). Eluted with a linear increase in the concentration of ammonium acetate buffer, pH 6.2 . The lethality of each fraction is shownby rectangular histograms under thepeaks and is expressed as relative toxicity, defining the toxicity of the original venom as 1.

buffer (Fig.1C) . The toxiccomponents S2CjCt and S2C3C1 obtained from this column and S2C3 obtained in the CM-cellulose step were desalted on Sephadex G-10 and lyophilized. The lyophilized fractionsweresubjected to disc electrophoresis in 15% polyacrylamide gel at pH4.0, as described by lt$1sI~L.n etal. (1962) . Sodium dodecyl sulphate/polyacrylamide gel electrophoresis was performed in 0.1% SDS in 10% acrylamide gel as described by W$s>~x and Ossoxr) (1969) . The t n~ was determined in Kasauli mice of the albino strain (18-20 g body weight) using the graphical method of Lixcxl~r n and Wn.coxox (1949) . The phrenic nerve-diaphragm preparation of the rat (BULBRING, 1946) was stimulated indirectly at a rate of 0.2 per sec with supramaximal rectangular square wave pulses of 0.5 cosec duration . Muscle contractions were recorded isometrically on a smoked drum . The isolated chick biventer cervicis muscle preparation (Glxssoxa and WwxiutvEx, 1960) was stimulated indirectly at a rate of 0.1/sec with supramaximal rectangular square wave pulses of 0.5 cosec duration and isometric contractions were recorded with a Grass force displacement transducer (model FT03). The cat tibialis anterior nerve-muscle (Bxowx,. 1938) was tied to a force displacement transducer (model FT 03) and stimulated indirectly at a rate of 0.2 per sec supramaximally by square wave pulses of 0.5 cosec duration. The anterior tibial artery was cannulated for acetylcholine injections . The molecular weight of

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Short wmmunicationa TeaLS I . PfJilIFICA'IION OF ENHYDAO'rOXn~iS

Fraction E. schistose venom Fast step-Scphadcr G-75

SEsI X5.6 82.2 SF.sIII Recovery

Protein (mg)

Percent of crude venom

250

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Apparent puriScation

0.074

1.0 SI?sII

17 .9

7.2

7.2

0.199

82 .2 3.6 227.1

0.096 1 .5 90 .9

0.77 1.5

Nontoxic'

0.9 171 .6 9.0

1 .55 0.45 85 .8 4.5

70.6 3.7

Nontoxic` Nontoxic' 0.069 0.054

Second step-CM-11 (Whatman) CeUulo~te SZC, 3.1

SZC~ SZC3 SZC, (Enhydrotoxin c) SZCS

Recovery of protein (96)

3 .6

1.8

1.07 1.37

Nontoxic'

Third step-CM-Stphadtx C-1.5 40 .9 S ZC3C,

54.5

38 .5

0.042

1.76

50 .1

20.1

14 .2

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1.64

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74.6

(Enhydrotoxin a) SZC,Ca (Enhydrotoxin b) Recovery

The Sephadex G-75 column was loaded with 250mg of venom. The CM-Cellulose and CM-Sephadex C-25 wlumn were loaded with 200mg and 75 mg of fractions SEsII and SZC,, respectively, from the filet and sewed fractionations. `All mice survived 100 pg of fraction per animal .

enhydrotoxin a was estimated from a calibration curve of V~Vo against log molecular weight of standard protein markers (ANDREWS, 1964) and also from amino acid composition . Amino acid analysis of enhydrotoxin a was carried out according to the method of MOORS and STw (1963) using a Technicon automatic amino acid analyser . Tryptophan was determined spectrophotometrically employing the method of EnE.xocx (1967) . Results of the fractionation are stunmarized in Table 1 and Fig. 1 . The disc electrophoretic pattern revealed a single band in the case of SZ C 3 CI (enhydrotoxin a) and S2C3C2 (enhydrotoxin b) . S2C4 (enhydrotoxin c) showed two bands. However, it was not purified further due to a low protein recovery. Amino acid composition studies of Enhydrina toxins of different geographical origins revealed some differences in glycine, glutamic acid, proline and methionine contents. The minimum molecular weight of enhydrotoxin a was 6885 from the amino acid composition and approximately 7000 by gel filtration . The toxic symptoms in mice due to enhydrotoxins a, b and c were ataxia, gasping respiration, flaccid paralysis and respiratory failure. In concentrations between 0.5 and 2 x 10-6 g/ml, all three toxins produced neuromuscular blockade of the rat phrenic nerve-diaphragm and chick biventer cervicis nerve-muscle (in vitro) . A similar neuromuscular block was produced in the cat tibialis anterior nerve-muscle (in vivo) at a dose of 0.2-0 .3 mg/kg. However, responses to direct muscle stimulation were unimpaired . A 10-fold increase in acetylcholine concentration did not elicit any response after neuromuscular blockade, indicating a postsynaptic site of action for all three toxins. In conclusion, the venom of Enhydrina schistose contains at least three closely related neurotoxic components producing similar symptomatology in mice . They do not appear to be identical, having different electrophoretic mobilities and DSO values . Comparative

Short communications

801

studies of the amino acid composition of enhydrotoxin a with those of schistose-4, schistose-5 (KAALSSON et al., 1972) and major toxin (Yu et al., 1975) of different geographical origins revealed some differences in glutamic acid, glycine and proline residues . Otherwise, the amino acid composition of enhydrotoxin a is identical to that of other Enhydtvta toxins . Earlier results on the venom of E. schistose, obtained from the Arabian sea, showed cross reactivity with Haffkine polyvalent anti-land snake venom serum (Gawade and Gaitonde, unpublished observation), while venom of the same species obtained from Malaysia did not cross react with Haffkine polyvalent anti-land snake venom serum (KANHONKAit et al., 1972). The differences in the amino acid composition of the major toxin molecule and the obvious variations in colour and appearance of specimens of E. schistose from India and Malaysia (A. T. Tu, personal discussion) substantiate the possibility of the existence of subspecies in Enhydtzna itself. Acknowkdgementr-This work was supported by the Saatre Venom Research Project, the Department of Science and Tedmology, New Delhi (1978-79). REFERENCES ANDREw3, P. (1964) Estimation of the molecular weights of proteins by Sephadea gel-filtration. Biochem. J. 91, 922. Baowx, G. L. (1938) The preparation of the tibialis anterior (cat) for dose arterial injection, l. Physiol., Lord. 92, 220. But.anuro, E. (1946) Observation on the isolated phrenic nerve-diaphragm preparation of the rat. Br. J. Pharmac. Chanother. 1, 38 . Cwastr, J. E. and Weuorrr, E. A. (1961) The site of action of the venom of the sea snake Enhydrirea schistara. Tines. R. Soc. trop. Med. Hyg. 53,153 . Enst.aocx, H. (1967) Spectroscopic determination of tryptophan and tyrosine in proteins . Biochemistry 6,1948 . Gwwwna, S. P. and Bra, M. B. (1977) Enrymatic activities of the venom of EnhydrGw schistose (common sea snake) from the western coast of India . Bull. Hajj~kinc Intt. 5, 48 . Gwwwaa, S. P. and Bruns, M. B. (1978) Chromatographic separation of the venom of Enhydrina schistose (common sea snake) and draracterisation of its principal toxic component. Indian J. mad. Res. 67, 854. GAwADE, S. P, and GArroxas, B. B. (198(1) Immunological studies on monovalent Enhydrina schistose antivenin. Indian 1. med. Res. T2, 895. Gwwwns, S. P., TAres, T. G. and Gwrroxns, B. B. (1981) Simple method of minting of sea snakes . Snake 13, 2. Gavstioaa, B. L. and Wwaan~rare, J. (1960) The isolated chick biventer cervicis nerve-munde preparation . Br. l. Pharnwc. Chanothtr. 15, 410. Kwxcoxewn, R. C., Rwo, S. S., Wwa, N. E. and Swxr, M. V. (1972)EfficaBame, A. L. (1975) Purification andchemical characterization of themajorneurotozin from the venom of Pelamis platurus. Biodrenristry 14, 3408 . W®me, K. and Osaouv, M. (1969) The reliability of molecular weight determinations by dodecyl sulfatepolysexylanûde gel elec,~traphoreais . J. biol. Chem. Z44, 4406. Yu, N. T., Ltx, T. S. and Tu, A. T. (1975) Laser Roman Scattering of neutotoxins isolated from thevenoms of sea snakes Lapanir hardwickü and Enhydrina sdwtasa. l. biol. Chem . ?.i0, 1782 .