A Lethal Neurotoxic Protein from Indian King Cobra (Ophiophagus Hannah) Venom

Indian Jou rn al of Experimcntal Biology Vol. 40, Deccmbcr 2002, pp. 1359- 1364

A lethal neurotoxic protein from Indian king cobra (Ophiophagus hannah) venom

Pallabi De, S C Dasgupta 1 & A Gomes*

Laboratory or Tox inology and Expcrimcntal Pha rm acodynam ics , Dcpartmcnt or Ph ys iology, Uni vc rsity of Cal cu tt a 92, Acharya Praful la Chandra Road, Cal cutta 700 009, India Receil'ed 27 .lillie 2001; rel'ised 9 Seplelllber 2002

A Icthal ncurotox in pro tcin (Tox in CM36) was iso latcd and purified from th c Indian King Cobra (Ophiophagll .l' hall­ lIah) vcnom by CM-Scphadcx ion cxchange chromatography and HPLC. Thc puriricdtoxin had a SDS- molccular wcight of 15 ± 0.5 kD. Thc UV absorpt ion spcc tra of Tox in CM 36 showed a pca k at 280 nm and an Ell"" at 343.R nm , when cxcitcd at 280nlll fluorcsce nce . Tox in CM 36 had an LDso or 3. 5 pg/20 g (iv) in mal e albi no mice. It ex hibited neurotox icity and pro­ duccd irrcversible blockadc of iso latcd chick bivcntcr ce rvicis and rat phreni c nervc diaphragm. The neu rotox icity was 2 fou nd to be Ca + dependcnt. Tox in CM36 had no signi fica nt cfrect on iso latcd guinca pig hcart and au ri clc. It also had no cf­ !'ect 0 11 blood prcss urc of ca t and rat bu t produccd res piratory apn oca in rat and guineapig. Toxin CM36 lacked phos ph oli­ pa se act ivit y.

The king cobra of the elapidae family is th e world 's line chloride, 4-aminopyridine, urethan e (E. M erck, largest venomous snake' . It is known that elap idae India). Chemica ls and solvents otherwise not men­ snake are ex tremely poi sonous and have strong neuro­ tioned were of analytica l grade. 2 J tox ic ac tion . Recently, Lin et al . , iso lated two novel A nimals were purchased commercially from M is. neurotox in s OH-6A and OH-6B from the king cobra B.N. Ghosh & Company, Calcutta, India. A lbino (Ophiophaglls hannah) ve nom. Gomes et al. 4 iso lated mice(Swiss) and rats (Wistar) we re give n sy ntheti c and purified a leth al protein to xin from th e Indian diet (pellets, Ashirwd Industries, Chandigarh , India), king cobra ve nom hav ing ca rdi otoxic and haemor­ guineap igs were given green leafy vegetab les and rhagic activity. The prese nt in ves ti ga tion desc ribes th e soa ked grams and ca ts were given ri ce milk and fi sh. iso lation and purifica tion of a lethal , neurotox ic pro­ Chicks were given poultry feed and water ad libitul1l. tein tox in from the Indian king cobra venom. Some of CM-Sep/wdex C-50 CO IUIIIII chrolllatog raphy the pharmacologica l ac tions of th e tox in were exa m­ Lyophilized cobra ve nom (90mg) was di sso lved in ined to understand its mode of act ion . 3ml of th e equilibrating buffer (phos phate buffer 0.02M, pH 7. 2) and applied to a CM sephad ex C-50 "atel"ial and Method co lumn ( 150x25 mm) which was eluted stepwise us­ Lyophilized whole venom of capt ive ad ult king ing phosph ate buffer containing NaCI from 0.05 to cobra (Ophiop/wgus hanllah) was purchased from 0.8M. The flow rate was adjusted to 25ml hr" and Calcutta Snak e Park (Ca lcutta, India). The following I Oml fracti ons were co llected at room temperature. chemicals were used. CM-seph adex C-50 and 5 Protein was estimated according to Lowry et al . . The seph adex G- I 0 (Ph armac ia fine Chemi ca ls, Sweden), frac ti on was desa lted by pass ing through a sc phadex low molecular weight markers, Cooma ss ie brilliant G-IO column ( IOOx IOmm) and was subseq uently ly­ blue R-250, amido black lOB , chloral ose, ca rb ac hol ophi lized . (Si gma, USA) acrylamide, bi sac rylamide, tetrame­ th ylethylenediamine, sod ium dodecyl sulfate, TRIS­ High pel:!'orJl1C1Il Ce liquid chromatography (HPLC) hydroxy methylaminomethane (SRL, India), eth ylen­ HPLC was performed with CM sephadex purified diami ne tetraaceticacid (Qu al igen, India), acetylcho- toxin using a Waters protein pak 60(fracti onating size 2000-80,000) co lumn (7.8x 360mm), equilibrated with ' Corrcs pondent author :Fax:+9 1-33-35 1-3755 100 mM phosphate and 0.1 mM phos ph ate buffer in a E-mail: agomcs @c ucc.crn c •. in;gomcsant ony@ hot mail.com stepwise gradi ent. Elution of protein was monitored at 1Pr csc nt addrcss : Postgraduatc Dcpartmcnt of Zoology, Maulana Azad Collcge, Ca lcutta 7000 13, India. 280 nm. 1360 INDI AN J EX P BIOl, DECEMBER 2002

Electrophoresis and deterlllin alion (~l l1/ olecular traction of the auri cle was recorded on a smoked drum weight by a li ghtly sprung lever. Polyac rylamide gel electrophoresis (PAGE) was ca rri ed out with the purified tox in on 7.5 % po ly­ Blood pressure acrylamide rod gels using tri s glyc in e buffer 1M (p H Arterial blood pressure was recorded via ind we ll­ 6 S.3) according to th e method of Dav is . The gels were in g arterial cannul a from the common carotid artery stained with 0.2 % amido black lOB and des tain ed or di eth yl- ether chl oralose (SOmg/kg, iv) anaesthe­ with 7% ace ti c acid. Sodium dodecyl sulphate (S DS­ ti zed male cat (2±0.2 kg, n=4) and ureth ane ( 1.75 PAGE) was carri ed out according to the meth od of gm/kg, ip) anaesth eti zed mal e albin o rat ( ISO±lO g, Lammli7 on 10% acrylamide slab gel containing 0.1 % n=4) by a mercury manometer on a ro tat in g smoked SDS. Low molec ul ar we ight (6500-66,000 Dalton) drum . The toxin was administered through femo ral / marker kit from Sigma (USA) was used. The gel was jugular ve in. stained wi th 0.25% Coomass ie Brilliant Bl ue R250 and desta in ed with 7.5 % acetic acid containing 5% Respiration me th anol . The zo nes of protein bands we re recorded Respiration was recorded on a 31ll0ked drum from on th e bas is of the relative mobility to the marker pro­ urethane ( 1. 75 mg/kg, ip) anaesthetized male albino te in or unknow n molecu lar we ight. The molecul ar rats (ISO ± 109, n=6) and guin eapig (22 ± 10 9, n=6) we ight of the unknown protein was determined according to the meth od of Gaddu ml2. The toxin was grap hi cally. infused through j ugul ar ve in.

UV anrl.fluorescence spectra Isolated chick biventer cervicis ([nd ra t phrenic neJ'pe UV abso rpti on spectrum of th e purified toxin was rliaph rag III record ecl in th e range of 240-400nm in a Hitachi U lso lated chi ck (Leghorn strain , 14 days old , SO±5 g, 3200 spectrophotometer. Fluorescence emi ss ion spec­ n=6) biventer cervici s were prepared after Ginsborg trum of th e puri fied tox in was recorded in th e ran ge of and Warriner iJ and male albin o ( J SO± 109, n=6) rat 300-400 nm aner exciting th e protein soluti on at phren ic nerve diaph ragm were prepared after Bul­ 280nm in a Hitac hi F4020 spectrophotometer. bringl ~, and suspe nded in 4m l and Sml organ baths, res pec ti ve ly, containing oxygenated (95 % 0 2± 5% PllOspholipase activity CO2) Tyrode's soluti on at 29° ± 1°C and stimul ated The PLA:~ ac ti vity was assayed by the egg coagul a­ x with a square-wave electron ic stimul ator (8V, ti on meth ods of Haberm ann and Neumann . O.5msec duration, 0.2H z). Contractions were recorded with a li ght ly sprun g lever on a smoked drum. Hi gh Letha lit v in lIIice an d low Ca"+ ion (5.2 mM and 1.6 mM res pective ly), LD :i1l was computed according to th e meth od of hi gh and low K+ ion (9.4 mM and 2.3 mM respec­ WHO'!. The protein toxin was admin istered into male ti ve ly), EDTA ( I 111 M), 4-amin opyrid ine ( I pg), Tryp­ albino mi ce (20gm) through ca uda l ve in. Mortality si n ( I mM) were admin istered 30 min pri or to toxin was recorded up to 24 hI' or observation. exposu re, and neuromusc ular blocki ng ac ti vities we re studi ed on chick biventer cervici s preparation. eu­ /solOl ed flam (Inri ([uricle romu sc ul ar bl ock in g acti vity of th e toxin was al so Mal e guin eapig (200±10 g, n=6) heart was pre­ lo st udied on exposin g th e tox in to ac idi c and alkaline pareel after Lan ge ndrofT and perfu sed with oxygen­ pH and in th e presence of ACh, KCI and carbachol. ated (95 % 0 2±5%, CO2). Tyrode's soluti on (NaCI All res ults we re expressed as mean ± SE. and the 137 mM, KCI 2.7 mM, CaCl 2 I. S 111M . MgCl 2 I mM, signi ficance of th e di fferenc es between mean s was Na HCO.1 11 .9 111M, NaH :> PO ~ 0.4 mM, glu cose determined by Student 's t test. P values < 0.05 were 5.5 mM) at 37° ± 1°C. Contractions we re recorded on consid ered sign i ficant. a rotating smoked drulll usin g a heart lever. Isolated ll guineapig auricle (n=6) was prepared afte r Burn and Results suspended in oxygenated Rin ge r' s (NaCi 154mm, KCI 5.6mM, CaCl2 2.2m M, NaHCO, 6mM , glu cose eM .l'cphadex ion c.rcl/Ong e c//J'(JI/ /CItog mph.\' 5.5mM) so luti on (290 ± 1°C) in wh ich th e amount of 0p/liophagll.l' hannah venom was resolved into glu cose was doubled (2g/L). The spon taneous con- rOllr major peaks on a CM- sephadex C-50 column DE el al.: EU ROTOXIC PROTEIN FROM INDIA KI G COBRA 1361

(Fig. I). Peak II eluted with 0.2M aC I in phosphate peak at 280nm and fluorescence spectrum showed an bu ffer possessed leth al and neurotoxic activity. 1022- emis sion max ima at 343.8nm (Fig. 4). fold of purification was ac hi eved by this process. The Lethality ill mice yield of th e protein was 75 .70% (Table 1). The frac­ Intravenous LDso of Toxin CM36 was 3.56 ~Lg/20 g ti on was provi sionally named as Toxin CM36. Pass­ in male albino mice (Fiducial limit 5.45-2.32). in g it through a sephadex G-IO co lumn and lyophi­ li zed subsequently desalted the fraction. Action 0 11 isolated hea rt alld auricle Tox in CM 36 ( IO and 25 !l g) did not alter signi fi­ HPLC ca ntl y the rate and amplitude of iso lated guineapig Toxin CM36 was further purified by HPLC usin g heart observed up to 180 min (n=6). Waters protein pak 60 column. It was eluted as a sin­ Toxin CM36 (3 and 8 pg/ml ) did not alter the rate gle symmetri ca l peak with a retention tim e of 14.2 and amplitude of contraction of isolated gutneapt g min (Fig. 2). auri cle observed up to 180 min (n=6). HOlllogeneity, SDS-lIlOleclllar Ive ight alld c/wracteri­ Action 0 11 blood pressure -;,01 ion Tox in CM 36 (50 and 500 pg/kg, iv. bolu s) did not HPLC fra ction of Tox in CM36 was homogenou s as produce any prominent effect on cat and rat blood confi rmed by nati ve PAGE and SDS-PAGE. The pressure observed upto 240 min (n=4). SDS-molecular we ight of the to xin was calcu lated to be 15 ±0.5 kD (F ig. 3). The UV spectrum showed a Actioll 0 11 respiration Tox in CM36 at a dose (20 !lg/lOO g, iv , bolu s) CM'S ' DhOdp C SO l 1S 0 X 7Smm ) prod uce a fa ll in respiratory rate in rat (50.2 ± 0.51 %, 0·50 005 ~ 0 - 10 * 0 -20,, 0 -50 xO-60 , NoCl l M) r--1 -4 ·76 Pili pf'l Gr od;

E c: VA E a PI c ~ 0 25 0 c ~ a '" 0 50

60 o ~==~====~~~~ -=~==~~ a I-a __ 2·0 Tub e no. 1 x 10 Fig. I - lon-exchan ge chromawgraph y or king cobra ve nom on CM -Scphadex C-50 co lumn. The Iyophilizcd vcnom (90 mg) lVas Fig. 2- I-1PLC purifica tion or Tox in CM36 on protein pak 60 dissolvcd in 3 ml or O.OIM phosphatc buITc r.I'I-17.2 and applied co lumn. Tox in CM36 was appl ied LO a protein pak 60 column \0 a co lumn ( 150x 25 mm ) or CM -Se phad cx cquili bl-a tcd with th c equ ilibrated with ph os phate bullcr. Succcss ive rraction;, wcre burre l'. Fractions ( 10 ml) IVcre co llectcd at 29°C. UA= Unab­ elut ed by step wise gradi ent at a rlow rat c or 0.5ml/min and lVas sorbed peak . PII =Lethalncurotoxic rract ion (Tox in CM 36) mon itored at 2RO nm .

Tab le I - Purirication orTox in CM 36 by CM -Sephad ex ion exchange chroillatogra ph y rrom Indian king cobra (Ol'iIiol'/wgll.l' iIlIIl//(/iI ) ve nom

PIII'iri cation Volume Protein Total protei n Ac ti vity MLD Total Yield Fold ur stcps

King cobra vcnom 3 30 l)() .+ 12 100

Toxin CM-J 6 10 0.5 5 4.5 .+5 7570 101 2

"' MLD=Minimum Ic th al dosc

n=6) and guineap ig (40,5 ± 0.20%, n=6) which gradu­ In guinea pi g, Toxin CM36 (40 I-lgl 100 g, iv, bolu ) all y ca me back to normal within 40.8±0.5 min and also produced a fa ll in res piratory rate (68 .8± 0.40%, 40.5 ± 0.3 min respec ti ve ly. However, at hi gher doses n=6). Thereafter, gasping was observed, fo llowed by (40 ,ug/ I OO g, iv, bo lus) it produ ce fa ll in respiratory resp iratory arrest within 15.8 ± 0.2min (Fi g. 5). rate (83.33 ±0.75%, n=6) and amplitude (60. 5 ±0.62%, Ph ospholipase activity n=6) and produced gasping in rats. The animal di ed Tox in CM36 was devoid of ph ospholipase ac ti vity. within 34.5 ±0.2 min (Fi g. 5) . Action 01/ isolated nerve lI1uscle preparation 80 On iso lated rat phreni c nerve diaphragm prepara­ t- A I 60 ti on, Toxin CM36 (8 ~t g/ ml ) produced 100% blockade '::? w 8 :!; 40 c a:: <{ o D .J ;:) E u 20 F 85 w 90 90 (s SO 90 .J ------G To xin eM 3& . . . 0 I t 15k Do) ::>: 10 » IUIh .ac <::> U., ~"1 • 0 .J T, 20 12 A ~ O 30 15 OL-______~~ ------L-~ 90 75 60 'is . o 0-5 1·0 . ~q 'I ' RELATI VE r-.W8111Ty(Cm) t .. Wzo. 4 , ---;> "~fu l ~~ T, LD Fig. 3- SDS- PAGE molecular \Vc igh t or Tox in CM36. f\ =Albumin, bo vinc Scrum (00.000 Dal ton). B=Ovalbumin (-+5.000 Dalton). C=Glyccraltlehydc 3- ph osphatc dchydrogcnasc &0 so . 5 35 4G ·5 iiO ( 36,OOOD~l lt o l1 ). D=Carboni c anh ydrase (29.000 Dalton). o • , . 0 . . 0 E=Trypsinogcn (24,000 Dalton). F=Trypsin inhibitor (20,000 .. ,~ Dalton). G=Alpha-LaclOalbulllin ( 14,200 Dalton)...... ~.~»f4 ~e1; r-;-o . ....,.*'~r .. ~ 15 12 II 75

E Fig. 5- Eflect of Toxin CM 36 on rcs piration. Ti mc sc alc in min : c uillcri ca l indicatcs rcspiratory rate/min. A=lbt rcspiration. 0 00 N :::i B=Guin ea pi g rcspi ration. T t=Tox in CM36 (20 pg/lOO g, iv. bo­ < .0'" lu s). T:!=Toxin CM 36 (40 pg/lOO g, iv, bolus) . <{ u:

A

'"10 OL-____~ ____~ ______240 280 320 300 350 400 450 Wavelength (nm) Wavelength (nm) 8 A II

Fig. -+ -(A) UV absorpt ion spec trum or Toxin CM36 and ( 13 ) iluorcsccnce abso rption spcc truill of Tox in CM 36. Toxin CM 36=l1.5 ~t g/ 1ll1. ~uffc r (ph os phatc) concc ntration=O.O I M. T hc Fig. 6-Effcct of Toxin CM36 on isol:ltcd skele tal mu sc le pn:: pa­ spect ra prcsc nted was not co rrec tcd for base line as the burfcr r1u o­ rat ion. Timc scalc in min. A=Chick bi ve l1lcr ce rvicis. B=Rat rcscc nce int cnsity was insignificant bctwccn 300-450 nill. phrcni c ncrvc diaphrag m. T 1= Tox in CM36 (i\ ~l g/ ml ) . DE el al.: EU ROTOX IC PROTEIN FROM INDIA KI G CO BRA 1363

centration (2.3 mM) of medium or the presence of voltage dependent K+ channel blocker, 4-aminopyri­ A __ dine ( I ~tM ) failed to alter toxin CM36 act ivity on thi s

A preparat ion. Doubling Ca2+ ion concentrati on (5.2mM) of th e medium had no effect on toxin CM36 induced neu­ romuscular block ing time. However, low C}+ ion concentration ( 1.6 mM) and prese nce of EDT A ( I mM) in the medium significa ntly redu ced the ncu­ romuscular blocking time ( IO.5 ±0.2 and 11. 6 ± OA A ... min) respectively (Fig. 7). T ox in CM36 lost its neuro­ Tl 'iCY B toxic activity ( 100%) when ex posed to enzyme acti on B_ (Trypsin I 111M). However, acidic (p H 3) and alkal ine .. '- (pH 10) medium did not alter toxin CM36 ac tion sig­ Tl 1W ni ficantly.

A DisclIssion ,.,... Venoms are complex mi xtures of proteins and pcp­ Tl 10' C tides possess ing a va ri ety of biolog ica l ac ti vities . Ela­ BP 1& pidae snakes are ex tremel y venomous and have strong neurotoxic acti ons. The hi gh tox icity of elap idae E Tl vcnom is apparently due to an unusual ly hi gh content of neurotox in s. Several to xins with neurotox icity have been iso lated from cobra venom. I aka i el 01.1 5 re­ ported th e presence of a neurotox in, toxin A rrom N. 1 ,.., noj(/ (Indi an co bra) of India. Karlsson el 01. (" rc­ 4' A K C ported th e prese nc e of a neurotox ins, tox i n 3 and toxin 4 from Naja lIC!io lIajo. (Indian spec tacled Fig,7-EITcct or ago ni st. and ions on Toxin CM36 acti on on cobra). T ox ina and bare two neurotox i n reported chick bivcnlcr ccrvicis prcparalion. Tilllc scale in min. A=EITect l 7 or Ca~+ i o n ( 1.6 mM) on Toxin CM 36 aClion. B=EfTcc l or K+ ion from King cobra from Thailand . Till now, no neu ro­ (9.4 mM) on Toxin CM 36 action. C=EITcct or EDTA ( I 111M) on tox in have been report ed from th e Indian King cobra Toxin CM36 aClion. D=EITecl or ace tylcholinc (A) carbachol (C) ve nom. In th e prese nt study. tox in CM 36 a lethal and KCI (K) bcron: and arlcr Toxin CM36 adm in istralion. neurotoxin was isolated and purified from the Indi,ln of elec tricall y induced tw itch res pon se within King cobra (Opliioplioglls 1101I1I ali) ve nom by a com­ I O.2±O.30 min (n=6). T hc block in g effec t remained bination of ion-exc hange chromatograph y and HPLC. unaltered up to 4±O.25 hI' insp ite of repeated was hing A single symmetrical peak in HPLC indica ted the (Fig. 6). On iso lated chick bivcnter ce rvicis prepara­ la ck of any other impurities. The SDS-MW of th e tion, Toxin CM36 (8 ~lg/m l ) bl oc ked thc electrically tox in ( 15 ±0.5kD) was fo un d to be higher than that induced twitch respon se w ithin 20 .0±OA8 min (n=6). normally ex hi bited by neurotox ins iso lated from co­ The bl ock ing effec t rema ined unaltered up to bras or kraits, whi ch show a range or 7000-8000 (reI". S ±O. I Shr inspite of repea ted wash in g (Fi g. 6). 18). UV and riuoresce nce emission spec tra resembled typica l of th at or a proteil\. Toxin C 136 had no sig­ EJlect oj' agonist, (/ntagonist, ions. en:YlIl es & p H on n i ficant effect on the ca rdi ovasc ul ar sys tem si nee. it Toxin CMJ6 aCii\'it v produced no efrect on blood pressure of ca tl rat. iso­ On iso lated chi ck bi vc nter cerv ices preparation, lated guinea pi g hea rt and auricle. However, thi s toxin Toxin CM 36 (8 pg/ml ) abol ished th c ACh, KCI, and like al l oth 'r neurotoxins produced ap noea and re sp i­ carbachol induced contractile respo nse (n=6). By ratory fa il ure in experimental an imals. It has been doub ling th e K+ ion conce ntration (9.4 mM) or th e repo rt ed th at th e main cause or deat h du e to elapi dac mcd ium, th ere was 40.8 ± 0.5% (n=6) increase of neu­ vc nom perhaps du e to th e pe ri pheral respiratory pa­ rom usc ul ar block in g time of chi ck bive nter ce rvices ralysis ca used by th e neurotox ic properties of the prcparation (fig. 7). However lowering K+ ion con- toxin IX. Besides its action on the respiratory system, the 1364 I DIA J EXP BIOL, DECEMBER 2002

most prominent acti on of Toxin CM36 was its effect Acknowledgement on neuromuscular preparation. It produced irreversible The authors are thankful to CSIR, New Delhi for blockade of chick biventer cervices (CSC) and rat partial financial assistance (Ref. No. 37(085 I )941 phrenic nerve diaphragm (RPND) preparations. Toxin EMRlldated 12. 12.94). CM36 was devoid of any PLA activity. The degree of 2 References sens itivity of the toxin to nerve- muscle preparation I Whitaker R, In COlllillon Indian snakes. A field gu ide (The was in th e following order: CSC> RPND. On CSC Macmillan Company of India Lilllited, New Delhi, India) preparati on, Toxi n CM36 abolished ACh, KCI and 197R. 2 Lee C Y, .In Advances in cytopharma : ology. Vol. 3, ed ited ca rbachol induced con tracti ons. This suggested that the by Ceccarelli B & C1 elllenti F (RavIndian cobra (Naja //aja ), ity. It has been shown earlier that post-synaptic neuro­ Biochelll Bio"h),s Res Commun, 44 ( 197 1) 893. toxins that are ex tremely tox ic limit th e immunizing 16 Karlsso n E, Amberg H & Eaker D, Isedation or the principal dose, thus co nferring to the low immunogenicity of th e neurotoxins of two Naja naja subspec ies, EliI' J Biochelll, 21 ( 1971) I. toxin as we ll as the whole venonlo. 17 Joubert F J, Snake vc nom tox ins. The am ino ac id sequences of two tox in s from O"hiol'haglls hall//a/I (King Cobra) Lethality contributing fac tors of Toxin CM36 is venom, Biochelll Biol'hvs Acra, 3 17 ( 1973) 85. probably the neuromuscular blocking activity. Respira­ 18 Yang C C, Chemistry and evolution or to xins in snake tory distress followed by resp iratory arrest induced by ve nom, Toxico// , 12 ( 1974) I. 19 Chang C C, Huang T Y, Kuo K W, Chen S W, Huang K F & Tox in CM36 may also probably due to respiratory mus­ Chiou S H, Sequence characteri zation of a no vel alpha- neu­ cle paralysis induced by neu romuscul ar blockade by the rotoxin from the king cobra (Ol'hiophaglls hallllah) ve nom , toxin. It is possible th at Tox in CM36 is one of the major lJiochelll Biol'h)'s Res COIIIIIIIIII, 191 ( 1993) 2 14. lethal components of king cobra venom. However, de­ 20 Chi navanig L, Billings P B, Matangkasombut P & Rat anaba­ nangkoon K Antigenic relationships .1nd relative immllno­ tailed structural characterization will enlighten the struc­ genicities of venom proteins from si K poi so nous snake of ture- function relation of the neurotoxin. Thailand , To.ricolI, 26 (1988) 883 .