venom purification in sea snakes

7/28/2019 Venom Purification in Sea Snakes.

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Toxfcon, 1977, VoL 13, pp38393PerBrmnPros . Pri nted i o t3reat Bri tai n

I SOLATIONANDCHARACTERZATIONOFALETHALMYOTOXICPHOSPHOLIPASEAFROMTHEVENOMOFTHECOMMONSEASNAKEENHYDRNASCHSTOSACAUSI NGMYOGLOBINURAINMCE

J aNFoi - u . n ~ax and Dev>DEAxExI n s t i t u t e of Bi ochem str y, Uppsal a Un i v e r s i t y , Box 376, 5- 751 23 Uppsal a, Sweden( Accept edj or publ tcatl on ZOJ anuary 1977)

J . Fot ~aswx andDFr+xEa . I sol at i on and characteri zati on of a l e t h al myotoxi c phospho-l i p a s e Af r om t he venomof t he commn sea snake Enhydrtna schi stose causi ng myogl o-bi nuri a i n m ce . Toxi con 15, 385- 393, 1977. -Astr ongl y ~yot oxi c phosphol i pase Awasi s o l a t e d fromt he venomf F, nhydrl na schi stose by ge l f i l t r a t i o n on Sephadex G- 73 and i onexchange chr omatogr aphy on Bi o- Rex 70 Themyot oxi n i s a basi c protei n contai ni ng 120amno aci d resi dues andseven d i s u l f i d e bri dgos . Thef ormul a wei ght i s 13, 500 Them ease ofmyogl obi n di agnosti c of muscl e necrosi s was moni t ored by pl aci ng i l t j e c t e d m ce on s h e e t s ofwhi t e paper . Af f ect ed m ce stai ned the paper r ed. The myot oxi n has an acute~so of 110ugl kgandcauses myogl obi nuri a at doses down t o 30 kg/ kg . Themol ecul e l acks t r ypt ophan,i nd i c at i ng t h at t h i s amno a c i d i s not requi red e i t h er f or t o x i c i t y or c at al y t i c ac t i v i t y .Ai n g l oh i s t i d i n e resi duewasmodi f i ed by reacti onw t hp- bromophenacyl brom de, r e s u l t i n g i n >9l o s s of enzymati c and toxi c a c t i v i t i e s . ThoN- term nal sequence shows homol ogy w th non-toxi c and neurot oxi c phosphol i pases AZof vertebrate o r i g i n . Themyotoxi ci ty my be ofgreater c l i n i c a l i mport ance t han t he c u r a r i m n l e t i c t o x i n s t h a t have recei ved s o mchatt enti onheretof oreINTRODUCTON

Sam sxnxFS abound i n t he warmer Asi an waters , and al t hough bathers are rarel y bi t tent he snakes are an occupat i onal hazard t o f i shermen (BAx~ 1963, 1968 ; R, 1961a) . Thecommn sea snake, Enhydr i na schi stose Daudi n i s t he most abundant , mst poi sonous,andaccounts f ormst sea snake bi tes i nvol vi nghumns ( R>uD, 1961a) . Theear l i est andmstconspi cuous symtomofEschi stosebi te i n mn i s general i zedmuscl e pai n f ol l owed 3- 6 hrl a t e r bymyogl obi nur i a, i ndi cati ve of ext ensi ve muscul ar damge(REm 1961a, b; M~sDErtand I tEI D, 1961) .I n e a r l i e r paper s from hi s l aborat ory, KAR . s soN et al . (1972) reported that about 60of t he protei n content of Enhydr i na schi stose venomconsi st ed of rura_ r i m me t i c post-synapt i c neur otoxi ns havi ng t . nao doses bel ow 10011g/ kg muse the val ue gi ven f or t hewhol e dri ed venombyCARSY and Wxi oi - rr (1960) . FYtYxt urmt al . (1972) reported t heamno aci d sequences of t he two pri nci pl e neur otoxi ns whi chtogether accounted f or about40~f t he venomprotei n . Athought he content of post- synapt i c neurot oxi ns i s adequatet o account f or t he i . n6o dose of t he venomn m ce, curari m meti c a c t i v i t y cannot be re-spons i bl e f or the c l i n i c a l pi cture i n humn vi cti ms .

HeRxts et al . (197 found that notexi n fromt he Aust ral i an t i g e r snake ( Notechi s s .scutatus) i nduced damge t o s k el e t a l muscl e. Fur t hermore, anti t i g e r snake serumgi vesgood pare- speci f i c protecti on agai nst sea snake venomMuvTON, 1967) . These two observat i ons suggested that t he myonecr ot i c factor of Enhydr i na schi stose venomm ght be astructural homl ogof notexi n, and t h i s i s borne out by t he r e s u l t s present ed i n t h i s paper .

385


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386 JANFOHLMANandDAVDFAKERMAgR A CANDMETHODS

CrudevenomDessi cated Eni r ydri na schi stose venomwas obt ai ned f romMs . Oo Sooi Li x, Department of Pharma-col ogy, Uni vers i ty of Mal aya, Kuala Lumpur 22- 11, Malaysi a.Gel f i l t r at i onTwogram of cr ude venomwas di ssol ved i n 10m of 0 2Mmmoni umacetatew thgentl e shaki ngonaWhirhni xer . Thef oamwasbroken w t h t he t i p of a toothpi ck moi st ened i n 1- octanol , i nsol ubl e debri swasremovedby centr i f ugat i on at 1000gf or 5mn f ol l owed byZOmnat 20, 000 x g, andt he cl ear, ochresuper -natant sol uti onwas gel f i l t e r e d onSephadexG75i n 0~2Mmmoni umacetateI on exchange chromatographyAbout 250mgof eeze-dr i ed protei n f romhemotoxic gel f i l t r a t i o n f ract ion VI was di ssol ved i n 7 mof 005Mmmoni umacetate and chromatographedon a col umn of Bio-Rex 70, -400mesh, equi l i bratedw t h 0 2Mmmoni umacetate at pH7 5 as descr i bedbyKwxi ssox et al . ( 1971) . Pr i or toappl yi ng t he sampl eabout 100m of 005Mmmoni umacetatewas pumped i n to t he col umn The col umn was el ut ed w th a2-1 concave gradi ent of 005Ms 1Mmmoni umacetate formedby2cyl i nders havi ngasur f ace area rat ioof 1 : 2, t he l arger bei ng t hemxi ng/ out put cyl i nder cont ai ni ng i n i t i a l l y t heweaker buf f er .Assay f or myogl obi nur l a

Myot oxi ci t y was r eveal ed by t he appearanceof mog obin i n t he ur i ne . I nj ected mcewere pl aced onwhi t e f i l t e r paper i n separat ecages . Control uri ne was col ourl ess or fa int ly yel l ow Sever el y i ntoxi cated mcestai ned t he paper red Theuri ne f romthese ani mal s was el ut ed f romhe paper w t h physi ol ogi cal sal i ne . Noerythrocytes were obser ved and t he presence of mog obi n rather thanhemogobin i n t he extracts wasconf i rmedby runni ngaspectr um si nce t he spectra of thetwo prot ei ns are d i s t i n c t l y di f f erent .Lethal i ty assay~sowasdetermnedby i . v . admni strat i on usi ng4 mce at eachdose l evel . The toxi n concent r ati on wasmeasured spect r ophot ometr i cal l y usi ng t ho mol ar absor pti vi ty val ue determnedi n conj uncti on w thamnoaci d anal ysi s .Phosphol i pase assayPhosphol i pase acti vi ty wasmeasured usi ng a modi f i cat i on of t hemethodof DEHwws et al . ( 1971) Onepar t egg yol kwasmxedw t h 1 par t 18mMaC, and 1 part 8 1mModiumdeoxychol ate . ThepHwasadj ust ed t o 8 " 0wthNaOHTwom of th is sol ut i onwas t i tr atedat roomt emper ature w t h 001MaOHna pH- st et under ni tr ogen af ter addi ti on of aknownamount of enzyme The l i berati on of f r ee fat ty aci dwascomputedas pmol e/mn/mg The i n i t i a l vel oci tywasmeasuredas t he i n i t i a l sl ope, si nce t he rate was l i neardur i ng the f i r s t three mnat t heenzymeconcentr at i ons usedModi f catton reacti onThemotoxinwas modi f i ed w t h p-bromophenacyl bromdeas descr i bed by VoLwsi uc et al . ( 1974) andbyHw reRT et al . (1976) f or porc i ne pancreat i c phosphol i pase AZand not exi n, respecti vel y . The protei n( 0"92umol e) was di ssol ved i n 2m 0 1Macodyl ate buf f er, pH60, cont ai ni ng 0~1MaC . p-Bromo-phenacyl bromde ( 5 umol e) wasaddedand al l owed t o react f or 18 hr at 30CThe reacti on mxture wasgel f i l teredona short col umnof SephadexG25andt he voi d peakwas col l ectedand l yophi l i zed . Aspectrumwasrun i n conj uncti on w thamnoaci d anal ysis to determne t hemol ar absorpt i vi ty, andt her eby, the degreeof reacti on .Amno aci d anal ysi sLyophi l i zed protei n sampl es were hydr ol yzed f or 24and 72 hr w t h 6NHC cont ai ni ng 10mg/mr eagent grade phenol i n t hor oughl y evacuated t ubes at 110C Total hal f -cysti ne and methi oni ne weredetermnedascystei c aci dandmethi oni nesul phone, respecti vel y, af ter perf orm c aci d oxi dati on . Tryptophanwas est i mated f romul tr avi olet spectra run i n conj uncti on w t h amnoaci d anal ysi s, usi ngmol ar absorpti vi tyval ues of 5554 f or t r ypt ophan, 1260 f or tyrosi ne and 150 f or di sul f i de br i dges at 278nmPreparati on of r educedandS- carboxymethyl ated der i vat i vesProtei n ( 2 umol e) was di ssol ved i n2m of 6Muani di ne hydrochl ori de contai ni ng 086Mr i s HC,pH8 6 and003~DTAD th ioerythr i to l ( ZZmg) was added and the reducti onwas al l owed t o proceedunder ni tr ogen at roomt emper ature f or 6hr . Then25 gl of a sol ut i on cont ai ni ng 2 3 mg/m13Hi odoacet at e( s p e c i f i c acti vi ty 90mC/mmol e) wasaddedandal l owed t o react f or 10mn, af ter whi ch compl ete al kylati onwasaccompl i shedby t he addi ti on of 61mg"col d" sodi umodoacetat e . Ther adi oacti ve l abel was i ntr oducedt o f a c i l i t a t e event ual pept i de i sol at i on and i denti f i cati on of cys tei ne resi dues i n t he sequence work nowi nprogress .N- t ermnal sequenceEdman degr adati on of t he reducedand S- carboxymethyl ated motoxinwas done by t he di rect pheny-l i sothi ocyanate method as descri bed by I wwxwawet al . ( 1969) andEnhrwNandHexscaEx ( 1975) . Af ter


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SeaSnakeMyotoxi n 387

spxtrophotometri cdetermnati onof theconcentrati on Snmoleportions of theethy acetate~o ubl oPTHderi vati ves were i dent i f i ed by thi n l ayer chromatographyon s i l i c a gel pl ates (Merck Fertigp attenF J i nso ventVJ EPPSONandSIOQuISr, 1967) andsol vents I I andI I I (BRENNEReta1. , 1962) . Thespotswere l ocatedvi s ua l l y under ul t r avi ol et (254nm i l l umnati onTheaqueous phases ware examned for PTH-arginineandPTHhi sti dine bypaper el ectrophoresis at

440Vor 2hr atpH65 i n sodi umphosphate buf f er contain ng0015Ma,HPO, 003MaHPO1 gof di sodi um13DTAandSgof so ubl estarchper l i t e r . PTHderi vati veswerevi sual i zedaswhi tespots onawfeo-co ouredbackground bymeansof thoiodineazidoreagent (EuMANandH ;NSCI I ert, 197S) .

RESULTSI so ationofmyotoxin YI : STheel ut i on pattern of Fal l tydri na schi stose venomprotei nonSephadexG-75 i s showni n

Fig 1 ( i nsert) .Aetarded peakof nucl eosi des i s not shown Nearl y a l l of the protei n con-st i tuents el ut e i n twos i z e f ract i ons : f ract i on VI I r epresent i ng about 70~f the total protei nandamo ecular weight of 7000, and f r act i on VI contai ni ng20~f the total protei nandcorresponding t o amo ecular weight of about 14, 000 . Fracti on VI I consi st s miny ofcurari mmeti c neurotoxins (KARssox et al . , 1972) .Fracti onVI showed phospho ipase acti vi ty, was l et hal t o mce, andproducedmyo-gl obi nuri a . I t was fur ther f r act i ona ted by i onexchange chromatography on the pol y-carboxyl i c res i nBo-Rex70 Sevenpeakswereobserved, as showni n Fig 1 , andaccountedf or 6, 2, 1 , 3, 7, 1 and 1~espect i vel y, of the total venomrote i n . TheunretardedpeakVI : 1 producedno symptom i nmce at adose l evel of 500Ftg/ kg VI : 5was hi ghl y l ethalandproducedmyog obinuria, andwas t heref ore st udi ed i n more det ai l . The other peakswerenot assayedCharact eri zati on ofmyotoxin VI : STheamnoa c id composi tion( Tabl e 1 ) i s very c l ose to i ntegral f or a l l amnoac i ds except

AsnandLeu, perhaps i ndi cat i ngadi screte Asn-Leesubst i t uti on. Thepost synapt i c t oxi ns 4ao

zoH0

FO 1 . IONEXCHANGECHOMATOORAPHY ( 1 b) OF3SOmgOFFRAC TONVI (~~PHOSPFI OLiPASEFRAC TO~~FROMTAE IN TIALOELFI I . TRATIONSTEP SHOWNNTOPINSERTThe32x30~mi onexchangecol umnwaspackedwthBo-Rex70 as describedi nMethodsThemai nmyotoxic pock emerges at an approximateammoni umacetate wncentrati on of0SMx s expressedi nm~' 1xcm1 Fracti onvol ume=m. I nsert near topof f i gure showstheprotein di s t r i but i on obtained i n aseparation of 2gcredovenomona32x 92S~mcol umnof SephadexG-75. Fracti onVI correspondstoaMWf 14, 000andVI I about 7000


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38 8 J ANFOHLMANandDAVI DERKERTAHLE1 . AMnvOACDCOMFO~IONOF F J J % y L l r l n a S C I l l S t O J a MYOT' OXIN

' Aver age of v a l u es f r S~rboxyme t hykyst ei neand c y s t e i c aci d.t 72 hr val ue onl y.and 5 fromt he samevenomdi er ed by a d i s c r e t e Pro- Ser subst i tut i on (FxYxLUxn et al . ,1972) . The spectrumhas a maximumat 276nm i ndi cat i ng a hi gh content of t yros i ne ( 12fromamno ac i d a n a l y s i s ) and t h i s , t ogether w t h t he mol ar absorpt i vi t y val ue 16, 900, i snot compat i bl e w t h t he presence of any t r ypt ophan . Themyotoxi n contai ns no amnosugars .N-termnal anal ysi sEl even turns of Edman- degr adat i on on t he reduced and S- carboxymethyl ated der i va-t i v e est abl i shed t he N- t erm nal sequence Asn-Leu- Val - Gl n- Phe- Ser - Tyr- Val - I I e- Gl n- Gj ~s,whi ch shows cl ose homol ogy w t h many ot her phosphol i pases A as i l l u s t r a t e d i n Fi g 2On t he basi s of t he obvi ous homol ogywe i nf er t hat t he 14 cys tei ne r esi dues are connect edby 7 d i s u l f i d e br i dges .Toxi c i t yAt doses above 100 F t g / k g t he mce seemt o di e fromrespi ratory f a i l ur e , t he symptoma-

Motaxi n Aan-Leu-Val -Gn-Phe-Ser -Tyr -Val - I l e-Gn-Cys-Notexi n Asn- Leu-Val -Gn-F; i e- Ser- Tyr -Leu- I l e-Gn-Cys-N. n g Pl ase Asn- Leu- Tyr-Gn-Phe- Lys-Asn-Met- I l e-Hs-CysTaipoxi n a Asn- Leu- Leu-Gn-Phe-Gy-Phe-Met- I l e-Arg-Cys-Taipoxi n ~ Asn- Leu-Val -Gn-Phe-Gy-Phe-Met- I l e-Gn-Cys-Pork p . Pl ase A a- Leu- Trp-Gn-Phe-Arg- Ser -Met- I l e- Lys-Cys-

Fra2NTERMNAL HOMOLOGY AMONGSOMETOXICANDNONTOXICFHOSPHOLIPASFS .Notexi n i s f r omAust ral i an t i g e r snake ( Not echi s s s c u t a t u s , HALPERTandEACER 1 975 ) , t heba s i c phosphol i pase f r om Af ri can s p i t t i n g cobra (NaJ a n l g r i c o l l l s , OHI)AROet al . , t o bep ub l i s h ed ) , t ai poxi n f r om the Austral i an t ai pan ( Oxyurant y s acut el l atw FOHLMANet a l . ,197 andpork pancreat i c phosphol i pase (DEHAASet a l . , 1970) .

Resi duoVI : SMol e r a t i o I nteger

Asx 206 21Thr 3 " 91 4Ser 5 " 24 SGk 5 " 90 6Pr o 297 3G y 9 09 9Al a 9 97 10Cys' 1 4 " 1 14Val t 592 6Mt 1 "96 2I I e t 314 3Leu 443 4Tyr 1 1 "7 1 2Phe 1 ~91 2Hi s 200 2Lys 10 "0 1 0Trp 0 0Arg 695 7No . of r e s i d ue s 120Formulawei ght 13, 500Mol ar absorpti on( 276 nm 16, 900As7e ( 1 mg/ m1) l


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SeaSnake Myotoxi n 38 9tol ogy c l o s e l y mmcki ng envenomat i on wi th presynapti c t ox i ns ( l i k e t a i p o x i n or n ot e xi n ) .I f t he mce do not di e wi thi n 1 2 hr they ei t h e r r ecover or go i n t o a s t a t e of catabol i smappar ent l y usi ng up al l energy s t o r e s . They l o s e wei ght, move about s h a k i l y and di e on the4th or 5th day i n an emaci ated s t a t e . Probabl ymuchof the s k el et al muscl e mas s i s s e ve r e l ydegr aded by t h i s s t a g e .The t o x i c i t y t h u s i nvol ved t wo sympt oms whi ch a r e appar ent l y due t o qui t e d i f f e r e n tphar macol ogi cal and pathogeni c mechani sms . W have accor di ngl y deter mned t wo1. D o ' S, one at 110 L I g J k g f or t he f a s t - e ns ui ng par a l y t i c death and one at 40 F t g J k g f or t h ewast i ng death . The c l i n i c a l i mpor t ance of t h i s f i n d i n g i s evi dent .Myotoxi ci t yThemyotoxi ci ty has so f ar been f ol l owedonl y by the si mpl e assay f or muscl e- dest r ucti onmani f ested by t h e appearance i n the serumof myogl obi n, whi ch i s r api dl y c l e ar e d t hr oughthe ki dneys and i s e a s i l y d e t e c t e d by the col our of the ur i ne Myogl obi nur i a appears atdose l e v e l s above about 30 l l g J k g , whi ch means t h a t thet oxi n i s extr emel y t o x i c f o r s k el et almuscl e . The 1 . n 6 o f or i nt r ape r i t o ne al i n j e c t i o n i s about ten t i me s t h a t obtai ned f o r i n t r a -venous i n j e c t i o n .Phosphol i pase a c t i v i t y

i : n t h e pr esence of sodi umdeoxychol ate a c a t a l y t i c a c t i v i t y of 200 l Tmol eJ m nJ mg wasdeter mned at roomtemper atur e usi ng egg yol k as s u b s t r a t e . The s u b s t r a t e s p e c i f i c i t y wasnot i nv es t i g at e d .Modi f i cati on r eacti onThemyotoxi c phosphol i pase r e a c t s wi th p- br omophenacyl br om de as evi denced by t h e2 ~5 - f ol d i n cr e a s e i n the mol ar a bs or p t i v i t y at 270 nm( Tabl e 2) . T hi s , t o g e t h e r wi th di s -appear ance of one h i s t i d i n e r es i d ue ( a s deter mned by am no a c i d a nal y s i s ) s ugg e s t s t h att h e modi f i cat i on i s the same as was observed wi th the pancr eat i c phosphol i pase (VOLWERKet a1 . , 1 97 4) andnotexi n (I I ALPERT e t al . , 197 . ThepAs,nval ue i s somewhat hi gher thant h a t r epor ted f or the protei n- conj ugated r eagent i n the l a t t e r r e f e r e n c e .Af ter modi f i cat i on the myotoxi n pr oduced no s i g n of myogl obi nur i a or any otherdi s t r e ss i n m ce at doses cor r espondi ng t o 50 x LDSo of then a t i v e protei n. Thephosphol i pasea c t i v i t y was 1 l Tmol e/ mnJ mg, or 0~5~h a t of t he v i r g i n protei n. By t h e s e measur es boththe l e t h a l i t y andthe enzymati c a c t i v i t y were decreased by 99~The p a r a l l e l i s m bet weent h e myotoxi c, neurotoxi c, and c a t a l y t i c a c t i v i t i e s i s d i s c us s e d bel owTAHEi . SOe~CHARAC EI tL4ITC8OFNATI VEANDP-HROMOFHENACYLHRO ~E-REACTEDbIYOTOX N

Nat i ve Modi f i edNo. o f h i s t i d i n e r esi dues (amno aci d a na l y s i s ) 200 1 " 09

`e , o ~ ( PBP- cor {j ugate) 17, 000 (HALPERTOt s i . , 1976) .

Mol ar e x t i n c t i o n c o e f f i c i e n t ( 270nm 13, 600 34, 600p- Br omophenacyl br om de r esi dues/ mol e protei n 0 1 " 2Phosphol i pase a c t i v i t y ( umol e f a t t y aci d/ mgxmn, seeMethods) 200 1~so ( ug/ kg) : Acute 110>000Longterm 40Smal l est myogl obi nur i c dose 30 >000


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390 JANFOHLMANandDAVIDERKERDSCUSSION

Phosphol i pases are al most i nvar i abl y pr esent i n snake venom ( I vhs , 1970) . Thei rf uncti on must be consi der ed t o be or i gi nal l y di ge st i v e, si nce these enzymes are acommonfeature of the al i ment ar y canal . I n r ecent years, however , i t has become evi dent that phos-phol i pases A have evol ved qui te speci al i zed toxi c pr oper t i es i n snake venom, as exemp l i f i e d by theneur ot oxi ci t y of t he structural homol ogs not exi n (HALPERTandEnxm 1975) ,tal poxi n (FoxLntAN et al . , 197, crotoxi n (Bx~s~urr et al . , 1975) , and pr obabl y ( 3 -bungar ot oxi n . H,~uus et al . (1975) showed that not exi n al so has myot oxi c pr oper t i es .Apparent l y t he Enhydr i na schi stosa myot oxi n VI : 5 al so has three bi ol ogi cal a c t i v i t i e s ,and t he r el ati onshi ps amongthemr emai n t o be el uci dated .Membrane s p e c i f i c i t yPhosphol i pases A hydr ol yze or gani zed l i p i d much more r api dl y than they attackmonomeri c substrate ( e. g . SLOTBOOMandDEHaas, 1975) . The l eakage of myogl obi n f romthe myof i br i l s l o g i c a l l y i mpl i es some ki nd of membrane damage Themembrane f uncti onhas been shown t o be i mpai r ed a f t e r poi soni ng w t h the pr esynapt i c neur ot oxi ns tal poxi nand not exi n (CULL-CANDY et al . , 1976) . I nter acti on w t h or gani zed l i p i d ( e , g . membranesor substrate mcel l es) i s thus a common f eat ur e of t he two qui te d i f f e r e n t tox i c a c t i v i t i e sand t he c a t a l y t i c a c t i v i t y as wel l . I n i t i a l l y we hoped t o f i n d an excl usi vel y myot oxi cphosphol i pase, but have i nstead f ound a mol ecul e w t h a d i f f e r e n t myot oxi c/ neur ot oxi cr a t io t han not exi n .

The N- t er mnal r egi on appear s t o pl ay an e s se nt i a l r ol e i n t he f or mat i on of t he i nter f acer ecognt i on s i t e (VAND~vt - MtExns et al . , 1975) , but as shown i n Fi g . 2there i s no s t r i k i n gdiss imlar i ty among s i x d i f f e r e n t phosphol i pases i n t h i s r egi on . Thus t he c e l l s p e c i f i c i t ymust r esi de somewhere e l s e i n t he mol ecul e .

The appar ent pr ef er ence f or d i f f e r e n t cel l - membr anes must have some structural basi s .Wecan thi nk of three p o s s i b i l i t i e s :( 1) Di f f er ent packi ng of phosphol i pi ds i n t he d i f f e r e n t c e l l membranes . I f c a t a l y s i s i si nvol ved i n t he bi ol ogi cal e f f e c t s t he surface t ensi on coul d be cr uci al (ZwaaL et al . , 1975 ;

DEALet al . , 197 .( 2 ) Di f f er ent c e l l membranesmayhave di f f e r ent 3-sn-phosphogl ycer i des, i . e . di f f er encesi n t he l ong~hai n fat ty aci d e s t e r s or t he al cohol par t . Thi s coul d account f or t he c e l l -di r ected s p e c i f i c i t y and shoul d beexper i mental l y tested usi ng d i f f e r e n t syntheti c der i vati ves .

( 3 ) Astructural or f unct i onal pr otei n i n t he membranemght be t he mai n tar get of t heattack . I t mght be a per mease or anenzyme t he pr oper f uncti on of whi ch i s dependent ont he i n t e g r i t y of the sur r oundi ng l i p i d bi l ayer .

The l o s s of both t he c a t a l y t i c and t he pathol ogi cal a c t i v i t i e s upon chemcal modi f i cat i onw t h p- br omophenacyl bromde cannot al one s u f f i c e as pr oof that t he phosphol i pasea c t i v i t y i s d i r e c t l y i nvol ved i n t he toxi c acti on(s) . Recent crystal l ographi c work w t h t hepancr eat i c prephosphol i pase shows h i s t i d i n e and tyr osi ne i n t he c a t a l y t i c c l e f t . I n t he nei gh-bourhoodi s anel ectr on densi ty whi ch coul d be aCa i on, perhaps coor di nat ed byan aspar ti caci d andan asparagi ne r esi due ( DxeN~r x et al . , 1976) . The i ntr oducti on of t he r ather bul kyp- br omophenacyl r esi due sever el y hampers t he a b i l i t y t o bi nd cal c i um and s t e r i c a l l ydi sturbs t he c a t a l y t i c s i t e . Appar ent l y t he i n t e g r i t y of t he c a t a l y t i c c l e f t i s i mpor t ant f orboth t he neur o- and myot oxi c a c t i v i t i e s , whet her c a t a l y s i s i s i nvol ved or not . Si nce themyot oxi n w t h i t s comparati vel y hi gh s p e c i f i c phosphol i pase a c t i v i t y i s devoi d of t r ypt ophant h i s amno aci d can haveno i mpor t ant r o le i n t he hydr ol ysi s of phosphogl ycer i des .

I t i s har d t o def i ne si mpl e end- poi nt s f or t he LDo det er mnat i ons si nce t he acute and


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Sea SnakeMyotoxi n 391chr oni c e f f e c t s over l ap . When t h e r e ar e l ong- t erme f f e c t s on l ocomot or organs one mus ta l s o take i n t o account s t a r v a t i o n or dehydr at i on. That i n f ac t t wo d i f f e r e n t a c t i v i t i e s a r epresent mus t be f ur t her i n v e s t i g a t e d by e l e c t r o phy s i o l og i c a l and h i s t o l o g i c a l t echni ques .Tai poxi n i s a poorer myot oxi n than not exi n but i s much mor e potent presynapt i cal l y( Fo fu. Max et a l . , 1976) . The Enhydr na s c h i s t o s a myot oxi n i s t he poorest pr e-synapt i cneur otoxi n of t he t h r e e , but i s t he onl y one of them t h a t pr oduces muscl e degenerat i onupon i ntr avenous admni strat i on ( Hnnx>s, personal communi cat i on) . However , h e r e wehave t he compl i cat i on t h a t t he dose of not exi n r equi r ed t o pr oduce muscl e degenerat i onupon i ntr avenous adm ni strat i on m ght be s e v e r a l t i mes the l e t ha l p a r a l y t i c dose . Theani mal t hus e xpi r e s bef ore t he sympt oms of myot oxi ci t y appear .Pathogeni c s o l e of t he myot oxi c phosphol l pasesWhet her sea snake poi soni ng i s pri mari l y myo- or neurotoxi c has been t he s u bj e c t ofsome d i s c us s i o n (R~ 1961b ; MnRSnsxand R>~ 1961) . Bi ochem cal separati on met hodshave demonst r ated t he pr esence of both myot oxi ns and neur otoxi ns . The pat hogeni cbal ance between t he t wo r emai ns t o be el uci dated andprobabl y depends on t he s p ec i e s oft he v i c t im For exampl e, manm ght be consi derabl y mor e s e n s i t i v e t o t he myot oxi n ( o ra l t e r n a t i v e l y , l e s s s e n s i t i v e t o t he curari mmet i c t o x i n s ) than are m ce . The f ol l ow ng f a c t sshoul d be consi der ed :( 1) M ce i n j e c t e d w t h the curar i mmeti c post - synapt i c neur otoxi ns di e wth i n 2- 4 hror recover compl etel y wth i n 12 hr .( 2 ) No i mpr ovement i n vi cti ms of sea snake b i t e i s obser ved a f t e r neost i gm ne ad-m n i s t r a t i o n ( l t ~ 1961a) . However , t h i s t r eatment m ght not be a s di agnost i c as i t a t f i r s tseemed, s i n c e i t i s d i f f i c u l t t o prove t h a t neost i gm ne ( or any a c et y l c ho l i n e - e s t e r a s e i n-h i b i t o r ) shoul d have s i g n i f i c a n t c l i n i c a l val ue i n humans , al t hough cl ai ms have been made(Bnt~~t a l . , 1974) .

( 3 ) Mort al i ty among s e a snake v i c t i ms i s a s hi gh as 17~of t he c as e s s ee n a t ho s pi t a l ,and 27~of t he deat hs occur a f t e r 2days . Theaverage deat h t i me i s 30 hr ( REm 1961a,25 c a s e s ) .( 4) The l e t h a l dose f o r one a dul t human i s est i mat ed a t 1 ~5 mg, or about 10~of t het o t a l venom i e l d ( Muv~ ox and Mnv~ ox, 1969) .(~ The l e t ha l dose f o r t he venomof t he Asi an cobra, Naj a r a j a , i s est i mat ed a t 15- 20mg, agai n about 10~f t he t o t a l gl and cont ent . The t o t a l amount of cura r i mme t i c neur o-t o x i n s i n j e c t e d i s per haps 5 t i mes t ha t i n j e c t e d i n a sea snake b i t e but >5~f t he v i c t i msrecover ( Mnaz~ox and Mt r r r oN, 1969) . Humans usual l y d i e wth i n 8 hr ( I t t ? r o , 1964) .These obser vat i ons suggest t h a t somet hi ng other than ( or i n a d d i t i o n t o ) curar i f ormneur otoxi ns i s r esponsi bl e f o r t he ul t i mate f a t a l out come or mm n g e f f e c t s of sea snakeb i t e . We suggest t h a t t h i s maybe the ba s i c myot oxi c phosphol i pase(s ) .Themode of acti on of t he myot oxi ns i s not known Nei t her not exi n nor t ai poxi ne xhi bi t myot oxi ci t y i n v i t r o , al t hough i n v i v o both ar e hi ghl y potent (Hnxxrs, per sonalcommuni cat i on) . I n d i r e c t evi dence s u g g e s t s t h a t musc l e damage i s f ol l owed by t he i nvasi onof phagocyt i c c e l l s , t he i nvadi ng c e l l s bei ng r i c h i n pept i de-hydr ol ase enz ymes . The " t r i g g e r "st i mul ati ng t he i nvasi on of t h e s e c e l l s maybe some f a c t o r r e l e a s ed by t he damaged muscl ef i b r e membr ane, or compounds r e l e a s e d by other c e l l s ( e . g . mast c e l l s ) t h a t ar e a l s o damagedby t he t oxi n (P i . usxnL e t a l . , i n p r e s s ) . Prel i m nary evi dence ( H~xxi s andJ oxrr sox, personalcommuni cat i on) s u g g e s t s t h a t muscl e damage f ol l ow ng seasnake myot oxi ns s h a r e s a l l t hef e a t u r e s so f a r descri bed f o r not exi n ( Hnxxi s et al . , 1975) .The modi f i ed myot oxi n shoul d be an e xc e l l e nt t oxoi d f o r t he product i on of a nt i s e r a,s i n c e very hi gh doses can be i n j e c t e d t o obt ai n agood response w t hout k i l l i n g t he ani mal .


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392 JANFOHLMANndDAVDBAKERThi s has al r eadybeen done wi t h t he p- br omophdenacyl bromde modi f i ed t a i pox i n (RAMLAUet al . , t o be publ i shed) . Thereason f or t he good par a s pec i f i c pr ot ect i on by t i g e r snake ant i -venomnowseems appar ent , s i n c e not exi n and t he myot oxi n ar e s i m l a r mol ecul es . Thi sa l s o i nd i c at e s t he pat hogeni c i mpor t ance of t he myot oxi n .Acknowedgements-Wearegreatl y i ndebtedtoMsOoSoozLrnr , Department of Pharmacol ogy, Uni versi tyof Mal aya, for supp yi ng the crudeErhydri naschi stosevenomWe thankMss MARAxxENoRnl nYC~ f ore nt h us i a s t i c hel pi n thei sol ati onof themyotoxi n, andval ue thehel pful c r i t i c i s ms of Dr. EvERZKAR ssor t .Thei nvesti gat i onwassupportedby theSwedi shNatural Sci enceResearch Counci l . dnr 2859-008

REFERENCESBANEREE,RN, SAExI,ALandCaAC co,KA(1974) Neost i gmne i n thetreatment of El ap dae b i t e .4th I nt. Synrp on Amral , Pl ant andMcrobi al Toxins, Tokyo, 8-13September, 1974.BARME,M(1963) Venomous sea snakes of Vi etNamnd thei r venons. I n: Venomous andPoi sonousAni rnaLsandNoxi ous Pl ants of thePaci f re Region, p 373, (KEEGAx,HLandMACFARLA aE,WV ,Eds l .Oxford : Pergamon Press .BARME,M(1968) Venomous sea snakes (Hydrophdae) . I n : Venomous Animal s oral Their Venons, Vol . I ,p 285, (BUCHERL,W, BucxI . EV,EandDEULOFEUV, Eds ) . NewYork : Academc Press .BREI THAUPT,H, OMORzSATOFI , T andLANG J . (1975) I sol ati onandcharacteri zati on of three phosphol i -pases fromthecrotoxi ncomp ex. B ochi mbiophys. Acta403, 355BRENNER,M, NEDERWEI3ERAandPATAK ,G(1962) I n : Daurschichtchromatographie, p 443, (SzAm,E,Ed ) . Wst Berl i n : Spri nger .CARET, J . CandWRGHT,EA(1960) The t oxi ci t y and i mmunol ogi cal properti es of some sea-snakevenons wthpart i cul ar referenceto that ofFnhydri naschi stose Trans .RSce tropMdHyg54, 50CLLrCANDY, SC, FOErc.MAN J . , GUSTAV930N,D, LuLL. MANN-RAUCH RandTHESLEFF, S (1976) Thee f f e c t s of tai poxi n andnotexi n onthe functi on andf i n e structure of themari neneuromuscul ar j uncti on .Neurosci ence 1, 175

DAMMmRASMCEvAN, SLOZaooMAJ . , PI EZERSONWAandHAAS,GHDE(1975) Thei nteracti onof phosphol i paseA, wthmcel l ar i nt e r f a ce s . Therol eof theN-termnal regi on . B ochemst ry 14, 5387DEMEL,RA, GEURTSvANKESSEI . ,WSM, ZWAAL,RFA, ROEIAFSEN BandDEENENLLMvnN(1975) Rel ati on between vari ous phosphol i paseacti ons onhumanredcel l membranesandthe i nt e r f a ci alphosphol i p dpressure i nmonol ayers. B ochi mbl ophys. Acta406, 97 .DRENTFi , J . , ENZ NC,C, KAtx,KandVESSI ES, J . (1976) Structure of porci nepancreati cprephosphol i paseA, . Nature 264, 373

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SeaSnakeMyot oxi n 393M$es,D( 1970)Aompar at i ve study of enzyme a c t i v i t i e s i n snake venom . I nt . 1 Bi ochem l , 335.Mcxrox, SA( 1967) P ar a s poc i f i c protect i onbyel api d andsea snake a nt i v e ni ns . Toxi conS, 47.Mxzox, SAandMxzox,MR( 1969) Venomous Re pt i l e s . NewYork ; Charl es Scri bner .P t . u s g. ~ . ,MG, H~xni s, J . B, PExxi xa~rox, RJ . and EucEx,D( 1976) Somebi ochemcal r esponses ofr a t s ke l e t a l muscl e t o as i n g l e subcut aneous i n j e c t i o n of a toxi n ( Not exi n) i s o l a t e d f romt hevenomf t heAustr al i an Ti ger snake ( Notechi s s s e u t a t u s ) . CJ tn Exp Pharnracol . Physi ol . To be publ i shed .REro,HA( 1961a) Di agnosi s, prognosi s andt r eatment of seasnake b i t e . Lancet , 399REI D,HA( 19616) Myogl obi nuri aand seasnake b i t e poi soni ng . Br . Medl . l , 1284 .R$mHA( 1964) Cobra b i t t y . Br . Med1 2 , 540Sr ateooMAJ . andHaves, GHna ( 1975) Spe c i f i c t r ansf ormati ons a t t he N- termnal r egi on of phos-phol i paseA, . Bi ochems tr y 14, 5394 .

VOWERIC J . J . , Pt$rexsox,WAandHoes, GHDE ( 1974) H st idi ne a t the a c t i v e s i t e of phosphol i pase A, .Bi ochem st r y 13, 1446 .Zweet . , RF A, Rortorssx, B, Coxroxi as, P andDraxax, L LMvex( 1975) Or gani zati on of phas-phol i pi ds i n human r ed c e l l membranes as detected by t he a c t i o n of var i ous p u r i f i e d phosphol i pases .Bt ochi m bi ophys . Acta 406, 83

venom purification in sea snakes

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