STONEFISH VENOM: SOME BIOCHEMICAL ANDCHEMICAL OBSERVATIONS"

hy L. AUSTIN, R. G. GILLIS AND G. Y O U A T T

{From the Defence Standards Laboratories, Maribyrnong, and theDepurtment of Biochemistry, Monash University, Victoria).

{Accepted for publication 23rd August, 1964.)

Summarr. Stoncfisb venom bas been fractionated on carboxymetbyl cellulose andScphadex columns; of the fractions ol)tained only one was toxic to rats. Theactive fraction contained a relatively high proportion of aromatic amino acids.Whole venom was examined for enzyme activity and enzyme inhibition; the onlysignificant aetivity found was tbat of hyaluronidase. The venom also eaused amarked increase in eapillary permeability. Ultracentrifuge measurements suggestthat tbe toxie material has a molecular weigbt of tbe order of 150,000.

INTRODUGTION.

The venom apparatus of stonefish (Synuneeja sp.) has been described byDuhig and Jones (1928) and Endean (1961). A number of cases of poisoningboth fatal and non-fatal have been described (Smith, 1951, 1957; Wiener, 1958;Phelps, 1960).

The Hrst effect of a stonefish sting is intense pain which may persist forseveral hours or even days. This is followed by shock, liyxiotension and occa-sionally death. The pharmacology of the venom has been examined (Wiener,1959; Saunders, 1959; Austin, Gairucross and McGallum, 1961), and these inves-tigations have shown that the venom has a direct paralysing effect on skeletal,cardiac and smooth muscle. This myotoxic action explains all the effects ofthe stonefish venom with the exception of the excruciating pain.

Saunders and Tokes (1961) have studied the heat stability and solubilityof the toxic fraction of S. horrlda which appears to be a globulin. In addition,they have separated this fraction from a number of other protein componentsof the venom by electrophoresis on starch gel.

This report describes the effects of the venom on a number of enzvmosystems and some attempts to fractionate the ven(jm by means of column chroma-tography; some chemical information is also reported.

' Some of these results were communicated to the 35th Congress of A.N.Z.A.A.S.,Brisbane, 1961.

Aust. J. exp. Biol. med. Sci. (1965), 43, pp. 79-90.

80 L. AUSTIN, R. G. GILLIS AND G. Y O U A T T

MATERIALS AND METHODS.

Extraction of the venom. Specimens of both S. trachynis and S. verrucosa were obtainedfrom Queensland and air freighted alive to Melbourne wrapped in damp muslin. In themajority of cases they arrived in jf"od condition. The fish were killed and venom wasextrat'tcd and lyophilised as described previons]>' (Anstin ct al., liJ61). Occasionally deadfish were obtained packed in dry ice. These were kept Irozen until required, tlic venomsacs were disseeted out and the venom was extracted and lyophilised. All coneen(rationsof venom refer to the lyophilised powder.

Biochemical activities of the venom. Acetylcholinesterase and psendo-cholinesterase activi-ties of venom solntioiis (1 mg./ml.) were measured manometrically in a bicarbonate medinm(Krehs and Henseleit, 1932). Aeet>'lcholine was used as the substrate; 6 niM for acetylcho-linestt'rase and 60 mM for pseudo-eholincsterase.

Protease activity of the venom was detected viscometrically during incubation wit!i asolution of casein (20 mg./ml.) at 37°.

To determine pliospholipase A-activity, 0-2 ml. Iccitliin .solution (1 per eent), 0-4 inl.human red cell suspension (1:40) and 0-2 ml. venom {2 mg./ml.) were incubated in 0-005 Mphosphate buffer pH 7-4 at 35° for 90 mimites. The solutions were eentrifnged at 4,000r.c.f. and examined for haemolysis (Bcrnheimer, 1947).

Stonefish venom was examined for calcium and magnesium-activated adenosine triphos-phatase activity (Perry, 1955), and also for inliibition of these enzymes (Kielly, 1955).

Hyaluronidase activity was determined by the albiunin precipitation method (Dorfmann,1955) using a commercial preparation of hyaluronidase (Berger) as a standard.

Capitltuy permeability after stont'fish venom poisoning was e.xamined following themethod of Diniz and Goncalves (1960).

The effect of stonefish venom on blood coagulation was examined as follows: 0-1 ml.oxalated human plasma was incubated with 0-1 ml. of venom (1 mg./ml.) for five minutesat 37°. At the end of this time O-l ml. calcium chloride (1 per cent) was added to thecontrol tube and the incubation continued. The tubes were examined at one minuteintervals for coagulation.

Oxidative phosphor>'latioii in rat heart sarcusomes was measured using «-ketoglutarateas substrate (Cleland and Slater, 1953).

After incubation of the venom with bo\ine plasma globulin, the presence of bradykininactivity or bradykinin releasing enzymes was detennined essentially as described by Rochae Silva, Beraldo and Rosenfeld (1949). Isolated guinea pig and rat ilea were used as testsystems for the determination of kinin activity.

Glycerinated rabbit psoas muscle fibres were prepared by the method of Szent-Gyiirgj'i(1951). Fibre bundles abont 40 mm. long and 100 n wide were suspended in an organbath containing 0-02 M sotlium phosphate pH 7-0, 0-001 M magnesium sulphate and 0-1 Mpotassium chloride, and were attached to a Phillips movement transducer. The contractionof the bundle after the addition of 0-002 M ATP was recorded on a Varian reeorder,model GIO.

Toxicity studies on the various separated fractions were made using anaesthetised ratsas described previously (Austin et al., 1961).

Column chromatoarapliy. Carboxymethy! cellulose (Serva, capacity 0-72 meq./g.) wastreated by the method described by O'Donnell and Thompson (1960) for DEAE cellulose.Golumns 0-9 cm. diameter and 12 cm. long were prepared and washed with 200 ml. of a

STONEFISH VENOM 81

solution containing (1-5 M polassiiiin ehloridn, 0-05 M sodiimi phosphate, and 0-01 per centTwcfii 20. The cohnnii was finally wiishi d with tin; buffer without potassium cliloridcuntil the pH was constant at 6-US and the absorbance was negligible at 230, 260 and 280 mii.

One ml. of solution containing 3 mg. of lyophili.sed venom in 0-1 M KCI was pipettedon to the column. The proteins were eluted with sodium phosphate buffer containinj; O-lM KCI, applied to the column under a pressure of 5-10 Ib./sq. in. of nitrogen, antl fractions(if 0-8 ml. were coilccted using a drop counter as volume hicHcator. Stepwise elution gave nobetter .separation. All the alKtvc operations were carried out in a cold room at 4' ' .

Columns of Sephadex G-75, 1 em. diameter and 27 cm. long were prepared similarly in0-9 per cent sodium chloride and a^ain washed until the absorbance was negligible. One ml.of solution containing 5-7 nig. of venom was applied to the column.

Immunoelectmphoresis. Inimunoelectrophoresis was carried out essentially in accordancewith the methods of Wundcrly (1960). The gel consisted of 1-5 per cent agar in 0-05 Ml)orate buffer pH 8-2 and the electrophoresis was carried out at 2 V/cm. for 3-4 hours at 0°.Stonefish anti\enine as supplied by the Common weal tb Scrum Laboratories was dilutedeight times before use as the antibotly component nnd diffusion was allowetl to proceed for48 hcmrs. Precipitin bands were stained with amido black.

Vltracentrifugf stutiies. Tbese were carried out on tbe Spinco analytical ultra-centrifugeat 59,780 r.p.m. and 25'^. The venom concentration was appro.xiinately 10 nig./ml. in 0-2 Msodium chloride.

Chemical determinations. Nitrogen was detennined by the miero-Kjeldalil procedure.Proteins were estimated by means of ultraviolet absorption measurements and Lowry detei-minations (Layne, 1955).

RESULTS.

Enzymic activity of stonefish venom.The only enzyme activity found was tliat of hyaluronidase. Tests for the

following enzymes showed no activity: protease, phospholipase A, acetylcholines-tcrasc, pscudo-cholincsterase, Ca and Mg activated adenosine tripho.sphiitase,blood clotting enzymes, haemolytic enzymes.

Enzyme-inhibiting activity of venom.

Human plasma psoudo-cholinestcrase was inhibited 20 per cent hy a con-centration of 1 mg./ml. of the lyophilisecl venom. At this concentration acetyl-cholinestcrase was unaffected. Neither Ca oi Mg activated adenosine triplios-phatase was inhibited at a concentration of 0-3 mg./ml. Oxidative phosphoryla-tion was also unaffected.

Kinin aetivity of the vejiom.

Although stonefish venom contracts isolated gut, the contraction is rapidand followed hy a loss of rhythmic activity of the gut. This contraction is fasterand less intense than that seen after the addition of bradykinin to the organbath. Further, if the gut is left in contact witli the venom for several minutes.

82 L. AUSTIN, R. G. GILLIS AND G. YOUATT

it becomes refractory to added bradykinin. In one experiment, bovine globulinwas incubated with venom and extracted with ethanol as described by Elliott,L,ewi.s and Horton (1960). The extract showed no kinin activity when testedagainst guinea pig ileum.

When glycerinated muscle fibre bundles were soaked in a solution of thevenom for several minutes prior to the addition of ATP the contraction develox^edwas as strong as that developed by control bundles indicating that the venomhas no inhibitory effect on tbis system.

Effect of the venom on capillary pcrmealnUiy.

Stonefish venom (100 ^g. in 0 02 ml. of saline) was injected intradermallyinto the hind paws of rats wliicJi had been dosed intravenously with EvansBlue 20 minutes eiirlier. Immediately after the injection of venom the ratswithdrew the affected foot. Within three to five minutes the dye extravasatedinto the treated foot whicli became rapidly oedeniatous. The area of stainingand oedema increased uver a period of about an hour. The staining spread upthe whole leg and in one case on to the belly surface. The other hind paw wassimilarly treated with saline but none of tlie above responses was seen.

Immunoelectrophoresis.

Fig. 1 shows the immunoelectrophoresis patterns obtained w itli the venomsfrom S. trachynis and S. verrucnm. Tiie two patterns show an overall similarity,

Fig. 1. Immunoelectrophoresis patterns obtained after electrophoresis of venom proteinsand diffusion against stonefish antivenine. Upper, S, traehtjnis; lower, S. t:errueo>ia.

STONEFISH VENOM S3

but there is a definite difference between the two species. At least seven com-ponents can be distinguished.

Ultracentrifuge analysis.The venoms from both S. trachynis and S. verrucosa showed two groups of

proteins which havt- S ,, vahies of 1 9 for the lighter group and 8-4 to 9 0 fortlio iieavier group. If a globular shape is a-ssirnied for the venom proteins, thesevalues indicate molecular weiglits around 15,000 and 150,000 respectively.

Separation on carhoxymelhyl cellulose columns.When run on a carboxymethyl cellulo.se column S, traehynis venom proteins

separated as shown in Fig. 2. Three major peaks were separated first and two

Fig. 2. Fractionatinn of S. Irachi/nis venom on CM cellulose. Upper curve, absorption at230 mm lower curve, absorption at 280 nifi. Peak C only was to.vic to rats.

strongly absorbed ininor peaks were ehited later. In the st'coiKl, andmore particularly in llic tliird. peak there is evidence of partial resolution intofurther fractions. Samples from each peak were tested for toxicity by measuringchanges in the blodd pressure and electrocardiogram of anaesthetised rats afterintravenous injection. It \\as found that the to.\ie fraction is located in the thirdpeak; other fractions showed no toxic activity. The fractions from each mainpeak were p(X)led. dialysed against plinsphate buffer and lyophilised. Theultraviolet spectrum of each of three fractions is shown in Fig. 3, together with

84 L. AUSTIN, R. G. CILLIS AND G. YOUATT

Wavelength

Fig. 3. Ultraviolet spectra of fractions 1, 2 and 3 from Fig. 2 and of whole venom (V).The curves are displaced vertically to avoid overlap.

tliat of the whole venom. Measurements of the absorption coefficients of the

whole venom gave values of E 2^0^^^" ~ ^ '^ '" *^^ ^^^^ "f •'• ^^f^ncosa

and E sMimu ~ ^'^ ^" *''^ ^^^^ °^ - f^<'<^hy^^s- These are both minimumvalues since in both species the venom contains a small amount of insolublematerial which was centrifuged out before absorption measurements were made.

The Brst component from the CM column showed strong absorption at short

wavelengths when compared with the second and third, whereas the third

component absorbed relatively strongly at 280 m/A. In Fig. 4 the ratios of the

absorbances at 230 and 280 m/i are plotted against fraction number. This curve

suggests that the toxic third fraction is rich in aromatic amino acids as compared

with the other main fractions. This conclusion is supported by the result of

determinations of protein by Lowry's method, which is dependent on the presence

of phenolic amino acids, and by ninhydrin estimations.

STONEFISH VENOM 85

Separation on Sephadex.

Fig. 5 shows the result of passing 7 mg. of S. verrucosa venom through aG.75 Sephadex column; three fractions were obtained. The first major peakprobably corresponds to the heavy fraction seen in the ultracentrifuge; it ishighly toxic to rats (60 /xg was lethal in 30 seconds). This peak contained

Tube Numbef

Fig. 4. Ratio of absorbances (A230/A280) ealeulated from Fig. 2.

strong capillary permeability activity, and the hyalurunidase activity was alsomainly in this fraction. The second fraction, which is the shoulder on the firstpeak, had no effect on the rat blood pressure or ECG at the same dose leveland there was little or no capillary permeability activity. The third fraction,which may correspond to the light fraction seen in the ultra centrifuge caused aslight rise in blood pressure and had a transient effect on the ECG at the samedose level.

Chemical analysis of the venom.

The nitrogen and sulphur contents, calculated on an ash-free basis, oflyophilised venom from S. trachynis were 12-9 per cent and 3-8 per cent respec-

86 L. AUSTIN, R. G. GILLIS AND G. YOUATT

tivcly. Venom from S. verrucosa showed nitrogen 12-4 per cent and sulphur3-0 per cent. Slotta and Fraenkel-Gonrat (1938) have reported a similar analysisfor rattlesnake venom (Crotalus terrificus), nitrogen 13-2 per eent and sulphur3-4 per cent.

1-6

-o 0 8'

10 15Tube Number

Fig. 5, Fractionation of S. verrucosa venom on G.75 Scpbadex. The toxic protein was foundonly in the first peak.

Analysis of the sulx^hated ash by atomic absorbtion spectroscopy showedsodium 2-42 per cent, potassium 0-5S per cent, calcium 0-097 per cent, magne-sium 0-60 per cent and zinc 0-036 per cent.

Neither ph<Dsphorus, nucleic acid nor carbohydrate was found.

STONEFISH VENOM 87

Stability of the venom.

Stonefish venom is very unstable in solution. A marked loss of activity isobserved within a few hours, even at 0°. Many attempts were made to stabilizethese solutions. These included the addition of botli thiol reducing and oxidizingagents, versene and BAL to remove heavy metals, hydrogen bond breaking sub-stances and buffers at a wide range of pH values. None of these were successful.Although no protease activity was found in fresh solutions of venom, this enzymewas detected in solutions left overnight at 0" . Presumably this was due tobacterial growth in the venom solutions, paiiicularly since the addition of amixture of chloromycctin and streptomycin to the solution suppressed the appear-ance of the protease aetivity. A small improvement in stability was also noticedwhen tho antibiotic was added to the venom solutions.

DISCUSSION.

Stonefish venom is comparable to scorpion (Tityus) venom in its limitedrange of biochemical activities. Both have a hyaluronidase and a capillarypermeabihty effect, both are intensely painful (Rocha e Silva ct al., 1949).However, scorpion venom also possesses kinin activity and its toxic fraction isof low molecular weight (Diniz and Goncalves, 19.56).

Stonefish venom is also similar to that of the lionfish or butterfly cod(Pterois volitan^); Savmders and Lifton (1960) consider that these venoms maybe identical. Stonefi h venom is unlike the venom of the wecver Hsh (Traclumisdraco) in that although the stoneHsh is extremely potent in the intact animal,preliminary experiments show that it has no effect on fibroblasts grown in tissueeulture as does the weever fish venom (Skeic, 1962). Stonefish venom also haslittle in common with snake venoms, except for a lower nitrogen and highersulphur content than normal proteins; in particular, snake ven(jms usually havea much more extensive variety of enzymes.

There appear to be fonr factors having biological activity present in stone-fish venom. These are:

1. Hyaluronidase. - - . - . . . . -

2. A capillary permeability factor which, also, is more stable than the

toxic factor.

88 L. AUSTIN, R. G. GILLIS AND G. YOUATT

3. A toxic material whieh, when injected intravenously into an anaesthe-tised rat, causes an initial rise followed by a marked fall in blood pressure andchanges in the ECG pattern such as an increase in the conduction time, a reversalof the T wave and an enhancement of the R-S segment (Austin et al., 1961).The initial rise in blood pressure may be caused hy a further component since,when the venom ages and toxicity disappears the rise in blood pressure stilloeeurs.

4. A factor which causes intense and prolonged pain. This may, of course,be identical with one of the above components.

Nothing is known of the significance of the other protein components whosepresence is detected during the various fracti<niation and separation procedures.

Although stonefish venom acts pharmacologically by paralysing muscle ofall types, it does not affect those enzyme systems which are normally regardedas being involved in muscle function. The results of Austin et al. (1961)indicate that the conductile rather than the contractile processes of muscle areaffected. This is supported hy our finding tliut tlie contraction of glycineratedpsoas musele by ATP is not inhibited by the prior addition of the venom. Stone-fish venom could, therefore, if it were readily available, be a useful tool for theinvestigation of muscle function.

Acknowledgments. Stonefisb were obtained by courtesy of Sir Edward Hallstroni andthe Taronga Zoological Park Trustees, tluough members of the North Queensland Naturalists'Club and from Dr. R. Endean, Department of Zoology, University of Brisbane. The atomicabsnrbtion spectra were obtained by Dr. J. B. Willis, Chemical Research Laboratories,C.S.I.R.O., Fisherman's Bend. The ultracentrifuge runs were carried out hy Dr. J. M.Cillespie, C.S.I.R.O. Division of Protein Chemistry. We are grateful to Mr. I. Chubb andMr. S. Barboutis for valuable technical assistance.

The permission of the Chief Scientist, Australian Defence Scientific Service, Departmentof Supply, Melbourne, to publish this communication is acknowledged by one of us (R.C.G.).

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90 L. AUSTIN, R. G. GILLIS AXD G. YOUATT

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