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Partial purification of Lophozozymus pictor toxin
r~. tsu. va. t; vv. t~o~-sta t~s.~ t~ trt.~.e
m a.u eduto.
PARTIAL PURIFICATION OF LOPHOZOZYMUS PICTOR TOXIN Y. F. TEtt and J. E. GA1tnIx~tt Department of Pharmacology, Faculty of Medicine, University of Singapore, Sepoy Lines, Republic of Singapore (Acceptedjor publieatfae 191ane 1974) Y. F. Tart and J. E. GxrrntNert . Partial purification of Lophozorynars pictor toxin. Toxkon 12, 603-610,1974 .-An aqueous extract of a coral reef crab Lophozozynacr pJctor was subjected to a 6-stage purification and chemical testing procedure. The semi-purißed toxin had a molecular weight between 1000 and 5000 and gave reactions that suggested it contained free amino and phenolic groups. The ta for mice was 377 pgJkg. The dace-death time relatiorrshipa for the crab toxin, saxitoxin and tetrodotoxin were determined . The relationship for the crab toxin differed markedly from those for the other two toxins, thecrab toxin being slower in itsaction .
Loplrozozynttrs pictor
INTRODUCTION
is a crab found on the coral reefs around Singapore. It has the common Chinese name 'lui-kong chim' meaning 'thunder crab'. Fishermen who collect edible crabs from the reefs know the animal to be noxious and avoid catching it, or reject any that are trapped. It is normally distinguished from otherwise similar crabs by its speckled appearance (Fig . 1), the pattern consisting of white spots on a unique maroon-coloured background. The body of an average specimen measures 8-9 cm across and about 6-7 cm antero-postcriorly. We have previously shown that an aqueous extract of Lop/rozozyntus pictor is lethal when injxted into mice (TETI and GARDiNGR, 1970). Animals injected with a fatal dose of the extract show partial paralysis which develops into laboured respiration, terminating up to 30 hr later in convulsions and death. The dose-death time relationship of the nude extract in mice, as well as its chemical properties, indicated that the crab toxin difïered from the more commonly known marine toxins such as tetrodotoxin, saxitoxin and ciguatoxin. The present paper reports a method for the purification of the toxin and for its assay during purification, together with further toxicological comparison of its properties with those of tetrodotoxin and saxitoxin. MATERIALS AND METHODS
Collection mrd ident~cation Live specimens of Lophozozymus pictor were purchased from fishermen who trapped them from coral reefs close to Singapore. Once landed, the crabs were transported without delay to the laboratory, killed by freezing and stored at -20°C until required . We are indebted to Dr. R. Serene, UNESCO Regional Marine Taxonomist and to Mr. E. R. Alfred, Curator of Zoology and Acting Director of the National Mtueum, Singapore, for identifying specimens of the animals investigated as Lophozozymus pictor. The characteristic maroon colour and the white speckling by which they are distinguished by the fishermen arc retained whether the crabs are alive or dead . TOXICON1971 Yd. t1
603
604
Y. F. TEH and J. E . GARDINER
Extraction andpurification of toxin
The purification procedure is shown diagrammatically in Fig. 2. Whole frozen crabs TOTAL AcTmTr
STAGE
1.
2.
Two extractions with baling wahr
I:ECOVeIn
CrusMd whote aab 100 g
Centrifuged 3000 y. 15 min. Filter through Whatman GF/A
Acidification with 2N HCI to pH 3 1 glass electrode ). Cenhifugad 3000 y. 15 min at 4~C Neutrahsod immediately to pH 7 with 2N NaOH 3,
Crude extract (100 mll
Clarified aqueous extract (95.6 ml )
CG900 MU
100'/.
Acid treated extract ( 93 ml 1
37100 MU
037.
Non-dialysable fractwn 1 BG ml 1
41700 MU
93'/.
Precipitate (discarded 1
Acid sùpernatant
Dialysed against 2 changes of 1 I, water owr 2t hr at G~C 4. Fraction of 100 mg batches on Sephadex G-50 gel column at 25~C 5.
Non toxic effluent i fractans ~containxrg lass than 20 MU/ml l discarded ) Toxic effluent (600 ml)
Freaza deed 6.
Purified toxin 0.112 g 1132000 MU/g )
18800 MU
i27.
FIO . T. SCHEMATIC DIAGRAM OF THE PARTIAL PURIFICATION PROCEDURE FOR iAp%WZOZytlftrs
The mouse unit of activity
pictor Toxttv. (MU) of the toxin is equivalent
to the ~xa in 20
g rttiott .
were crushed and extracted twice with boiling water as previously described (TEti and GARDINGR, 1970) . The crude extinct so obtained was clarified by centrifugation and filtration through a glass fibre filter. The dialysed extract (stage 4) was evaporated in vacuo in a rotary evaporator and the residue dried to constant weight over CaCI, in a desiccator. The dried residue was not normally stored but was redissolved in water to give a concentration of 50 mg/ml for use in stage 5. The column, used in stage 5 (diameter 2~8 cm) contained Scphadex G-50 suspended in water to give a packed height of 40 cm. Batches of the stage 4 toxin in 2 ml water were put on the column and using water as elutant, fractions (150 drops/tube, about 5 ml) were collected using a LKB UItroVac Fraction Collector TOXICON I97I Yot. lI
f=u~ . I . -I~na. r rmnr . ew .r cenr+, Luplnr_u_ynru.v pir~rur.
ro.t-lc-nxly -{ v .,l . r~
c~. coi
LoPliomsynne Pktar Toxic
tî05
Type 7000. The flow rate through the column was approximately 20 ml/hr. The u .v, absorption of the e®went at 280 nm was monitored with an LKB Type 8300 Uvicord II Absorptiometer . The toxin was eluted between two bands of material absorbing at 280 nm (Fig. 3). Tubes containing toxin activity above 20 MU/ml and showing no u.v. absorption at 280 nm were pooled together . In stage 6 the solution was concentrated in vacuo in a rotary evaporator and freeze dried. The residue was a fluffy buff powder . It was usually dissolved in water to give a solution 10 mg/ml and stored in sealed ampoules at 4°C.
ELUTION
FIa. 3 . ELUTION PROFILI? OF 100
mg
TUBES
DIALYSED CRAB TOXIN CIIROMATOGRAPItED ON SEPHADhJC
G-SO cta The circles indicate the amounts of toxin in the elution tuba as quantitatcd and located by the mouse bioassay in a typical chromatography run . The continuous line represents the elution profile of dcxtran bloc whilo the dashed line represents the components of the crude extract which absorbed strongly at 280 nm . MU = mouse unit of lethality.
Battchlvise absorption tests
Solutions of stage 6 toxin 1 mg/ml were festal for removal of toxic activity by various absorbents. To the toxin solution at room temperature (24-30°C) the absorbent was addt;d in the proportion 25 mg to 1 mg toxin. After shaking for 15 min the mixture was centri fug~d at 2500 e for 10 min and the supernatant assayed for its toxin content. Ifthe toxic activity was removed from the solution, the absorbent was extracted with the appropriate elucnt to recover the toxin if possible. Absorbents tested were charcoal, weak cation exchange resin Zeo-Karb 226, strong cation exchange resin Zeo-Karb 225, strong anion exchange resin, De-acidite FF-IP (The Permutit Co., London). Paper chromatography
Paper chromatogrnphy was performed on portions of the solution from stage 6 applied to Whatman No. 3 paper. Solvent mixtures used for ascending development of the sheets at 24-30°C wet+e : water saturated benryl alcohol; n-butanol-acetic acid-water (12 :3:5); methanol-water-pyridine (20 :5:1). Spray reagents for the detection of the following groups were applied to the chromatograms and, separately, to spots of the extract on paper. Ninhydrin-primary amine groups, diazotized sulphanilic acid and diazotized 4nitranilinephenolic groups and aromatic nuclei, aniline hydrogen phthalate--sugar rings, Dragendorfl"s reagent-tertiary bases and quaternary ammonium compounds, Ehrlich's reagentindole compounds, molybdatc/hydrazine reagent-phosphate esters, silver nitrate-reducing substances. 710XlCON 1971 Yd. !1
606
Y. F. TEH and J. E. GARDINER
lethality measurements For the determination of the t.D s , of the crab toxin, saxitoxin and tetrodotoxin the method of McFAnREN (1966) was followed . White mice of either sex, weighing 19-21 g were used. The animals were bred and supplied by the University of Singapore Laboratory Animal Centre, Sembawang, Singapore. Toxin solution was injected intraperitoneally, the injection volume in all cases being 025 ml. The animals were observed for 72 hr and the number of mice in a group dead at the end of the period was recorded. The t.D ao was calculated from the results by the probit method (Ftst~R and YATES, 1963) and was arbitrarily used to define a unit of activity, the mouse unit (MU), for the particular toxin . Dose--death time relationships The relationship between the dose of crab toxin and the time taken for the animal to die was investigated as follows . Doses of 2, 4, 5, 7, I5, 23 or 30 MU of the crab toxin (stage 6) were administered to groups of 6 mice as in the determination of the t .Da a. The time elapsing between the injection and the time of death was recorded for each animal . A HewlettPackard Calculator Type 9810A and standard programmes available for the machine were used to apply curve fitting tests to the results and thus determine the relationships which best correlated the dose and time of death. Similar experiments with saxitoxin and tetrodotoxin were performed in which doses of I, 125, L~S, 2~0, 2~5, 5~0 or 7~5 MU were used. Bioassay ofcrab to xin A bioassay method based on the dose-death time relationship for the crab toxin was used to monitor the purification procedure and to estimate concentration of toxin in the various fractions. Groups of 10 mice were injected with suitable volumes of an unknown toxin solution to kill the mice within 2 hr. For each mouse the death time was noted and the corresponding dose in M U calculated from the dose-death time relationship determined about. The mean of the l0 values was taken as toxin content of the dose of unknown solution injected . Clremicals Wherever possible, chemicals were of analytical reagent quality . Tetrodotoxin was purchased from Sankyo Co., Tokyo and saxitoxin obtained from Dr. E. J. Schantz, U.S. Army Biological Laboratories, Fort Detrick, Maryland. RESULTS
Purification procedure The steps in the extraction and purification of the toxin together with the potencies and yields at the various stages of a run are shown in Fig. 2. Throughout the purification procedure the toxin was detected by lethality tests in mice as described in the Methods because, so far, no physico-chemical parameter has been found to be associated with the presence of the toxin in a solution . The starting material was crushed whole crabs since we had previously shown (TEtt and GARDINER, 1970) that the toxin was not confined to or localized in any particular part of the animal . Compared with our previous extraction method the filtration through the glass fibre (stage 2) served to remove suspended inactive material not readily sedimented by centrifugation . TOXJCON J97I YoJ. J2
Lopbozorrnue p~tw Toxin
6D7
Although the toxin is known to be labile under acid conditions it was found that brief and convolkd acidification in the cold could affect a useful purification with only a small loss of activity . Further purification was achieved by dialysis which removed inorganic salts from the extract and produced an apparent increase in lethality . This increase may have been due to the removal of an antagonist or it may, like the slight loss at stage 3, have been due to the imprecision of the mouse kthality assay technique. The behaviour of the toxin during dialysis indicated that its molecular weight was 10' greater or ; this was confirmed by the fractionation of the extract on Sephadex gels . Figure 3 shows the location of the toxin and two non-lethal but u .v. absorbing bands in the fractions collected from the Sephadex G-50 gel filtration column. Material that absorbed light at 280 nm was eluted from the column between fractions 7 and 30 (120 ml). Two bands of strong absorption were observed, the first, fractions 7-10, corresponded to elution of the void volume of the column as determined by Dextran Blue ; the second exhibited peak u.v. absorption with fraction 25. The toxin was eluted between these bands with fraction 17 containing the highest lethality. The variation in the toxin content of the fractions was not paralleled by comparable variation in the u.v. absorption of the fractions at 280 nm. To obtain better separation of the toxin from inactive components of the extract only fractions containing 20 MU/ml or above were retained. This procedure accounted for much of the activity loss at this stage of the purification since there were low levels of toxin activity in the discarded fractions preceding and following those that were selected . A similar fractionation of the dialysed extract using Sephadex G-25 indicated the toxin had a molecular weight between 1 and S x l0' since it was eluted between vitamin Bl, (molecular weight 1355) and inulin (molecular weight 5000). It was not practicable to use Sephadex G-25 for the purification since the toxin was eluted from it together with the slower moving u.v. absorbing band that is shown in Fig. 3. The final product obtained on freeze drying the pooled toxic fractions from the Sephadex G-50 was a butt amorphous powder. Chemical nature ojloxln The freeze-dried stage 6 toxin was stable without deliquescence towards the warm damp air of Singapore. It freely redissolved in water and, for convenience, the toxin was normally kept in solution. Stored at 0~°C the solution was stable and retained its lethal activity unchanged for at least 12 months . The toxin could not be induced to crystallize ; a solution if allowed to evaporate slowly in a desiccator left a thin uniform amorphous glassy deposit ; similarly no crystallization of strong solutions could be produced by cooling or `scratching' . Depending on the rate of heating the stage 6 product melted, with decomposition, within the range 19a-205°C. Various other procedures were investigated in an effort to further purify the toxin but none proved practicable owing to large losses of activity that occurred . Batch testing showed that the toxin was readily removed from solution by charcoal or svong cation exchange resins, however, from neither could appreciable lethal activity be removed other than by reagents such as strong acids in which the toxin is unstable . Similarly, although it could be shown with location reagent sprays that various components had been separated when paper chromatography was carried out, no lethal activity could be eluted from any part of duplicate unsprayed sheets. Figure 4 shows the u.v. absorption spectrum of the toxin at stages 2 and 6 of the purification procedure. The purified toxin at concentration of 0~1 or 10 mgJml shows no charaoTOXlCON 1971 Yol. !?
608
Y. F. TEH and J. E. GARDINER
tetistic strong bands of absorption which may be used to detect the toxin nor to give direct indication as to its chemical nature . I. .. . .. L2
am Lo 0m a~ m o.s a e4 0.2 0 .0
FIa . 4 . ABSORPTION SPECTRA OF CRUDE AND PARTIALLY PURIFIED CRAB TOXIN . The absorption spectrum of the crude toxin (stage 2) was measured at 100 pg/ml (p) while that of ehc partially purified toxin (stage 6) was measured at 100 pgJml (Q) and 10 mgJml (~ . All dilutions were made with distilled water which was also used as the blank .
The stage 6 toxin, SO leg on Whatman No. 3 MM paper, gave positive reactions when sprayed with ninhydrin, diazotized sulphanilic acid or diazotized p-nitraniline indicating the presence of free amino groups and probably phcnolic hydroxyl groups. No reaction was obtained towards sprays to detect sugar rings, tertiary bases or quaternary ammonium compounds, indolc compounds, phosph:etc esters or reducing substances . The ninhydrin spray showed the most components when used to spray the chromatograms. The toxic material was not soluble in n-pentane, benzene or diethyl ether; absolute ethanol extracted a small (4 per cent) amount of toxin but after successive extractions with all these solvents the bulk of the toxic activity (96 per cent) remained in the water-soluble residue. Biological tests Determination of the t.O su in mice of the crab toxin, tetrodotoxin and saxitoxin gave the following values (t.nsQ, 95 per cent confidence limits), stags: 6 toxin 377"4, 304"9-467"2 Ftg/kg ; saxitoxin 1 l"2, 10"7-11 "8 leg/kg and tetrodotoxin 105, 102-108 leg/kg. Each value is based upon results obtained using six dilTerent doses and 10 animals per dose. Mice given minimally efïective doses of saxitoxin or tetrodotoxin died with 0"S hr whereas mice given low doses of the crab toxin took up to 4 hr before they died. This observation indicated that it should be possible to distinguish the toxin in the stage 6 product from either saxitoxin or tetrodotoxin by comparing the dose-death time relationship for the three toxins . Figure 5 shows the results of theca experiments. The results were analysed mathematically with an electronic calculator and the curves shown in the figure are those drawn from the equations which were found to give the best statistical correlation of the death time and the dose. For each of the three toxins the relationship was that of a power caries (y = axb) rather than an exponential (y = ae°x) or linear regression (y = a -}- bx) where x = time for animals to die when given a dose y and a and b are constants. The equation for the three 71DXICON 1971 YoL !1
609
Lopbomsynurr pktor Toxin 3a~
10~
O
e
e~
2
-T
°
50
~
100
DEATH
200 150 T11AE5 (min /
250
Fla. S. Daas-ueA~rEt rwe CtnevFS r°oa
DEATH
Loplmzozynrrrs ptctor
ro~mv.
TIMES Imln /
~m~art, sAra~roxnv AND isneooo-
The curves drawn are those which show the best statistical correlation between the death time and the done of each of the three toxiTls. Their equatioro aro given in the text. MU mouse unit of lethality . " Lopho:orymrcr pictor toxin. ,~, Tetrodotoxin. " Saxitoxin . toxins are crab toxin y = 225-19c °'", saxitoxin y = 13-31x-1'" and tetrodotoxin y = 11-79.x'°''". The relationship For the crab toxin is clearly different from the other two toxins. DISCUSSION
The purification procedure described in this paper has produced a concentrated extract of the toxin from Lophozozynras plctor which has enabled a comparison of its lethality to be made with tetrodotoxin and saxitoxin . The t.n6° value for mice (377 Ng/kg) of the final solid shows that at its present degrce of purification it is comparable in potency with Naja naja or cobra toxin (approximately 340 Ftg/kg, Ftscttett and KAnARA, 1966) and more potent than samandarin the biotoxin from the salamander Sala»rantlra nraculosa (1500 Frg/kg, Mosttett et al., 1964) . Toxins from a number of aquatic animals have been shown to be either tetrodotoxin or saxitoxin (MosttEtt et al., 1964 ; Korrosu et al., 1968 ; Nocucltt et al., 1969) . It was necessary therefore to distinguish the crab toxin from them since they both have high potencies and an impure preparation of either could therefore exhibit a similar potency to that determined for the crab toxin . Against such an explanation must be set the differences observed in the doso-death time relationships for the three toxins which clearly show the crab toxin to be different. An identity different from saxitoxin and tetrodotoxin is also suggested by the chemical and chromatographic tests that have been applied in this and the previous paper (Tart and GARDINER, 1970) ; however, at the present time chemical tests cannot be conclusive since if the toxic material were to have an intrinsic lethality of the order of 10 Ng/kg it would only constitute about 2-5 per cent of the stage 6 product . Although the results of the chemical tests applied to the stage 6 product can only give hints of the nature of the toxin, from the positive reaction with ninhydrin and the attachment of the toxin to strong cation exchange resin it would appear that it is a basic substance containing free primary amino groups. The lack of reactivity towards the aniline phthalate or Dragendorff's reagents suggests that neither sugar rings nor quaternary ammonium groups are responsible for the hydrophilic character the toxin exhibited . rnxtconr t~r+ v~r. u
610
Y. F. TEH and J. E. GARDINER
Further purification of the stage 6 material has not, so far, been practicable. There are two main reasons for this ; the first is the chemical lability of the toxin either in solution or once it has become absorbed onto a chromatographic absorbent ; the second is the slowness of assay method together with the large quantities of toxin that are required . Investigations into the pharmacological activity of the stage 6 material are being continued to seek another assay system . One possibility is to test the action of the toxin on cells in tissue culture (TAx and TEx, 1972), which requires less toxin than the mouse assay although it is procedurally more inconvenient and costly . Acknowledgtment,}--We thank the China Medical Board of New Yorr, Inc. for supporting this study under Grant No . 72-290 and Dr. E. J. Scrrernz for gifts of saxitoxin. REFERENCES Ftsc~rt, G. A. and K~eau, J. J. (196 Low molecular weight toxins isolated from elapidae venoms . In : Animal Tasirts, p. 283, (Russet.t., F. E. and S~rlrvneas, P. R., Eds.) . Oxford : Pergamon Press. Ftsrrea, R. A. and YerFS, F. (1%3) In : Statirtlcal Tables jor Biologkal, Agrkultural and Medical Rr'.rterch, Sixth Edition, p. 10. London : Oliver do Boyd . Kor+oacr, S., hroue, A., Nocucrn, T. and H~srrrMOro, Y. (1968) Comparison of crab toxin with saxitoxin and tetrodotoxin. Toxicon 6, 113 . McF~Raetv, E. F. (1966) Differentiation of the poisons of fish, shellfish and plankton . In : Arrinral Toxins, p. 85, (Rt13sEU., F. E. and S~rttwEas, P. R., Eds.) . Oxford : Pergamon Pass . Mosrrert, H. S., Futrasun, F. A., Buc~rw~w, H. D. and Flaren, H. G. (1964) Tarichatoxin-tetrodotoxin : a potent neurotoxin. Sciurn 144, 1100 . Nocucrrr, T., KoNastr, S. and H~srrnroro, Y. (1%9)ldentity of the crab toxin with saxitoxin. Toxkon7, 323. TArv, C. H. and TeH, Y. F. (1972) The effect of Lopho:o :ynuu pictor toxin on HeLa cells. Erpaitntla 28, 46 . Terr, Y. F. Snd GARDINER, J. E. (1970) Toxin from the coral r~ccfcrab, Lopho:osymur pictor. Pharraac. Rer. Conrrnwr. 2, 251 .
7UXICON 1971 YoL l2
m a.u eduto.
PARTIAL PURIFICATION OF LOPHOZOZYMUS PICTOR TOXIN Y. F. TEtt and J. E. GA1tnIx~tt Department of Pharmacology, Faculty of Medicine, University of Singapore, Sepoy Lines, Republic of Singapore (Acceptedjor publieatfae 191ane 1974) Y. F. Tart and J. E. GxrrntNert . Partial purification of Lophozorynars pictor toxin. Toxkon 12, 603-610,1974 .-An aqueous extract of a coral reef crab Lophozozynacr pJctor was subjected to a 6-stage purification and chemical testing procedure. The semi-purißed toxin had a molecular weight between 1000 and 5000 and gave reactions that suggested it contained free amino and phenolic groups. The ta for mice was 377 pgJkg. The dace-death time relatiorrshipa for the crab toxin, saxitoxin and tetrodotoxin were determined . The relationship for the crab toxin differed markedly from those for the other two toxins, thecrab toxin being slower in itsaction .
Loplrozozynttrs pictor
INTRODUCTION
is a crab found on the coral reefs around Singapore. It has the common Chinese name 'lui-kong chim' meaning 'thunder crab'. Fishermen who collect edible crabs from the reefs know the animal to be noxious and avoid catching it, or reject any that are trapped. It is normally distinguished from otherwise similar crabs by its speckled appearance (Fig . 1), the pattern consisting of white spots on a unique maroon-coloured background. The body of an average specimen measures 8-9 cm across and about 6-7 cm antero-postcriorly. We have previously shown that an aqueous extract of Lop/rozozyntus pictor is lethal when injxted into mice (TETI and GARDiNGR, 1970). Animals injected with a fatal dose of the extract show partial paralysis which develops into laboured respiration, terminating up to 30 hr later in convulsions and death. The dose-death time relationship of the nude extract in mice, as well as its chemical properties, indicated that the crab toxin difïered from the more commonly known marine toxins such as tetrodotoxin, saxitoxin and ciguatoxin. The present paper reports a method for the purification of the toxin and for its assay during purification, together with further toxicological comparison of its properties with those of tetrodotoxin and saxitoxin. MATERIALS AND METHODS
Collection mrd ident~cation Live specimens of Lophozozymus pictor were purchased from fishermen who trapped them from coral reefs close to Singapore. Once landed, the crabs were transported without delay to the laboratory, killed by freezing and stored at -20°C until required . We are indebted to Dr. R. Serene, UNESCO Regional Marine Taxonomist and to Mr. E. R. Alfred, Curator of Zoology and Acting Director of the National Mtueum, Singapore, for identifying specimens of the animals investigated as Lophozozymus pictor. The characteristic maroon colour and the white speckling by which they are distinguished by the fishermen arc retained whether the crabs are alive or dead . TOXICON1971 Yd. t1
603
604
Y. F. TEH and J. E . GARDINER
Extraction andpurification of toxin
The purification procedure is shown diagrammatically in Fig. 2. Whole frozen crabs TOTAL AcTmTr
STAGE
1.
2.
Two extractions with baling wahr
I:ECOVeIn
CrusMd whote aab 100 g
Centrifuged 3000 y. 15 min. Filter through Whatman GF/A
Acidification with 2N HCI to pH 3 1 glass electrode ). Cenhifugad 3000 y. 15 min at 4~C Neutrahsod immediately to pH 7 with 2N NaOH 3,
Crude extract (100 mll
Clarified aqueous extract (95.6 ml )
CG900 MU
100'/.
Acid treated extract ( 93 ml 1
37100 MU
037.
Non-dialysable fractwn 1 BG ml 1
41700 MU
93'/.
Precipitate (discarded 1
Acid sùpernatant
Dialysed against 2 changes of 1 I, water owr 2t hr at G~C 4. Fraction of 100 mg batches on Sephadex G-50 gel column at 25~C 5.
Non toxic effluent i fractans ~containxrg lass than 20 MU/ml l discarded ) Toxic effluent (600 ml)
Freaza deed 6.
Purified toxin 0.112 g 1132000 MU/g )
18800 MU
i27.
FIO . T. SCHEMATIC DIAGRAM OF THE PARTIAL PURIFICATION PROCEDURE FOR iAp%WZOZytlftrs
The mouse unit of activity
pictor Toxttv. (MU) of the toxin is equivalent
to the ~xa in 20
g rttiott .
were crushed and extracted twice with boiling water as previously described (TEti and GARDINGR, 1970) . The crude extinct so obtained was clarified by centrifugation and filtration through a glass fibre filter. The dialysed extract (stage 4) was evaporated in vacuo in a rotary evaporator and the residue dried to constant weight over CaCI, in a desiccator. The dried residue was not normally stored but was redissolved in water to give a concentration of 50 mg/ml for use in stage 5. The column, used in stage 5 (diameter 2~8 cm) contained Scphadex G-50 suspended in water to give a packed height of 40 cm. Batches of the stage 4 toxin in 2 ml water were put on the column and using water as elutant, fractions (150 drops/tube, about 5 ml) were collected using a LKB UItroVac Fraction Collector TOXICON I97I Yot. lI
f=u~ . I . -I~na. r rmnr . ew .r cenr+, Luplnr_u_ynru.v pir~rur.
ro.t-lc-nxly -{ v .,l . r~
c~. coi
LoPliomsynne Pktar Toxic
tî05
Type 7000. The flow rate through the column was approximately 20 ml/hr. The u .v, absorption of the e®went at 280 nm was monitored with an LKB Type 8300 Uvicord II Absorptiometer . The toxin was eluted between two bands of material absorbing at 280 nm (Fig. 3). Tubes containing toxin activity above 20 MU/ml and showing no u.v. absorption at 280 nm were pooled together . In stage 6 the solution was concentrated in vacuo in a rotary evaporator and freeze dried. The residue was a fluffy buff powder . It was usually dissolved in water to give a solution 10 mg/ml and stored in sealed ampoules at 4°C.
ELUTION
FIa. 3 . ELUTION PROFILI? OF 100
mg
TUBES
DIALYSED CRAB TOXIN CIIROMATOGRAPItED ON SEPHADhJC
G-SO cta The circles indicate the amounts of toxin in the elution tuba as quantitatcd and located by the mouse bioassay in a typical chromatography run . The continuous line represents the elution profile of dcxtran bloc whilo the dashed line represents the components of the crude extract which absorbed strongly at 280 nm . MU = mouse unit of lethality.
Battchlvise absorption tests
Solutions of stage 6 toxin 1 mg/ml were festal for removal of toxic activity by various absorbents. To the toxin solution at room temperature (24-30°C) the absorbent was addt;d in the proportion 25 mg to 1 mg toxin. After shaking for 15 min the mixture was centri fug~d at 2500 e for 10 min and the supernatant assayed for its toxin content. Ifthe toxic activity was removed from the solution, the absorbent was extracted with the appropriate elucnt to recover the toxin if possible. Absorbents tested were charcoal, weak cation exchange resin Zeo-Karb 226, strong cation exchange resin Zeo-Karb 225, strong anion exchange resin, De-acidite FF-IP (The Permutit Co., London). Paper chromatography
Paper chromatogrnphy was performed on portions of the solution from stage 6 applied to Whatman No. 3 paper. Solvent mixtures used for ascending development of the sheets at 24-30°C wet+e : water saturated benryl alcohol; n-butanol-acetic acid-water (12 :3:5); methanol-water-pyridine (20 :5:1). Spray reagents for the detection of the following groups were applied to the chromatograms and, separately, to spots of the extract on paper. Ninhydrin-primary amine groups, diazotized sulphanilic acid and diazotized 4nitranilinephenolic groups and aromatic nuclei, aniline hydrogen phthalate--sugar rings, Dragendorfl"s reagent-tertiary bases and quaternary ammonium compounds, Ehrlich's reagentindole compounds, molybdatc/hydrazine reagent-phosphate esters, silver nitrate-reducing substances. 710XlCON 1971 Yd. !1
606
Y. F. TEH and J. E. GARDINER
lethality measurements For the determination of the t.D s , of the crab toxin, saxitoxin and tetrodotoxin the method of McFAnREN (1966) was followed . White mice of either sex, weighing 19-21 g were used. The animals were bred and supplied by the University of Singapore Laboratory Animal Centre, Sembawang, Singapore. Toxin solution was injected intraperitoneally, the injection volume in all cases being 025 ml. The animals were observed for 72 hr and the number of mice in a group dead at the end of the period was recorded. The t.D ao was calculated from the results by the probit method (Ftst~R and YATES, 1963) and was arbitrarily used to define a unit of activity, the mouse unit (MU), for the particular toxin . Dose--death time relationships The relationship between the dose of crab toxin and the time taken for the animal to die was investigated as follows . Doses of 2, 4, 5, 7, I5, 23 or 30 MU of the crab toxin (stage 6) were administered to groups of 6 mice as in the determination of the t .Da a. The time elapsing between the injection and the time of death was recorded for each animal . A HewlettPackard Calculator Type 9810A and standard programmes available for the machine were used to apply curve fitting tests to the results and thus determine the relationships which best correlated the dose and time of death. Similar experiments with saxitoxin and tetrodotoxin were performed in which doses of I, 125, L~S, 2~0, 2~5, 5~0 or 7~5 MU were used. Bioassay ofcrab to xin A bioassay method based on the dose-death time relationship for the crab toxin was used to monitor the purification procedure and to estimate concentration of toxin in the various fractions. Groups of 10 mice were injected with suitable volumes of an unknown toxin solution to kill the mice within 2 hr. For each mouse the death time was noted and the corresponding dose in M U calculated from the dose-death time relationship determined about. The mean of the l0 values was taken as toxin content of the dose of unknown solution injected . Clremicals Wherever possible, chemicals were of analytical reagent quality . Tetrodotoxin was purchased from Sankyo Co., Tokyo and saxitoxin obtained from Dr. E. J. Schantz, U.S. Army Biological Laboratories, Fort Detrick, Maryland. RESULTS
Purification procedure The steps in the extraction and purification of the toxin together with the potencies and yields at the various stages of a run are shown in Fig. 2. Throughout the purification procedure the toxin was detected by lethality tests in mice as described in the Methods because, so far, no physico-chemical parameter has been found to be associated with the presence of the toxin in a solution . The starting material was crushed whole crabs since we had previously shown (TEtt and GARDINER, 1970) that the toxin was not confined to or localized in any particular part of the animal . Compared with our previous extraction method the filtration through the glass fibre (stage 2) served to remove suspended inactive material not readily sedimented by centrifugation . TOXJCON J97I YoJ. J2
Lopbozorrnue p~tw Toxin
6D7
Although the toxin is known to be labile under acid conditions it was found that brief and convolkd acidification in the cold could affect a useful purification with only a small loss of activity . Further purification was achieved by dialysis which removed inorganic salts from the extract and produced an apparent increase in lethality . This increase may have been due to the removal of an antagonist or it may, like the slight loss at stage 3, have been due to the imprecision of the mouse kthality assay technique. The behaviour of the toxin during dialysis indicated that its molecular weight was 10' greater or ; this was confirmed by the fractionation of the extract on Sephadex gels . Figure 3 shows the location of the toxin and two non-lethal but u .v. absorbing bands in the fractions collected from the Sephadex G-50 gel filtration column. Material that absorbed light at 280 nm was eluted from the column between fractions 7 and 30 (120 ml). Two bands of strong absorption were observed, the first, fractions 7-10, corresponded to elution of the void volume of the column as determined by Dextran Blue ; the second exhibited peak u.v. absorption with fraction 25. The toxin was eluted between these bands with fraction 17 containing the highest lethality. The variation in the toxin content of the fractions was not paralleled by comparable variation in the u.v. absorption of the fractions at 280 nm. To obtain better separation of the toxin from inactive components of the extract only fractions containing 20 MU/ml or above were retained. This procedure accounted for much of the activity loss at this stage of the purification since there were low levels of toxin activity in the discarded fractions preceding and following those that were selected . A similar fractionation of the dialysed extract using Sephadex G-25 indicated the toxin had a molecular weight between 1 and S x l0' since it was eluted between vitamin Bl, (molecular weight 1355) and inulin (molecular weight 5000). It was not practicable to use Sephadex G-25 for the purification since the toxin was eluted from it together with the slower moving u.v. absorbing band that is shown in Fig. 3. The final product obtained on freeze drying the pooled toxic fractions from the Sephadex G-50 was a butt amorphous powder. Chemical nature ojloxln The freeze-dried stage 6 toxin was stable without deliquescence towards the warm damp air of Singapore. It freely redissolved in water and, for convenience, the toxin was normally kept in solution. Stored at 0~°C the solution was stable and retained its lethal activity unchanged for at least 12 months . The toxin could not be induced to crystallize ; a solution if allowed to evaporate slowly in a desiccator left a thin uniform amorphous glassy deposit ; similarly no crystallization of strong solutions could be produced by cooling or `scratching' . Depending on the rate of heating the stage 6 product melted, with decomposition, within the range 19a-205°C. Various other procedures were investigated in an effort to further purify the toxin but none proved practicable owing to large losses of activity that occurred . Batch testing showed that the toxin was readily removed from solution by charcoal or svong cation exchange resins, however, from neither could appreciable lethal activity be removed other than by reagents such as strong acids in which the toxin is unstable . Similarly, although it could be shown with location reagent sprays that various components had been separated when paper chromatography was carried out, no lethal activity could be eluted from any part of duplicate unsprayed sheets. Figure 4 shows the u.v. absorption spectrum of the toxin at stages 2 and 6 of the purification procedure. The purified toxin at concentration of 0~1 or 10 mgJml shows no charaoTOXlCON 1971 Yol. !?
608
Y. F. TEH and J. E. GARDINER
tetistic strong bands of absorption which may be used to detect the toxin nor to give direct indication as to its chemical nature . I. .. . .. L2
am Lo 0m a~ m o.s a e4 0.2 0 .0
FIa . 4 . ABSORPTION SPECTRA OF CRUDE AND PARTIALLY PURIFIED CRAB TOXIN . The absorption spectrum of the crude toxin (stage 2) was measured at 100 pg/ml (p) while that of ehc partially purified toxin (stage 6) was measured at 100 pgJml (Q) and 10 mgJml (~ . All dilutions were made with distilled water which was also used as the blank .
The stage 6 toxin, SO leg on Whatman No. 3 MM paper, gave positive reactions when sprayed with ninhydrin, diazotized sulphanilic acid or diazotized p-nitraniline indicating the presence of free amino groups and probably phcnolic hydroxyl groups. No reaction was obtained towards sprays to detect sugar rings, tertiary bases or quaternary ammonium compounds, indolc compounds, phosph:etc esters or reducing substances . The ninhydrin spray showed the most components when used to spray the chromatograms. The toxic material was not soluble in n-pentane, benzene or diethyl ether; absolute ethanol extracted a small (4 per cent) amount of toxin but after successive extractions with all these solvents the bulk of the toxic activity (96 per cent) remained in the water-soluble residue. Biological tests Determination of the t.O su in mice of the crab toxin, tetrodotoxin and saxitoxin gave the following values (t.nsQ, 95 per cent confidence limits), stags: 6 toxin 377"4, 304"9-467"2 Ftg/kg ; saxitoxin 1 l"2, 10"7-11 "8 leg/kg and tetrodotoxin 105, 102-108 leg/kg. Each value is based upon results obtained using six dilTerent doses and 10 animals per dose. Mice given minimally efïective doses of saxitoxin or tetrodotoxin died with 0"S hr whereas mice given low doses of the crab toxin took up to 4 hr before they died. This observation indicated that it should be possible to distinguish the toxin in the stage 6 product from either saxitoxin or tetrodotoxin by comparing the dose-death time relationship for the three toxins . Figure 5 shows the results of theca experiments. The results were analysed mathematically with an electronic calculator and the curves shown in the figure are those drawn from the equations which were found to give the best statistical correlation of the death time and the dose. For each of the three toxins the relationship was that of a power caries (y = axb) rather than an exponential (y = ae°x) or linear regression (y = a -}- bx) where x = time for animals to die when given a dose y and a and b are constants. The equation for the three 71DXICON 1971 YoL !1
609
Lopbomsynurr pktor Toxin 3a~
10~
O
e
e~
2
-T
°
50
~
100
DEATH
200 150 T11AE5 (min /
250
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DEATH
Loplmzozynrrrs ptctor
ro~mv.
TIMES Imln /
~m~art, sAra~roxnv AND isneooo-
The curves drawn are those which show the best statistical correlation between the death time and the done of each of the three toxiTls. Their equatioro aro given in the text. MU mouse unit of lethality . " Lopho:orymrcr pictor toxin. ,~, Tetrodotoxin. " Saxitoxin . toxins are crab toxin y = 225-19c °'", saxitoxin y = 13-31x-1'" and tetrodotoxin y = 11-79.x'°''". The relationship For the crab toxin is clearly different from the other two toxins. DISCUSSION
The purification procedure described in this paper has produced a concentrated extract of the toxin from Lophozozynras plctor which has enabled a comparison of its lethality to be made with tetrodotoxin and saxitoxin . The t.n6° value for mice (377 Ng/kg) of the final solid shows that at its present degrce of purification it is comparable in potency with Naja naja or cobra toxin (approximately 340 Ftg/kg, Ftscttett and KAnARA, 1966) and more potent than samandarin the biotoxin from the salamander Sala»rantlra nraculosa (1500 Frg/kg, Mosttett et al., 1964) . Toxins from a number of aquatic animals have been shown to be either tetrodotoxin or saxitoxin (MosttEtt et al., 1964 ; Korrosu et al., 1968 ; Nocucltt et al., 1969) . It was necessary therefore to distinguish the crab toxin from them since they both have high potencies and an impure preparation of either could therefore exhibit a similar potency to that determined for the crab toxin . Against such an explanation must be set the differences observed in the doso-death time relationships for the three toxins which clearly show the crab toxin to be different. An identity different from saxitoxin and tetrodotoxin is also suggested by the chemical and chromatographic tests that have been applied in this and the previous paper (Tart and GARDINER, 1970) ; however, at the present time chemical tests cannot be conclusive since if the toxic material were to have an intrinsic lethality of the order of 10 Ng/kg it would only constitute about 2-5 per cent of the stage 6 product . Although the results of the chemical tests applied to the stage 6 product can only give hints of the nature of the toxin, from the positive reaction with ninhydrin and the attachment of the toxin to strong cation exchange resin it would appear that it is a basic substance containing free primary amino groups. The lack of reactivity towards the aniline phthalate or Dragendorff's reagents suggests that neither sugar rings nor quaternary ammonium groups are responsible for the hydrophilic character the toxin exhibited . rnxtconr t~r+ v~r. u
610
Y. F. TEH and J. E. GARDINER
Further purification of the stage 6 material has not, so far, been practicable. There are two main reasons for this ; the first is the chemical lability of the toxin either in solution or once it has become absorbed onto a chromatographic absorbent ; the second is the slowness of assay method together with the large quantities of toxin that are required . Investigations into the pharmacological activity of the stage 6 material are being continued to seek another assay system . One possibility is to test the action of the toxin on cells in tissue culture (TAx and TEx, 1972), which requires less toxin than the mouse assay although it is procedurally more inconvenient and costly . Acknowledgtment,}--We thank the China Medical Board of New Yorr, Inc. for supporting this study under Grant No . 72-290 and Dr. E. J. Scrrernz for gifts of saxitoxin. REFERENCES Ftsc~rt, G. A. and K~eau, J. J. (196 Low molecular weight toxins isolated from elapidae venoms . In : Animal Tasirts, p. 283, (Russet.t., F. E. and S~rlrvneas, P. R., Eds.) . Oxford : Pergamon Press. Ftsrrea, R. A. and YerFS, F. (1%3) In : Statirtlcal Tables jor Biologkal, Agrkultural and Medical Rr'.rterch, Sixth Edition, p. 10. London : Oliver do Boyd . Kor+oacr, S., hroue, A., Nocucrn, T. and H~srrrMOro, Y. (1968) Comparison of crab toxin with saxitoxin and tetrodotoxin. Toxicon 6, 113 . McF~Raetv, E. F. (1966) Differentiation of the poisons of fish, shellfish and plankton . In : Arrinral Toxins, p. 85, (Rt13sEU., F. E. and S~rttwEas, P. R., Eds.) . Oxford : Pergamon Pass . Mosrrert, H. S., Futrasun, F. A., Buc~rw~w, H. D. and Flaren, H. G. (1964) Tarichatoxin-tetrodotoxin : a potent neurotoxin. Sciurn 144, 1100 . Nocucrrr, T., KoNastr, S. and H~srrnroro, Y. (1%9)ldentity of the crab toxin with saxitoxin. Toxkon7, 323. TArv, C. H. and TeH, Y. F. (1972) The effect of Lopho:o :ynuu pictor toxin on HeLa cells. Erpaitntla 28, 46 . Terr, Y. F. Snd GARDINER, J. E. (1970) Toxin from the coral r~ccfcrab, Lopho:osymur pictor. Pharraac. Rer. Conrrnwr. 2, 251 .
7UXICON 1971 YoL l2