Isolation and characterization of sea urchin toxin

Toxteon, 1963 . Vol . ~ PP . D-17. PerQamon Pnu Ltd ., Printed in C'mat Britain

ISOLATION AND CHARACTERIZATION OF SEA URCHIN TOXIN* CHARLES B . ALE1vDERt, GEORGE A. FEIGEN,

and JosErx T. TOrurA

Department of Zoology, University of Hawaii and Department of Physiology, Stanford University (Acceptedjorpublication 16 May 1963) Abstract-From the present results it is evident that the active principle of sea urchin toxin is a non-dialy7able, thermolabile protein. It is pH-stable, almost completely soluble in distilled water, and can be precipitated at relatively high potency in the presence of 2/3 saturated ammonium sulfate in a yield amounting to 26 per cent of the dry weight of the starting material . The potency tends to decline slowly at -20° with a half-life of approximately 3 years. Absorption peaks at 278 mts and 323 mp are reduood by maneuvers which remove inert materials . On the basis of an ultracentrifugal analysis, the most active fraction, which has approximately 60 per cent of the lethal toxicity of the parent material, appears to be a single molecular species having a sedimentation coefficient of 2"6 Svedberg units at 20°.

TxE sEA

INTRODUCTION

urchin, Tripneustes gratilla (Linnaeus), a member of the family Toxopneustidae, is a common littoral inhabitant of the Indo-Pacific area . It possesses, as do all members of this family, globiferous pedicellariae which in certain cases have been shown capable of inflicting puncture wounds with the concomitant introduction of toxin . Stings received by humans, attributable to the globiferous pedicellariae of Tripneustes gratilla (Linnaeus) and Toxopneustes pileolus (hamarck), have been described and shown to cause serious local and systemic effects [l, 2, 3] but no documentation is available to substantiate reports of human fatalities by these or other members of the family . Except for reports that globiferous pedicellarial homogenates ofSphaerechinus grattularis (Lamarck) are thermostable [4, 5], of Echittus esculentus (Linna,eus), Paracentrotus lividus (Lamarck), and Psammechirrus miliaris (Mullet) are hemolytic [6], and that homogenates ofLytechinus variegatus contain a dialyzable, acetylcholine-like material [7], little is known about the chemical nature of pedicellariae poison . This investigation was undertaken primarily to isolate and characterize the active material obtained from the globiferous pedicellariae of Tripneustes gratilla (Linnaeus) . "Supported by N6 ONR 225(46) between Stanford University and the Office of Naval Research, NR 107342 between the University of Southern California and the Office of Naval Research, and by HE-03693-07 from the National Institutes of Health . Contribution No . 228, Hawaii Marine Laboratory, Honolulu, Hawaii . tPresent address: Laboratory of Neurological Research, Loma Linda University School of Medicine, Los Angeles County General Hospital .

10

CHARLFS H. ALENDER, GEORGE A. FEIGEN and JOSEPH T. TOMITA MATERIALS AND METHODS

Collection andprocessing Specimens of Tripneustes gratüla

(Linnaeus) were obtained from the flats and slopes of a series of coral reefs in Kaneohe Bay, Oahu, Hawaii, during Augast and September, 1960 and September, 1964. The heads of globiferous pedicellariae used for extraction of the toxin were obtained from 645 sea urchins. They were removed from the animals immediately after each collecting trip. All animals were thoroughly cleaned with a strong sea water spray, the wash water passed through two successive stainless steel sieves with mesh openings of 1 ~95 mm (13 meshes/in.) and 0123 mm (120 meshes/in.), respectively, and the contents of the latter sieve transferred to a large volume of filtered sea water. The pedicellariae and debris were allowed to settle by gravity until detritus and other particulate matter were the only materials remaining suspended, and the latter were removed by decantation. This procedure was repeated several times until the rinse water was free of all suspended matter . Finally, the isolated heads were transferred to fared polyethylene bottles, the excess sea water removed after a period of settling, and the contents frozen and stored at -20°. The pedicellarise from each succeeding batch were overlaid on the previously frozen suspension in the plastic bottles until sufficient material was accumulated for large scale extraction . Bioassay Preliminary studies ofthe biological activity of sea urchin toxin consisted of intravenous, intraperitoneal, and subcutaneous injections, or oral administration of preparations into male albino mice (closed colony, Carworth Farms, Webster strain) weighing 18-28 g. The biological activity of the major preparations described in this report is expressed as the specific biological potency, i.e., the LD6o/mg of precipitable protein. The LD6o values were derived from the von Krogh transformation of dos~mortality data according to the method of FSICiEN et al. [8] in which at least five animals per dose are used . Physical acrd chemical methods

The protein content of toxin preparations obtained in preliminary experiments was determined by the biuret reaction as standardized by C3oxxni r, et al. [9]. A crystalline bovine serum albumin (Armour and Company) was used as a reference protein. Estima tion of protein nitrogen contents of toxin preparations obtained in later experiments was based upon tungstate precipitates according to the micro-modification of the Kjeldahl method of LAxtvc et al. [10] and FSICiSN et al. [1l]. Absorbance determinations of toxin preparations were routinely made at a wavelength of 278 m~. in a Beckman DU spectrophotometer, while toxin solutions were analyzed in a B and L Spectronic 505 spectrophotometer over a wavelength range of 200 m~. to 650 m~. The sedimentation behavior of saline solutions of crude toxin [SUT (64)] and of a 65 per cent-saturated ammonium sulfate fraction was determined on a model E analytical ultracentrifuge (Spinco). The protein concentration used in the experiments was 4 ~ 15 mg/ml in the first case and 266 mg/ml in the second . All measurements were made at 20° in a centrifugal field of 201,400 g. Crude sea urchin toxin [SUT (61)] was subjected to electrophoresis on polyacrylamide gel by the procedure of OxxsT~rr [l2] and DAVrs [13] . Toxin samples were prepared in an

Isolation and Cheracterimtion of Sea Urchin Toxin

11

acrylamide reagent sohrtion (3 per ant acrylamide, pH=6 "7) and this solution photopolymerized in small diameter glass tubes (0 "635 em dice. x 7 "62 em), each containing recently polymerized `spear' and `separator' gels (3 per cent acrylamide, pH=6"7 and 7" 5 per cent acrylamide, pH=8 "9, respectively). The total quantity of protein did not exceed 200 Ng/gel. Generally, seven toxin~ontaining gels and one without toxin were run simultaneously in a vertical position with the tubes suspended between two reservoirs of 0 "0178 M TRISglycine buffer at a constant current of 2 mA/gel. At 25° the runs usually required 25 to 30 min for maximum separation . After electrophoresis was completed the gels were removed from their containers, and six toxin-containing gels were stained with 1 per ant Amido black IOB in 7"5 per cent acetic acid. One toxin-containing gel and one containing no toxin were separately homogenized in sodium phosphate buffer (0"2 M) and the two filtered solutions injected intravenously into min. The effect of temperature on the time-course of inactivation of the-toxin [SUT (61)] was studied by subjecting aliquots of an assayed saline stock solution of toxin (0"2 mg protein/ml, pH=7 "0), each sealed in a glass container, to six temperatures : 40 "0, 42 "S, 45"0, 47" 5, 50 ~0, and 60 "0°, in a constant temperature water-bath. At temperatures between 42 "S and 60 "0°, inclusive, a container was withdrawn every 5 min up to a maximum exposure of 20 min. At 40° exposure time was extended to 1 hr, and the samples were withdrawn every 5 min to the 30 min point, and every 10 min thereafter . Each withdrawn sample was immediately transferred to an is-water bath and all solutions subsequently assayed for activity at a dose of 9"60 x 10-' mg N/kg (6 "03 LD as) . In those cases in which inactivation had occurred, the dose injected was increased to 19 "20 x 10-~ mg N per kg (12"07 LD6~ to assure that inactivation was indeed complete . Finally, the stability of the toxin as a function of pH was determined over a pH range of 4"3-10"6. Seven aliquots of an assayed saline stock solution of toxin (3"32 ing protein per ml, pH=7 "0) were each diluted with an equal volume of 0 "2 M sodium phosphate buffer solution at pH values of 4"3, 5 "0, 6 "0, 7 "0, 8"0, 9 "0, and 10"6, while a control aliquot was diluted with saline. The samples were incubated at 8° for 24 hr, and final pH determinations were made just prior to the assay for activity in mice . The toxin solutions were injected intravenously at a dose of 53 "00 x 10-' mg N/kg (33 "3 LD as) of mouse weight . PREPARATION AND PURIFICATION OF TOXIN Crude extract [SUT (61), SUT (64), and .SUT (64-2)] Sea water extraction . An extraction was carried out with the pediallarial

heads obtained from 310 sea urchins. The frozen mass of heads was homogenized with 4 vol offiltered sea water for 5 min in a Waring Blender, and the resultinghomogenate centrifuged at 10;000 g for 15 min. After the supernatant had been removed the sediment was twice resuspended in cold sea water, homogenized, and centrifuged as above. The supernatants from the three extractions were combined and stored at 5°, while the residue was dried to constant weight . The pooled supernatant was then dialyzed against distilled water for 18 hr at 5°, lyophilized, and the dried material stored at-20° for subsequent analysis . This extract was designated SUT (61). Distilled water extraction . An extraction of the pedicellarial heads from 100 sea urchins was carried out by the procedure outlined above using doubly distilled water in place of sea water as the extractint . The pooled supernatant was saturated with crystalline ammon ium sulfate. The precipitate was removed by centrifugation at 10,000g for 1 hr, dissolved

12

CHARLES B. ALENDER, GEORGE A. FEIGEN and JOSEPH T. TOMITA

in doubly distilled water and the resulting solution filtered and dialyzed first against 4 vol portions ofsodium phosphate buffer (E~=O ~Ol, pH=7 ~0) for 24 hr and then against doubly distilled water for 4 hr. The toxin dislysate was lyophilized, and the friable powder stored at -20°. This extract was designated SUT (64) . A second distilled water extraction of sea urchin toxin was made from pedicellarial heads obtained from 235 specimens by means of the technique outlined above. Since this material was to be used for subsequent fractionation it was reprecipitated with crystalline ammonium sulfate, exhaustively dialyzed against doubly distilled water, and then frozen before fractionation. This preparation was designated SUT (64-2) . The yields of dry material obtained from the 3 preparations are shown in Table 1 . TABLE I . YIELDS OF CRUDE SEA URCHIN TOXIN

Extraction Solvent No . Sea Urchins Yields (g dry woight) Alive extract Extract/animal Residue Residue/animal

SUT(61) Sea Water

SUT(64) Distilled Water

SUT(64-2) Distilled Water

310

100

235

1 ~22 0 012 3 38 0~03

1 ~68 0 007 6 ~75 0~03

3~64 0 012 16 ~17 0 ~OS

Carbon treatment

The sea water and distilled water extracts were colored. Several attempts were made to remove these contaminants by adsorption on carbon . Activated carbon (Darco G-60) was added to extracts of sea urchin toxin in a ratio of 10 mg of charcoal to 1 mg of protein . The mixture was stirred intermittently for 5 min at 25°, centrifuged at 10,000 g for 5 min, and the supernatant removed. The parent extract and the clarified material were .then compared with respect to their spectrophotometric and toxicological properties . Ammonium sulfate fractionation

Solutions of SUT (64) and (64-2) were fractionated by being brought sequentially to 033, 065, and full saturation with solid ammonium sulfate at room temperature. After each addition of salt the suspension was incubated for 1 hr at 37°, to induce coacervation, and then cooled to room temperature. The precipitate was separated by filtration and the filtrate was treated with the requisite amount of solid salt to bring it to the desired increment of saturation . The precipitates obtained at each step were dissolved in doubly distilled water, cleared of residual salt by exhaustive dialysis (over 40 hr) against several changes of distilled water, and dried from the frozen state . The relative effectiveness of sea water and distilled water as extractants can be judged only grossly by comparing the amount of extractable toxin obtained per animal in the two cases. The yield of active toxin per animal was 0 012 g by both methods of extraction, but the insoluble residue obtained overall by the sea water method was 53 per cent greater than that produced by the distilled water technique. It is not possible to judge the effectiveness of the two procedures by comparing the ratios of yield-to-residue because the distilled water extract was precipitated before the final lyophilization whereas the sea water extract was freeze-dried immediately after the last dialysis. Preparation SUT (64-2)

Isolation and Characterization of Sea Urchin Toxin

13

yielded about one-half the amount of toxin per animal obtained in the earlier preparation. Since this preparation was frozen before fractionation, it is possible that some of the active material was denatured by the maneuvers of freezing and thawing. The amount of protein precipitated at various ammonium sulfate concentrations is shown in Table 2 for SUT (61) and SUT (64) . The results show that 68 per cent of the Txate 2.

AMMONNM SULFATE FRACRONATION OF

Conc . (NHJ,SO, (% saturation) SUT(61)

20 40 60 80 100

0"S g s,~eteLSq oF sE~ vRCgmv Toxiv Amount of protein precipitated (~ of original wt) (e) 0 "1200 0 "1281 0 "0303 0 "0233 0"0343

24 "4 25 "8 S "90 4 "68 6 "82 Total

SUT(64)

33 63 100

0"0418 0"1295 0"0104

67 "70 8 "36 23 "90 2-08

Total

36 "34

Unprecipitable material from SUT(61) at full saturation amounted to 0"0051 g or 1 "10 ~ of crude weight .

original weight of SUT (61) was recovered in cumulative precipitations between 20 and 100 per cent saturation with ammonium sulfate. Preparation SUT (64) was fractionated by 3 cumulative saturations with ammonium sulfate instead of five, as in the case of SUT (61) . In the latter preparation 36 per cent of the original weight of material was recovered by precipitation, with the major portion precipitating when the solution was 65 per cent saturated with ammonium sulfate. ANALYTICAL PROPERTIES OF TOXIN PREPARATIONS Protein content

The total and precipitable N contents of the two major preparations are exhibited in Table 3 along with their corresponding specific optical densities at 278 m~. Neither of the lyophilized preparations was completely soluble in 1 per cent NaCI solution. Based on 1 mg of dry weight the ratios of precipitable to total N of the soluble material were found to be 86 and 87 per cent for SUT (61) and (64) respectively, and the corresponding extractable protein contents were 29 and 58 per cent of the dry weight for the earlier and later preparations, respectively. In comparing SUT (64) and (64-2) the greatest yield of extractable protein was in both cases found in the fractions precipitating at 65 per cent saturation with ammonium sulfate. The ratios between the 65 per cent and 33 per cent fractions were 2 "6 and 3 "7 in the case of preparations SUT (64) and (64-2), respectively. The 65 per cent fractions had a significantly lower quantity of insoluble residue and in both cases the specific absorption at 278 m~ was higher in the 65 per cent than the 33 per cent frâction .

14

CHARLE.S H . ALSNDi3R, Ci130R(313 A. FSIC3IBN aad JOSEPH T. TOMITA TeetB 3 .

NITROCiSN

ANALYSIB

I~IUßAA1s OF ~A URCSRQ 70XIIV FHBFAitATi01~

YBR

Solubb fractiont

Sediment

N/dry toxin (mg)

P/T

OD , of soluble extractl

0 " 0114

0 " 0653

70 " 83

1 "067

0 "584 0 " 184 0 "486 -

0 "0141 0 " 0434 0 " 0180 -

0 "1214 0 " l 100 0 " 1269 0 " 1050

77 "02 26 "40 61 " 23 -

2 "020 1 "255 1 " 390 1 " 161

0 " 159 0 " 589 0 "339

0 " 0422 0 " 0095 0

0 " 1187 0 " 1260 0 " 1609

21 "40 74 " 76 33 "7S

1 " 301 1 "694 1 "030

Malarial Prep " analysed

Total N Pptbla N (m8J (mg)

PjT

61

Parant

0-0541

0 "0464

85 "77

0 "290

64

Parent 33 ~ 65~ 100

0 " 1073 0 "0676 0 " 1089 -

0-0935 0 "0294 0-0777 -

87 " 14 43 "34 71 "35 -

33~ 6S~ 100~

0 "0766 0 " 1166 0 " 1609

0 "0254 0 "0942 0 " 0543

33 " 16 ~ "79 33 "75

64-2

Whole preparation

prat "/dry Total N toxin (mBJ (m~

~N x 6 "25 ti3xtraction of 1 ~ dry material with 1 ml l ~ NaCI

Light absorption

Absorption curves of the crude extract [SUT (61), 1 mg dry weight/ml] before and after carbon treatment are shown in Fig. l . Theparent material has absorption maxima at 278 m~. and at 323 "6 m~. The effect of treatment with carbon was to reduce both peaks. This change probably reflects a simple loss by absorption on carbon of one or more constituents that absorb at these wavelengths.

i'aV .

1 . ULnuvsot$r AHSOAP'rfL1N aFßCl"RA OF CAUDS At~ CARHON1rABATBD ~A UaCHn41~O)ON ~~svr(61) ; H-svr(61), ra8at~

SUT (64)

SUT (64}--65 ~ a3

Pi(3. i. ULTRACBNTRIFUOB PATTERNS OF CRUDE SBA URCHII3 TOXIN AND AN AAß~fONIUM SULFATE FRAGTION. (LBPC TO RIßHT)

Ieolstion and C~aracteri7sdon of Sea Urchin To:;n

1S

Ultracentrifugation

Records are shown of ultracentrifugation patterns of the parent material and the 65 per cent ammonium sulfate fraction, each made up to an initial concentration of .20 mg of dry material/ml in 1 per cent NaC1 . It is evident from Fig. 2 that the principal difference between the parent material and the fraction is the elimination of the 6 "2S component by fractionation leaving only a 2 ~6S component. It appears from this determination that the active toxin is a relatively small molecule . Gel electrophoresis

Figure 3 shows that the parent toxin can be separated into 6 components on an acrylamide gel. The migration pattern was obliterated by diSi~sion during the 2 hr period

Fx~. 3.

Dm~rRIC TRACE OF AN ON ACAYLAI®S CiBt .

PATT®tN OF ®A URCSRQ 1bXRi

required for staining and clearing of a reference gel, even though gels containing venom were maintained at a low temperature ; for this reason assignment of activity to one or more of these components could not be made . The electrophoresis procedure, however, caused no detectable change in potency between the originally applied toxin sample and that removed from the gel by homogenization in phosphate buffer . BIOLOGICAL POTENCY OF TOXIN PREPARATIONS

The effects of temperature on SLIT (61) are illustrated by the results given in Table 4. The effects of time and treatmént will become apparent by a discussion of the data given in Table 5.

l6

CHARLES B. ALENDER, QEORaE A. FEIQEN and 30SEPH T. TOMITA TABLE

4.

THE STAHII.ITY OF 9EA URCHIN TOXIN

Temperature (°C)

SUT(61) AS

A FUNCTION OF TEMPERATURE

Exposure time (min) 0 -~ -~ -i-f-I-1-

40-0 42 "5 45-0 47 "S 50 "0 60 "0

S -I-~ + -

10 -~ ~-~ _

15 -l-I_

20 ~-

2S ~-

30 -I-

40 ~-

50 -i-

60 -

-

-l= active at dosag~e of 9 "60 x 10- ' mg N/kilo (6 ~03 LD,~ - = inactive at dosage of 19 " 20 x 10 - ' mg N/kilo (12-07 LD,~ TABLE S .

SPECIFIC HIOLOOICAL POTENCY OF SEA URCHIN TOXIN PRFPARATION9

No. Mice

LD Protein/20 g mouse x l0~mg

LD /mg Protein

SUT'(61~61 SLrI'(61}64 Carbon

40 47 45

11 "06 19 "88 17 "SO

903 S03 S71

SUT'(64) Carbon 33 ~ as (Fr 1) 6S ~ aa (Fr ld] 100~ as (Fr IIi)

33 42 9 2S 24

12 "88 777 1800 556 Non-toxic at 100 LD  457 21 "88 211 27 "50

SUT(64-2) 33 ~ as (Fr n 6S ~ as (Fr >n 100~ ee (Fr l0>)

27 40 38

Toxin Preparation

117 "SO 14 "SO SS "50

85 690 180

as=ammonium sulfate Effect ofpH

Experiments carried out as described under `Methods' showed no detectable loss in potency of preparations maintained for 24 hr in the cold at pH values ranging from 4 "3 to 10 "6. Effect of temperature

The interaction of incubation time and temperature upon the inactivation of toxin is apparent from the results displayed in Table 4. The critical point for 20 min inactivation of 6 LD6 u doses appears to lie between 42 "5 and 45 "0°. At 45 "0° full inactivation was achieved after 15 min of incubation, and at temperatures of 47 "5° the toxicity was destroyed in 5 min. Effect of storage time

The sea urchin toxin prepared in 1961 [SUT (61)] and stored continuously at -20° was reassayed in 1964 after 39 months of storage. By reference to Table 5 it is evident that the specific activity declined by about 44 per cent-from 903 to 503 LD s ~/mg-wring that interval .

]isolation and Characterization ofSea Uranhin Toxin

17

E~`ect of carbon treatment The usefulness of carbon as a decolorizing agent is offset by the unpredictability of its effect upon the activity of the toxin. Although there was a significant change in the absorption spectrum as shown in Fig. 1, the treatment resulted in the increase of specific activity of SUT (61) from 503 to 571 LD 6o/mg but a decrease from 777 to 556 in the case of SUT (64) . Fractionation with ammonium sulfate Preliminary tests carried out on 5 fractions of SiJT (61) showed that the preparations possessing the highest activity were those obtained at ammonium sulfate concentrations corresponding to 40 and to 60 per cent saturation. Owing to the nature of the preparative details involved in the isolation of SUT (64`2) (i.e. the elimination of the lyophili7ation step before fractionation) the potency of the starting material was not estimated; hence, the change in activity resulting from fractionation can be assessed only among the fractions and not between each fraction and the parent extract. In the case of SUT (64) an aliquot sample of the lyophilized powder was taken for bioassay making it possible to compare the activity of fractions both with respect to that of the starting material as well as to that of other fractions in the set. The specific potency of SUT (64) was 777 LD se/mg ; none of the fractions pre~red from it had an activity of that order; the most active pre~ratio~Fraction II had a potency amounting to less than 60 per cent of that shown by the parent material. The most active fraction in either set was the one precipitating at 65 per cent salt saturation . The least active fraction was precipitated at 33 per axnt saturation which, in the case of SUT (64), was inactive in a dose amounting to 100 LD aa

[1] [2] [3] [4] [S] [6] [7] [8] [9] [10] [11] [12] [13]

REFSRBNCBS Mo~~r~ora, T., A Monograph of the Echhioldea. Copanhaeen : Reitzel,1943, Vo13, Pt 2, pS53 Fwiw~, T., Atatot . Zool.,Ja~my 1S, 62,1935 . Ai srro~, C. H., irh.D. Thesis, Univ. of Hawaü,1964, (DLssertatlon Abstra. 2S, 3023). I3~, V. and lKAYALOF, E., C. r. soc. blol., Paris, 60, 884,1906 . P~, J., Arch. Zool. F.xp . Gen., 86,118,1950 . L~v~r, R., C. r. Soc. blol., Parls,181, 690,1935 . M~es, B. a., AHSim, L., aad Uaan, S., Sdence,139, 408,1963. F~as~, C3. A., Veuasa~x Wnii"va , lü. M., PsrB