Toxiovn, 1973, VoL 13, pp. 31-3ti . Perpmon Press. Printed in Great Britain.

ISOLATION AND PARTIAL CHARACTERIZATION OF PELAMITOXIN A FROM PELAMIS PLATURUS VENOM* C.-S. Lu, C.-L. WANG and R. Q. BLACKWELLt

Department of Biochemistry, U.S. Naval Medical Research Unit No. 2, Taipei, Taiwan, Republic of China (Acceptedfor publication 30 July 1974) C.-S . Llu, C:L. WaNa and R. Q. Bt.pcxwt=,u.. Isolation and partial characterization of pelatnitoxin a from Pelamis platurus venom. Toxicon 13, 31-36, 1975 .-The principal toxin, named pelamitoxin a, was isolated from venom of the sea snake Pelamis platurus caught in Northern Formosan waters . Pelamitoxin a is a polypeptide containing 60 amino acid moieties, including 9 cysteines, with N-terminal metllionine and C-terminal asparagine. Its amino acid composition appears to be identical to that of hydrophitoxin b (Llu and BL~cgwEl l., Toxicon 12, 543, 1974) from venom of the sea snake Hydrophis cyanocinctus as well as schistose 5 toxin from venom Enhydrina schistose (FttYxlvrm et al., Biochemistry 11, 4633, 1972). These results indicate excellent prospects for a rational approach to production of a common antivenin effective against major toxins from several of the common sea snakes . INTRODUCTION

snake Pelamis platurus is known to be widely distributed in regions of the Pacific and Indian oceans extending from the west coast of Central America westward to the east coast of Africa and from southeastern Siberia southward to Tasmania (HALSTEAD, 1970). Pelamis platurus, commonly called the yellow-bellied sea snake, belongs to the subfamily Hydrophünae of the family Hydrophüdaes (HÀLSTEAD, 1970). Chemical studies of toxins from several sea snakes in the Hydrophünae subfamily including Enhydrit:a schistose, Hydrophis cyanoclnctus, and Lapemis hardwickü have been reported recently (KA1tISSOrr et al., 1972 ; F~Ytci tnvn et al., 1972 ; LIU et al., 1973 ; Tu and HoxG, 1971 ; Lu and BLACI{wsLL, 1974) but only a preliminary report has appeared concerning Pelamis platurus (SHIPMAN ând PICRWELL, 1973). The present study describes isolation of some toxins from Pelamis platurus venom and partial chemical characterization of the major toxin which we have called pelamitoxin a. Tt1E sEa

MATERIALS AND METHODS

Specimens of sea snakes Pelmnis platurus were captured by Chinese fishermen from northern Formosan waters . Crude venom was obtained either by milking or by extraction with 0"1 M acetic acid from minced venom glands . For fractionation purposes, venom obtained by milking was used directly after lyophilization whereas venom material obtained by extraction was partially purified prior to further use by chromatography with 0"2 M ammonium acetate through a 3" 5 x 64 cm Sephadex G-50 column (KARr.ssox et al., 1972). 'This work was supported by the Bureau of Medicine and Surgery, Department of the Navy, Washington, D.C . The opinions and assertions contained herein are those of the authors and are not to be construed as official or reflecting the views of the U.S . Navy Department or the U.S. Naval Service at large . tPublications Office : U.S . Naval Medical Research Unit No . 2, Box 14, APO San Francisco 96263, U.S .A. 31 710XICON 1975 Yol. I3

32

C.-S. LIU, C:L. WANG and R. Q. BLACKWELL

Lyophilized crude venom was subjected to Whatman CM52 column chromatography in ammonium acetate solution. For example, 70 mg crude venom was applied to the Whatman CM52 column, 0~9 x 23 cm, and separated using a linear gradient produced with 150 ml each of 001, 005, 009, 013, and 0'17 M ammonium acetate solutions at pH 6~5 . Flow rate was approximately 28 ml per hr. Column ei$uent was monitored with an ISCO UAW absorbance monitor at 280 nm. The major fraction was checked for homogeneity by cellogel electrophoresis as previously described (Liu et al., 1973) . LDsu of the major toxin, pelamitoxin a, was determined by injecting intraperitoneally 0~1 Inl of appropriate amounts into 20 g white mice. Six mice were used for each dilution and numbers surviving in each group were observed after 22 hr (RED and MUENCH, 1938) . Reduction and carboxymethylation of pelamitoxin a was carried out by the method of CÀESTFIELD et al. (1963) . Amino acid determination of reduced and carboxymethylated toxin and its tryptic peptides, peptide mapping, as well as N-terminal and C-terminal amino acid determinations were performed essentially as described previously (Lu et al., 1973). Tryptophan content was measured according to the method of GATTONDE and Dovsx (1970) . Quantitative determination of free sulfhydryl groups in pelamitoxin a was made with 5,5'-dithiobis (2-nitrobenzoic acid), DTNB, by the method Of ELLrtAN (1959) as modified by FERNANDEZ D~z et al. (1964) . One mg of pelamitoxin a was dissolved in 10 ml of008 M sodium phosphate buffer, pH 8~0 . To 3 ml of the resulting solution, 60 mg sodium dodecylsulfate was dissolved followed by 0~1 ml of DTNB solution made with 4 mg DTNB dissolved in 1 ml of 0'1 M sodium phosphate buffer, pH 8~0. After 10 min of reaction at room temperature, the reaction mixture was tested for absorbance at 410 nm. Amino acid sequences were determined by Edman degradation as modified by P~eTSxsoN et al. (1972) . The resulting PTH-amino acids were identified by thin layer chromatography with Eastman No. 6060 silica, gel chromatogram sheets using the chromatographic systems of INAGAMI and MURAKAMI (1972) and J~pssox and SJöQuisr (1967) . RESULTS AND DLSCUSSION

The purified major toxin from venom of Pelamis platurus, named pelamitoxin a, was obtained by column chromatography. For example, from 70 mg of dried crude venom O.a

O.A

N ô0.2 Ô O. I

0.0~

~., 10' 2

65

70

80

50

100

Fraction Number (I Fraction=3.7m1)

I10

-L IIS

I . FYtACrIONATION OF PGIamtS platurus VENOM BY CARBOXYMEI'HYLCELLULOSE COLUMN CHROMATOGRAPHY W1TH A LINEAR CONCENTRATION GRADIENT OF AMMONIUM ACETATE AT pH 6~5. FYO.

Pelamitoxin awas obtained from the peak emerging between fractions 97 and 104.

TOXICON 1975 YoL 13

f ~., _. ~ -- T

.~ .~'R.~a:~.ti t S~

~F

1~

~tït~,~

~

~f t!:

!

.

~ .

;.

_

N

FIG. T. PATTERNS OBTAINED HY CELLOGEL ELECTROPHORESIS OF SAMPLE3 IN O'OS M AMMONIUM ACETATE BUFFER, pH 6~5, AT 2SO VFOR 3O MIN.

Crude venom obtained by milking Pelamis platurus (bottom) is compared with pelamitoxin a (top) which was obtained by column chromatography in fractions 97-104 as illustrated in Fig. 1.

FIG. 3. MAP OF PEPTII)E3 PRODUCED BY TRYPTTC DIGESTION OF PELAMITOXIN a. HLACKWELL,

Comparison with peptide map of hydrophitoxin b (in Fig. 3, Llu and indicates the two patterns are identical.

TOXICON 1975 YoI. 13

1974)

f.p. 32

Pelmnis platunrs toxin

33

obtained from live snakes, 12 mg (17 per cent) of pelamitoxin a was recovered by column chromatography using CM-52 carboxymethylcellulose and the ammonium acetate buffer system at pH 6"5. Results are illustrated in Fig. 1 ; pelamitoxin a comprised the major component which emerged from the column approximately between fractions 97 and 104. As shown in Fig. 2, only one band appeared when this material was examined by cellogel electrophoresis . Larger amounts of pelamitoxin a for analysis were obtained by acetic acid extraction of dissected venom glanda . For example, 412 mg dried crude venom, obtained by extraction of dissected glands from approximately 180 snakes, was dissolved in 0"2 M ammonium acetate and passed through a Sephadex G-50 column . The resulting main desalted fraction containing toxin weighed 130 mg. When 100 mg of that material was passed through the CM-52 carboxymethylcellulose column as described above, 35 mg of pelamitoxin a was recovered. The i.n6o of the pelamitoxin a component was 1 "4 ltg per 20 g mouse as compared to 3~61tg per 20 g mouse reported for crude venom of Pelamis plattuvs (Tu and GANTHAVORN, 1969) . Amino acid composition of pelamitoxin a is shown in Table 1 and the composition of its tryptic peptides are summarized in Table 2. With due regard for overlapping peptides, TAHLS

Amino acid residue CM-cys Asp Thr Scr Glu

Pro

Gly

Ala Val Met Ile Leu Tyr Lys His Arg Trp

1. AüffNO

ACII~ OO~rION OF PELAI~flTOX1N

nmoles 106 74 84 71 100 23 45 12 9"2 8"5 19 11 11 62 24 33

Molar ratio 8"8 6"I 7~0 5"9 8ß 1"9 3"7 1~0 0"76 0"70 1 "6 I "0 I "0 5" 1 2"0 2~7

a

Nearest integer 9 6 7 6 8 2 4 1 1 1 2 1 1 5 2 3 l'

'Tryptophan content was estimated spectrophotometrically by the method of GArroNDE and Dov$Y (1970) ; the amount found was 0"8 moles per mole of toxin.

the sum of amino acid components of the tryptic peptides agrees with the composition of the complete molecule which appears to contain 60 amino acid moieties. The pattern of tryptic peptides from pelamitoxin a, shown on the peptide map in Fig. 3, was quite similar to that reported previously for hydrophitoxin a (LIU et al., 1973) and identical to that of hydrophitoxin b (LN and BLACICWELL, 1974). Tryptophan content of pelamitoxin a, determined by the method of GAITONDB and Dovsr (1970), was found to be approximately 0"8 mole per mole of pelamitoxin a. The C-terminal amino acid of pelamitoxin a was asparagine which also occupies that position T10XICON 1975 Vol. 13

34

C.-S. LIU, C.-L . WANG and R. Q. BLACKWELL

TABLE 2. COMPARISON OF NUMBSR3 OF AMIIJO ACIDS IN PELAMITOXIN

Amino acid Pelamitoxin residue a CM-cys 9 Asp 6 Thr 7 Ser 6 Glu 8 Pro 2 Gly 4 Ala 1 Val 1 Met 1 Ilc 2 Leu 1 Tyr 1 Lys 5 His 2 Arg 3 Trp 1 Total 60

T-1 3 3 1 2

(T-2) 2

1 3 2 1 1

1 1 11

1 1

T-3 2

1 1 2 1

1

a

WTIH THOSE IN IT3 TR .YPIIC PEPI~ES

Tryptic peptides' T-4 T-5 T-6 T-7 2

l 1 2 3 1

2

1 3 2 1

1

1 1

8

+T-2 ~- Lys = T-5; T-11 contained only lysine . tPositive to Ehrlich stain .

12

(T'-11) T-12

1

1

1

1

1

1 1 2 1

(12)

1 1 1

T-8

13

~3

1 1 1 lt 6

1

1

(1)

4

1 3

in numerous other snake toxins ; methionine was found at the N-terminal position. Amino acid sequence studies of pelamitoxin a currently are in progress . At present only the initial seven positions of the N-terminal region have been identified : Met-Thr-Cj~s-Gds-AsnGln-Gln. This sequence is identical to that reported in toxins 4 and 5 from EJ :hydrina SChlStOSa

(FRYRLUIVD

et al., 1972) .

Since amino acid analysis results indicated nine cysteine residues in the pelamitoxin a molecule, at least one cysteine residue should be unpaired and have a free sulfhydryl group. This was verified when the free sulfhydryl group content of pelamitoxin a was determined by the method Of FERNANDEZ DrEZ et al. (1964) . Assuming molar absorbance of 13,600 for the reaction product and a molecular weight of 7000 for pelamitoxin a, it was found that each mole of the toxin contained 095 moles of free sulfhydryl group. The remaining 8 cysteine residues presumably are linked into 4 pairs by disulfide bridges. From the present results it appears that pelamitoxin a may be identical in structure to hydrophitoxin b. As mentioned above the peptide maps for the two toxins are identical and the amino acid compositions of respective tryptic peptides from the two toxins are identical (compare present Table 2 with Table 1 in Liv and BLACKWELL, 1974) . It was reported previously (Liu and BLACKWELL, 1974) that the amino acid compositions ofhydrophitoxin b and schistosa 5 toxin from Enhydrina schistosa are identical ; therefore, there now are three toxins, one each from three sea snakes in the Hydrophünae subfamily, known to have identical amino acid compositions . These and other relationships are summarized in Table 3. Further sequence studies are required for hydrophitoxin b and pelamitoxin a to determine whether one or both are identical to schistosa 5. The extremely similar chemical structures of major toxins from several of the more common sea snakes as outlined above indicate excellent prospects for rational production of effective common antivenins (OICONOGI et al., 1972) . TOXJCON 1975 VoL J3

Pelamis platurus toxin

a000OOd'1~Nh

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TOXlCON 1975 Vol. J3

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36

C:S. LIU, C:L, WANG and R. Q. BLACKWELL

Acknowledgement-We thank Miss Jaxe Y.-O. HvNa for amino acid analyses . REFERENCES CAREY, J. E. and Wtuct~, E. A. (1960) The toxicity and immunological properties of some sea-snake venoms with particular reference to that of Enhydrina schistosa. Traps. R. Soc. trop . Med. Hyg. 54, 50 . C1tESCIr[ELD, A. M., Moor, S, and STtart, W. H. (1963) The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins . J. biol. Chem. 238, 622. Et .t .nfax, G. L. (1959) Tissue sulfhydryl groups. Archs Biochem. Biophys . 82, 70. FERNANDEZ DI>;z, M. J., Osvca, D. T. and FEexev, R. E. (1964) The sulfhydryls of avian ovalbumins, bovine ß-lactoglobulin and bovine serum albumin. Archs Biochem. Biophys. 107, 449. FRYRLUI~, L., Ewxex, D. and Kwtus4otv, E. (1972) Amino acid sequences of the two principal ncurotoxins of F.nhydrina schistosa venom. Biochemistry 11, 4633 . CAITOt~E, M. K. and DOVEY, T. (1970) A rapid and direct method for the quantitative determination of tryptophan in the intact protein. Biochem. J. 117, 907. Hntst~wn, B. W. (1970) Poisonous and Venomous Marine Animals ojthe World, Vol . 3, p. 617. Washington, D.C . : U.S. Government Printing office . hvna.~nu, T. and MuxntutNt, K. (1972) Identification of phenylthiohydantoins of amino acids by thin-las°er chromatography on a plastio-backed silica-gel plate. Analyt . Biochem. 47, 501 . JEPP330N, J.-O. and S.ttiQvtsT, J. (1967) Thin-layer chromatography of PTH amino acids. Analyt. Biochem . 18, 264. Kno, C,-S ., Ltu, C: S, and Br nctcwt:rt, R. Q. (1973) Presence of Erabutoxins a and b in venom of the sea snake Ltrticauda semifasciata from Taiwan . Toxicon 11, 383. Kaxt .ssort, E., Enxex, D., FRYKLUND, L. and Kantx, S. (1972) Chromatographic separation of Enhydrina schistosa (common sea snake) venom and the characterization of two principal neurotoxins . Biochemistry 11, 4628 . Ltu, C.-S . and Bt,nctcwet,t., R. Q. (1974) Nydrophitoxin b from Hydrophis cyanocinctus venom . Toxicon 12, 543. Ltu, C.~ ., HUIiER, G. S., Lttv, C.-S. and Bt,ncxwta.t,, R. Q. (1973) Fractionation of toxins from Hydrophis cyanoci'nctus venom and determination of amino acid composition and end groups of hydrophitoxin a. Toxicon 11, 73 . Oxoxoct, T., HwTrotu, Z., AtN.act, E., Snwnt, Y. and Kawaravxa, Y. (1972) Studies of immunity against the venom of Laperais hardwicki. The Snake 4, 84. Prrsttsort, J. D., NetmLtcx, S., DYER, P. E. and $71~NER, D. F. (1972) Determination of the amino acid sequence of the monkey, sheep, and dog proinsulin C-peptides by a semi-micro Edman degradation procedure. J. biol . Chem. 247, 4866 . Reso, L. J, and MttExcx, H. (1938) A simple method of estimating fifty per cent endpoints. Am . J. Hyg. 27, 493. SwTO, S ., YosHmn, H., .AHe, H. and TnnttYn, N. (1969) Properties and biosynthesis of a neurotoxic protein of the venoms of sea snakes Laticauda laticaudata and Laticauda colubrina. Biochem. J. 115, 85 . SHIPMAN, W. H. and Ptcxwet.t., G. V. (1973) Venom of the yellow-bellied sea snake (Pelamis platurus) : some physigl and chemical properties . Toxicon 11, 375. TAMIYA, N. and Meat, H. (1966) Studies on sea-snake venoms . Crystallization of Erabutoxins a and b from Laticauda semifasciata venom. Biochem. J. 99, 624. TAbtIYA, N. and AaE, H. (1972) The isolation, properties and amino acid sequence of Erabutoxin c, a minor neurotoxic component of the venom of a sea snake Laticauda semijasciata, Biochem . J. 130, 547, Tu, A. T, and Gaxrxevoteta, S. (1969) Immttnological properties and neutralization of sea-snake venoms from Southeast Asia. An:. J. trop . Med. Hyg. 18, 151 . Tu, A. T. and Hoxc, B:S. (1971) Purification and chemical studies of a toxin from the venom of Lapentis hardwickü(Hardwick's sea snake) . J. biol. Chem. 246, 2772 . Tu, A. T., Hortc, B.-S. and Sot.tE, T. N. (1971) Characterization and chemical modifications of toxins isolated from the venoms of the sea snake, Loticauda semifasciata, from Philippines. Biochemistry 10, 1295 . Y.+rtc, C. C., CrtatJC, C. C., HAYASHI, K. and Suzuxt, T. (1969) Amino acid composition and end group analysis of cobrotoxin . Toxicon 7, 43 .

TOX/CON 1975 Vol. IJ