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Detection of erabutoxins in the venom of sea snake Laticauda semifasciata from The Philippines
199 Biochimica et Biophysica Acta, 532 (1978) 199--201 © Elsevier/North-Holland Biomedical Press
BBA R e p o r t BBA 31236 DETECTION OF E R A B U T O X I N S IN THE VENOM OF SEA SNAKE LA TICA UDA S E M I F A S C I A T A F R O M THE PHILIPPINES
NOBUO TAMIYA and CHIKAHISA TAKASAKI Department of Chemistry, Tohoku University, Aobayama, Sendai 980 (Japan) (Received August 29th, 1977)
Summary Erabutoxins a, b and c were detected in the venom of sea snake Laticauda semifasciata from The Philippines.
Erabutoxins a and b are the main neurotoxic components of the venom of the sea snake Laticauda semifasciata from Japan [1]. Both of them consist of 62 amino acid residues. Their original results of amino acid analysis were corrected for the presence of another serine residue (8 instead of 7) by the results of shorter period (12 h) of hydrolysis [2]. Erabutoxin c is a minor toxic c o m p o n e n t in the same venom [ 3 ]. The amino acid sequences [3,4] and the positions of disulfide bridges [5] of erabutoxins were elucidated. The presence of serine and glutamic acid at positions 21 and 22 w a s corrected to glutamic acid and serine b y Maeda and Tamiya [6]. The three dimensional structure of the peptide chain of erabutoxin b was clarified [ 7]. On the other hand, Tu et al. [8] isolated two main toxic components from the venom of L. sernifasciata from The Philippines by the same method as the above authors and named them toxins a and b. The only differences between erabutoxins a and b and toxins a and b were their amino acid compositions: a consisted of 62 and b of 61 amino acid residues. Tsernoglou and Petsko [9,10] presented the three dimensional structure of the peptide chains of toxins a and b. The present study was carried out to examine the possible identity of toxins a and b with erabutoxins. Sea snakes (L. sernifasciata) were collected at Gigantes Islands, The Philippines, during the Visayan Sea Expedition on the research vessel Alpha Helix of the Scripps Institution of Oceanography, in September, 1975. The methods of isolation of erabutoxins, reduction, S-carboxymethylation and tryptic digestion of erabutoxin b, separation of the resulting tryptic frag-
200 ments, and the amino acid analysis of the toxins and the fragments were the same as described previously [1,4]. The whole profile of the elution pattern from the CM-cellulose column of the venom gland extract of sea snake L. semifasciata from The Philippines was the same as that from Japan. In addition to erabutoxins a, b and c, L. semifasciata III [ 11] and other components were also detected. The results of amino acid analysis of erabutoxins from L. semifasciata from The Philippines are shown in Table I. The results by Sato et al. [2] and Tu et al. [8] are also shown for comparison. The results of amino acid analysis of the tryptic fragments from reduced and S-carboxymethylated erabutoxin b are shown in Table II. The Gigantes Islands, where the snakes were collected, are located at less than 100 km to the west of Gato Islands, where Tu collected his snakes [ 8 ] , and about 2000 km away from Okinawa Islands, Japan, where Tamiya and Arai [1] collected their snakes. It is concluded, therefore, that the same erabutoxins are present in the venoms of L. semifasciata from Japan and from The Philippines, and that there are no subspecies as far as the venom components are concerned. The authors propose to use the original names of erabutoxins a, b and c to avoid further confusion. The work was supported by NSF grants DES 74-24129 from Pennsylvania State University and OFS 74-02888 and OFS 74-01830 from the
TABLE I A M I N O A C I D C O M P O S I T I O N O F E R A B U T O X I N a. b A N D c O F S E A S N A K E L S E M I F A S C I A T A FROM THE PHILIPPINES T h e values axe given in m o l a m i n o a m d / m o l p r o t e i n in 24 h h y d r o l y s a t e . Erabutoxin a Integer valuer Asp Thr SertT Glu Pro
Gly V~ Cys** Val Ue¶ ¶ Leu Tyr Phe Lys H~s Arg Trp Total
5.0 4.9 7.9 8.1 4.2 5.0 7.4 2.1 3.8 1.1 0.9 2.0 3.9 1.1 3.0 *
Erabutoxin b Toxin a¶
5 5 8 8 4 5 8 2 4 1 1 2 4 1 3 1
5 6 7 8 4 5 8 2 4 1 1 2 4 1 3 1
62
62
Integer value'[" 4.0 4.9 7.8 8.2 4.1 5.0 6.0 2.0 3.1 1.1 1.0 1.9 4.0 2.1 3.0 *
Erabutoxin c Toxin b¶
4 5 8 8 4 ,5 8 2 4 1 1 2 4 2 3 1
4 5 6 8 4 6 8 3 4 1 1 2 5 1 2 1
62
61
Integer value § 5.0 4.8 7.7 8.4 4.3 5.0 7.2 1.9 3.3 1.1 1.1 2.0 2.7 1.9 2.9 *
5 5 8 8 4 5 8 2 4 1 1 2 3 2 3 1 62
t T a k e n f r o m S a t o e t al. [ 2 ] . ¶ T a k e n f r o m T u e t al. [ 8 ] . § T a k e n f r o m T a m i y a and A b e [ 3 ] . ~ " C o r r e c t e d for d e s t r u c t i o n by 12 h h y d r o l y s i s values. * Not determined. * * C y s t i n e t e n d s t o give s m a l l values. ¶ ¶ I s o l e u c i n e t e n d s to give l o w values due to t h e p r e s e n c e o f Ile-Ile s e q u e n c e .
201
TABLE II AMINO ACID COMPOSITION OF TRYPTIC PEPTIDES FROM REDUCED AND S-CARBOXYMETHYLAT E D ERABUTOXIN b FROM L. S E M I F A S C I A T A FROM THE PHILIPPINES The values show molar ratio of amino acids in 24 h hydrolysate. The names of the peptides and their amino acid c o m p o s i t i o n (figures in parentheses) are t a k e n from Sato and Ta mi ya [ 4 ] . T-1 Cmc Asp Thr Set GIu
2.4 (3) 2.0 (2)
Total
T-2b
1.8 (2)
T-4a
T-4b¶
1.0t(1) 1.0 (1)
1.1 (1)
T-3 1.6 (2) 0.9 (1) 2.9 (3) 1.0t(1)
1.9 (2) 2.2 (2)
Pro Gly Val Iie Leu Tyr Phe Lys His Arg Trp
T-2a
2.3 (2) 3.0"F(3) 0.9 (1)
1.0 (1) 0.9 (1) 1.0 (1)
0.8 (1) 1.3 (1)
1.1 (1)
T-5
0.7 (1) 1.0"I"(1) 1.8 (2) 2.1 (2) 3.1 (3) 1.3 (1)
1.01"(1)
0.8 (1)
1.7 (2)
0.6 (1)
1.0t(1) 0.9 (1) 0.9 (1) 1.2(1) 1.2 (1)
1.1 (1) 2.1 (2)
1.3 (1) 1.0 (1) 1 (1)
11
12
1
12
0.9 (1) 0.9"(1)
1.0 (1)
6
6
14
t The n umb ers of these amino acids axe t a k e n as standard. The acid h ydro lysis was carried o u t for 60 h. * The acid hydrolysis was carried out in the presence of 4% (v/v) thloglycolhc acid [ 1 2 ] .
Scripps Institution of Oceanography for operation of the R/V Alpha Helix. It was supported also by the Yoshida Science Foundation for the travelling expenses to join the program. The authors are grateful to the members of the Visayan Sea Expedition (Chief Scientist Dr. W.A. Dunson) of the research vessel Alpha Helix for their co-operation in the collection of sea snakes.
References 1 2 3 4 5 6 7 8 9 10 11 12
Tamiya, N. and Arai, H. (1966) Biochem. J. 9 9 , 6 2 4 .--~630 Sato, S., Yoshida, H., Abe, H. and Tamlya, N. (1969) Biochem. J. 115, 85--90 Tamiya, N. and Abe, H. (1972) Biochem. J. 130, 547---555 Sato, S. and Tamiya, N. (1971) Biochem. J. 122, 453--461 Endo, Y., Sato, S., Ishii, S. and Tamiya, N. (1971) Biochem. J. 1 2 2 , 4 6 3 - - 4 6 7 Maeda, N. and Tamiya, N. (1977) Bloehem. J. 167, 289--291 Low, B.W., Preston, H.S., Sato, A., Rosen, L.S., Seasl, J.E., R udko, A.D. and Richaxdson, J.S. (1976) Proc. Natl. Acad. Sci. U.S. 73, 2 9 9 1 - - 2 9 9 4 Tu, A.T., Hong, B.S. and Solie, T.N. (1971) Biochemistry 10, 1 2 9 5 - - 1 3 0 4 Tsernoglou, D. and Petsko, G.A. (1976) FEBS Lett. 68, 1---4 Tsernoglou, D. and Pets]co, G.A. (1977) Proc. Natl. Acad. SeL U.S. 74,971---974 Maeda, N., Takagl, K., Tamiya, N., Chen, Y.M. and Lee, C.Y. (1974) Biochem. J. 1 4 1 , 3 8 3 - - 3 8 7 Matsubaxa, H. and Sasakl, R. (1969) Biochem. Biophys. Res. Commtm. 35, 175--181
BBA R e p o r t BBA 31236 DETECTION OF E R A B U T O X I N S IN THE VENOM OF SEA SNAKE LA TICA UDA S E M I F A S C I A T A F R O M THE PHILIPPINES
NOBUO TAMIYA and CHIKAHISA TAKASAKI Department of Chemistry, Tohoku University, Aobayama, Sendai 980 (Japan) (Received August 29th, 1977)
Summary Erabutoxins a, b and c were detected in the venom of sea snake Laticauda semifasciata from The Philippines.
Erabutoxins a and b are the main neurotoxic components of the venom of the sea snake Laticauda semifasciata from Japan [1]. Both of them consist of 62 amino acid residues. Their original results of amino acid analysis were corrected for the presence of another serine residue (8 instead of 7) by the results of shorter period (12 h) of hydrolysis [2]. Erabutoxin c is a minor toxic c o m p o n e n t in the same venom [ 3 ]. The amino acid sequences [3,4] and the positions of disulfide bridges [5] of erabutoxins were elucidated. The presence of serine and glutamic acid at positions 21 and 22 w a s corrected to glutamic acid and serine b y Maeda and Tamiya [6]. The three dimensional structure of the peptide chain of erabutoxin b was clarified [ 7]. On the other hand, Tu et al. [8] isolated two main toxic components from the venom of L. sernifasciata from The Philippines by the same method as the above authors and named them toxins a and b. The only differences between erabutoxins a and b and toxins a and b were their amino acid compositions: a consisted of 62 and b of 61 amino acid residues. Tsernoglou and Petsko [9,10] presented the three dimensional structure of the peptide chains of toxins a and b. The present study was carried out to examine the possible identity of toxins a and b with erabutoxins. Sea snakes (L. sernifasciata) were collected at Gigantes Islands, The Philippines, during the Visayan Sea Expedition on the research vessel Alpha Helix of the Scripps Institution of Oceanography, in September, 1975. The methods of isolation of erabutoxins, reduction, S-carboxymethylation and tryptic digestion of erabutoxin b, separation of the resulting tryptic frag-
200 ments, and the amino acid analysis of the toxins and the fragments were the same as described previously [1,4]. The whole profile of the elution pattern from the CM-cellulose column of the venom gland extract of sea snake L. semifasciata from The Philippines was the same as that from Japan. In addition to erabutoxins a, b and c, L. semifasciata III [ 11] and other components were also detected. The results of amino acid analysis of erabutoxins from L. semifasciata from The Philippines are shown in Table I. The results by Sato et al. [2] and Tu et al. [8] are also shown for comparison. The results of amino acid analysis of the tryptic fragments from reduced and S-carboxymethylated erabutoxin b are shown in Table II. The Gigantes Islands, where the snakes were collected, are located at less than 100 km to the west of Gato Islands, where Tu collected his snakes [ 8 ] , and about 2000 km away from Okinawa Islands, Japan, where Tamiya and Arai [1] collected their snakes. It is concluded, therefore, that the same erabutoxins are present in the venoms of L. semifasciata from Japan and from The Philippines, and that there are no subspecies as far as the venom components are concerned. The authors propose to use the original names of erabutoxins a, b and c to avoid further confusion. The work was supported by NSF grants DES 74-24129 from Pennsylvania State University and OFS 74-02888 and OFS 74-01830 from the
TABLE I A M I N O A C I D C O M P O S I T I O N O F E R A B U T O X I N a. b A N D c O F S E A S N A K E L S E M I F A S C I A T A FROM THE PHILIPPINES T h e values axe given in m o l a m i n o a m d / m o l p r o t e i n in 24 h h y d r o l y s a t e . Erabutoxin a Integer valuer Asp Thr SertT Glu Pro
Gly V~ Cys** Val Ue¶ ¶ Leu Tyr Phe Lys H~s Arg Trp Total
5.0 4.9 7.9 8.1 4.2 5.0 7.4 2.1 3.8 1.1 0.9 2.0 3.9 1.1 3.0 *
Erabutoxin b Toxin a¶
5 5 8 8 4 5 8 2 4 1 1 2 4 1 3 1
5 6 7 8 4 5 8 2 4 1 1 2 4 1 3 1
62
62
Integer value'[" 4.0 4.9 7.8 8.2 4.1 5.0 6.0 2.0 3.1 1.1 1.0 1.9 4.0 2.1 3.0 *
Erabutoxin c Toxin b¶
4 5 8 8 4 ,5 8 2 4 1 1 2 4 2 3 1
4 5 6 8 4 6 8 3 4 1 1 2 5 1 2 1
62
61
Integer value § 5.0 4.8 7.7 8.4 4.3 5.0 7.2 1.9 3.3 1.1 1.1 2.0 2.7 1.9 2.9 *
5 5 8 8 4 5 8 2 4 1 1 2 3 2 3 1 62
t T a k e n f r o m S a t o e t al. [ 2 ] . ¶ T a k e n f r o m T u e t al. [ 8 ] . § T a k e n f r o m T a m i y a and A b e [ 3 ] . ~ " C o r r e c t e d for d e s t r u c t i o n by 12 h h y d r o l y s i s values. * Not determined. * * C y s t i n e t e n d s t o give s m a l l values. ¶ ¶ I s o l e u c i n e t e n d s to give l o w values due to t h e p r e s e n c e o f Ile-Ile s e q u e n c e .
201
TABLE II AMINO ACID COMPOSITION OF TRYPTIC PEPTIDES FROM REDUCED AND S-CARBOXYMETHYLAT E D ERABUTOXIN b FROM L. S E M I F A S C I A T A FROM THE PHILIPPINES The values show molar ratio of amino acids in 24 h hydrolysate. The names of the peptides and their amino acid c o m p o s i t i o n (figures in parentheses) are t a k e n from Sato and Ta mi ya [ 4 ] . T-1 Cmc Asp Thr Set GIu
2.4 (3) 2.0 (2)
Total
T-2b
1.8 (2)
T-4a
T-4b¶
1.0t(1) 1.0 (1)
1.1 (1)
T-3 1.6 (2) 0.9 (1) 2.9 (3) 1.0t(1)
1.9 (2) 2.2 (2)
Pro Gly Val Iie Leu Tyr Phe Lys His Arg Trp
T-2a
2.3 (2) 3.0"F(3) 0.9 (1)
1.0 (1) 0.9 (1) 1.0 (1)
0.8 (1) 1.3 (1)
1.1 (1)
T-5
0.7 (1) 1.0"I"(1) 1.8 (2) 2.1 (2) 3.1 (3) 1.3 (1)
1.01"(1)
0.8 (1)
1.7 (2)
0.6 (1)
1.0t(1) 0.9 (1) 0.9 (1) 1.2(1) 1.2 (1)
1.1 (1) 2.1 (2)
1.3 (1) 1.0 (1) 1 (1)
11
12
1
12
0.9 (1) 0.9"(1)
1.0 (1)
6
6
14
t The n umb ers of these amino acids axe t a k e n as standard. The acid h ydro lysis was carried o u t for 60 h. * The acid hydrolysis was carried out in the presence of 4% (v/v) thloglycolhc acid [ 1 2 ] .
Scripps Institution of Oceanography for operation of the R/V Alpha Helix. It was supported also by the Yoshida Science Foundation for the travelling expenses to join the program. The authors are grateful to the members of the Visayan Sea Expedition (Chief Scientist Dr. W.A. Dunson) of the research vessel Alpha Helix for their co-operation in the collection of sea snakes.
References 1 2 3 4 5 6 7 8 9 10 11 12
Tamiya, N. and Arai, H. (1966) Biochem. J. 9 9 , 6 2 4 .--~630 Sato, S., Yoshida, H., Abe, H. and Tamlya, N. (1969) Biochem. J. 115, 85--90 Tamiya, N. and Abe, H. (1972) Biochem. J. 130, 547---555 Sato, S. and Tamiya, N. (1971) Biochem. J. 122, 453--461 Endo, Y., Sato, S., Ishii, S. and Tamiya, N. (1971) Biochem. J. 1 2 2 , 4 6 3 - - 4 6 7 Maeda, N. and Tamiya, N. (1977) Bloehem. J. 167, 289--291 Low, B.W., Preston, H.S., Sato, A., Rosen, L.S., Seasl, J.E., R udko, A.D. and Richaxdson, J.S. (1976) Proc. Natl. Acad. Sci. U.S. 73, 2 9 9 1 - - 2 9 9 4 Tu, A.T., Hong, B.S. and Solie, T.N. (1971) Biochemistry 10, 1 2 9 5 - - 1 3 0 4 Tsernoglou, D. and Petsko, G.A. (1976) FEBS Lett. 68, 1---4 Tsernoglou, D. and Pets]co, G.A. (1977) Proc. Natl. Acad. SeL U.S. 74,971---974 Maeda, N., Takagl, K., Tamiya, N., Chen, Y.M. and Lee, C.Y. (1974) Biochem. J. 1 4 1 , 3 8 3 - - 3 8 7 Matsubaxa, H. and Sasakl, R. (1969) Biochem. Biophys. Res. Commtm. 35, 175--181