Hoppe-Seyler's Z. Physiol. Chem. Bd. 356, S. 1893 - 1900, Dezember 1975
Snake Venom Toxins
The Amino Acid Sequence of Toxin V l! 2, a Cytotoxin Homologue from Banded Egyptian Cobra (Naja haje annufifera) Venom Francois J. Joubert
(Received 22 September 1975)
Summary: Toxin V n 2 comprises 60 amino acid residues and is cross-linked by four disulphide bridges. The complete amino acid sequence of this toxin was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides were purified by ion-exchange chromatography and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the
automatic sequenator or by manual manipulation, was employed to obtain the sequence of the intact toxin and the pure peptides. The chymotryptic digest provided the necessary overlapping peptides which allowed the alignment of tryptic peptides. The amino acid sequence of Naja haje annulifera toxin V n 2 shows a high degree of homology with cytotoxin V111 of the same venom.
Toxine aus Schlangengift: Die Aminosäuresequenz von Toxin V112 aus dem Gift von Naja haje annulifera Zusammenfassung: Toxin V11 2 enthält 60 Aminosäurereste und jeweils vier Disulfidbrücken. Die vollständige Aminosäuresequenz des Toxin V11 2
wurde aufgeklärt. Die Aminosäuresequenz von Naja-haje-annulifera^o\in V11 2 zeigt einen hohen Grad von Homologie mit Zytotoxin V11 1 .
The primary structure of cytotoxins of a number of species in the genera Naja^1'9^ has been reported. In addition Fryklund and Eakerl10! described the amino acid sequence of a non-neurotoxic hemolytic basic protein (cytotoxin) from the venom of Hemachatus haemachatus (Ringhals). Recently, two toxin homologues V111 and V n 2 fiomNaja haje annulifera venom were purified by a combination of gel filtration and chromatography on carboxymethyl-cellulose by Weise et . I11!. The same authors reported also the complete amino
acid sequence of the major cytotoxin V111. This cytotoxin comprises 60 amino acid residues and is cross-linked by four disulphide bridges. The present communication describes the complete amino acid sequence of toxin V H 2 fiomNaja haje annulifera venom. Experimental Procedure Toxin V H 2 was purified from Egyptian cobra venom as described^111. The physico-chemical methods; the tox-
Address: Dr. F. J. Joubert, National Chemical Research Laboratory, Council for Scientific and Industrial Research, P.O. Box 395, Pretoria 0001, Republic of South Africa.
Brought to you by | Purdue University Li Authenticated Download Date | 6/8/15 1:54 AM
1894
F.J. Joubert
icity determinations by intravenous injection; the immuno-chemical examination by gel diffusion; the reduction and S-carboxymethylation; the digestion with trypsin and chymotrypsin; the fractionation of enzyme digests by chromatography on DEAE-cellulose and by paper chromatography and high-voltage paper electrophoresis; the amino acid analyses of peptides of toxin V"2; the sequence determination of reduced and S-carboxymethylated toxin V^2 and peptides by Edman degradation with a Beckman Sequencer or manually; and the nomenclature of the toxin and peptides have been described previously l 1 1 » 1 2 1. Table 1. Some properties of toxin VU2. Property Molecular weight (i) Gel filtration21 (ii) Calculated from the amino acid sequence Toxicity, LD50 [Mg/g of mouse] Serological group a
Toxin V n 2 8600 6850
1.98 ±0.12 II
In 0.05M Tris + 0.2M NaCl at pH 7.3
Results Properties of toxin VH2 Some of the properties of toxin V n 2 are summarised in Table 1. The molecular weight obtained by gel filtration is in good agreement with the molecular weight calculated from the amino acid composition. The toxicity (LD50 value) of toxin VH2 is of the same order as that of cytotoxin VH1. The immunochemical properties of toxin V n 2 were investigated using polyvalent antiserum. Although the antiserum was not prepared against Naja haje annulifera venom, only a single distinct precipitin band was found for toxin V H 2. Naja nivea cytotoxin V n l* showed complete coalescence when it was compared with toxin VH2. Therefore, the immunochemical properties revealed thatTVfl/tf haje annulifera toxin V H 2 is related to the cytotoxin (cardiotoxin) serological group, as classified by Bouqet et al. t13!. The amino acid composition of toxin V H 2 is given in Table 3. The toxin is particulary rich in lysine, half-cystine, aspartic acid, proline, valine
Bd. 356 (1975)
and leucine. No free sulphydryl groups could be detected in the intact toxin. Amino acid sequence of reduced and S-carboxymethylated toxin VU2 The reduced and S-carboxymethylated toxin V112 was subjected to 40 steps of sequential Edman degradation, employing the Beckman automatic Sequencer, and the sequence is shown in Table 2. Fig. 1 shows the elution pattern of the tryptic peptides fractionated by DEAE-cellulose chromatography. The peaks were collected as indicated, lyophilised and further separated by paper chromatography or electrophoresis as presented in Table 3. This table also lists the amino acid composition of the pure peptides. Sequence studies on the tryptic peptides are summarised in Table 4. The sequences of peptides T-l (residues 1 - 5), T-2 (residues 6-12), T-3 (residues 13-18), T-4 (residues 19 - 23) and partial sequence of peptide T-5 (residues 24 - 36) and T-6 (residues 36 - 44) were inferred from the amino-terminal segment of the intact toxin as shown in Table 2. The sequences of peptides T-6a (residues 37 - 44), T-7 (residues 45 - 50) and T-8 (residues 51 - 60) were also entirely elucidated. Thus, apart from residues 33 and 36, the complete sequence of the reduced and S-carboxymethylated toxin chain was established. Table 2. TV-Terminal sequence of reduced and S-carboxymethylated toxin Vn2. The upper half-arrows show the residues identified by using the automatic Beckman Sequencer. Parentheses indicate that the residue in that position could not be identified.
Leu-Lys-Cys-His-Lys-Leu-Val-Pro-Pro-Phe15
Gly-Lys-Thr-Cys-Pro-Glu-Gly-Lys-Asn-Leu25
30
Cys-Tyr-Lys-Met-Tyr-Met-Val-Ala-Thr-Pro-
_
* Botes, D.P.,Viljoen, C. C. & Kruger, H., to be published.
20
35
40
Met-Leu-( )-Val-Lys-( )-Gly-Cys-Ile-Asp-
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Snake Venom Toxins
Bd.. 356 (1975)
30
60
90 Fract. no. —^
1895
120
150
Fig. 1. Chromatography of the tryptic digest of reduced and S-carboxymethylated toxin V H 2 on DEAE-cellulose (0.9 χ 150 cm). Gradient elution from 0.025M to 0.6M NH4HCO3 solution with a 2-1 gradient at a flow rate of 50 m//h was used, the column temperature was 20 °C and the eluate was monitored at 230 nm.
90
120
Fract. no. Fig. 2. Chromatography of the chymotryptic digest of reduced and S-carboxymethylated toxin cellulose (0.9 χ 150 cm). Experimental conditions the same as in Fig. 1.
2 on DEAE-
Brought to you by | Purdue University Li Authenticated Download Date | 6/8/15 1:54 AM
1896
F.J. Joubert
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Snake Venom Toxins
The Amino Acid Sequence of Toxin V l! 2, a Cytotoxin Homologue from Banded Egyptian Cobra (Naja haje annufifera) Venom Francois J. Joubert
(Received 22 September 1975)
Summary: Toxin V n 2 comprises 60 amino acid residues and is cross-linked by four disulphide bridges. The complete amino acid sequence of this toxin was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides were purified by ion-exchange chromatography and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the
automatic sequenator or by manual manipulation, was employed to obtain the sequence of the intact toxin and the pure peptides. The chymotryptic digest provided the necessary overlapping peptides which allowed the alignment of tryptic peptides. The amino acid sequence of Naja haje annulifera toxin V n 2 shows a high degree of homology with cytotoxin V111 of the same venom.
Toxine aus Schlangengift: Die Aminosäuresequenz von Toxin V112 aus dem Gift von Naja haje annulifera Zusammenfassung: Toxin V11 2 enthält 60 Aminosäurereste und jeweils vier Disulfidbrücken. Die vollständige Aminosäuresequenz des Toxin V11 2
wurde aufgeklärt. Die Aminosäuresequenz von Naja-haje-annulifera^o\in V11 2 zeigt einen hohen Grad von Homologie mit Zytotoxin V11 1 .
The primary structure of cytotoxins of a number of species in the genera Naja^1'9^ has been reported. In addition Fryklund and Eakerl10! described the amino acid sequence of a non-neurotoxic hemolytic basic protein (cytotoxin) from the venom of Hemachatus haemachatus (Ringhals). Recently, two toxin homologues V111 and V n 2 fiomNaja haje annulifera venom were purified by a combination of gel filtration and chromatography on carboxymethyl-cellulose by Weise et . I11!. The same authors reported also the complete amino
acid sequence of the major cytotoxin V111. This cytotoxin comprises 60 amino acid residues and is cross-linked by four disulphide bridges. The present communication describes the complete amino acid sequence of toxin V H 2 fiomNaja haje annulifera venom. Experimental Procedure Toxin V H 2 was purified from Egyptian cobra venom as described^111. The physico-chemical methods; the tox-
Address: Dr. F. J. Joubert, National Chemical Research Laboratory, Council for Scientific and Industrial Research, P.O. Box 395, Pretoria 0001, Republic of South Africa.
Brought to you by | Purdue University Li Authenticated Download Date | 6/8/15 1:54 AM
1894
F.J. Joubert
icity determinations by intravenous injection; the immuno-chemical examination by gel diffusion; the reduction and S-carboxymethylation; the digestion with trypsin and chymotrypsin; the fractionation of enzyme digests by chromatography on DEAE-cellulose and by paper chromatography and high-voltage paper electrophoresis; the amino acid analyses of peptides of toxin V"2; the sequence determination of reduced and S-carboxymethylated toxin V^2 and peptides by Edman degradation with a Beckman Sequencer or manually; and the nomenclature of the toxin and peptides have been described previously l 1 1 » 1 2 1. Table 1. Some properties of toxin VU2. Property Molecular weight (i) Gel filtration21 (ii) Calculated from the amino acid sequence Toxicity, LD50 [Mg/g of mouse] Serological group a
Toxin V n 2 8600 6850
1.98 ±0.12 II
In 0.05M Tris + 0.2M NaCl at pH 7.3
Results Properties of toxin VH2 Some of the properties of toxin V n 2 are summarised in Table 1. The molecular weight obtained by gel filtration is in good agreement with the molecular weight calculated from the amino acid composition. The toxicity (LD50 value) of toxin VH2 is of the same order as that of cytotoxin VH1. The immunochemical properties of toxin V n 2 were investigated using polyvalent antiserum. Although the antiserum was not prepared against Naja haje annulifera venom, only a single distinct precipitin band was found for toxin V H 2. Naja nivea cytotoxin V n l* showed complete coalescence when it was compared with toxin VH2. Therefore, the immunochemical properties revealed thatTVfl/tf haje annulifera toxin V H 2 is related to the cytotoxin (cardiotoxin) serological group, as classified by Bouqet et al. t13!. The amino acid composition of toxin V H 2 is given in Table 3. The toxin is particulary rich in lysine, half-cystine, aspartic acid, proline, valine
Bd. 356 (1975)
and leucine. No free sulphydryl groups could be detected in the intact toxin. Amino acid sequence of reduced and S-carboxymethylated toxin VU2 The reduced and S-carboxymethylated toxin V112 was subjected to 40 steps of sequential Edman degradation, employing the Beckman automatic Sequencer, and the sequence is shown in Table 2. Fig. 1 shows the elution pattern of the tryptic peptides fractionated by DEAE-cellulose chromatography. The peaks were collected as indicated, lyophilised and further separated by paper chromatography or electrophoresis as presented in Table 3. This table also lists the amino acid composition of the pure peptides. Sequence studies on the tryptic peptides are summarised in Table 4. The sequences of peptides T-l (residues 1 - 5), T-2 (residues 6-12), T-3 (residues 13-18), T-4 (residues 19 - 23) and partial sequence of peptide T-5 (residues 24 - 36) and T-6 (residues 36 - 44) were inferred from the amino-terminal segment of the intact toxin as shown in Table 2. The sequences of peptides T-6a (residues 37 - 44), T-7 (residues 45 - 50) and T-8 (residues 51 - 60) were also entirely elucidated. Thus, apart from residues 33 and 36, the complete sequence of the reduced and S-carboxymethylated toxin chain was established. Table 2. TV-Terminal sequence of reduced and S-carboxymethylated toxin Vn2. The upper half-arrows show the residues identified by using the automatic Beckman Sequencer. Parentheses indicate that the residue in that position could not be identified.
Leu-Lys-Cys-His-Lys-Leu-Val-Pro-Pro-Phe15
Gly-Lys-Thr-Cys-Pro-Glu-Gly-Lys-Asn-Leu25
30
Cys-Tyr-Lys-Met-Tyr-Met-Val-Ala-Thr-Pro-
_
* Botes, D.P.,Viljoen, C. C. & Kruger, H., to be published.
20
35
40
Met-Leu-( )-Val-Lys-( )-Gly-Cys-Ile-Asp-
Brought to you by | Purdue University Lib Authenticated Download Date | 6/8/15 1:54 AM
Snake Venom Toxins
Bd.. 356 (1975)
30
60
90 Fract. no. —^
1895
120
150
Fig. 1. Chromatography of the tryptic digest of reduced and S-carboxymethylated toxin V H 2 on DEAE-cellulose (0.9 χ 150 cm). Gradient elution from 0.025M to 0.6M NH4HCO3 solution with a 2-1 gradient at a flow rate of 50 m//h was used, the column temperature was 20 °C and the eluate was monitored at 230 nm.
90
120
Fract. no. Fig. 2. Chromatography of the chymotryptic digest of reduced and S-carboxymethylated toxin cellulose (0.9 χ 150 cm). Experimental conditions the same as in Fig. 1.
2 on DEAE-
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1896
F.J. Joubert
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