Eur. J. Biochem. 58. 117 - 122 ( 1975)
Amino-Acid Sequence of Toxin 111 of Nuju haje C’liatlcs KOPEYAN. Fritnqois MIRANDA, and Hervi ROCHAT
Lahoratoirc de Biochimie. Faculte de MCdecine. Secteur Nord et LInitC 38 dc I’lnstiiut National de la Sante ct de la Rzchrrchc Mkdicalc. h l a rsci Ilc.
(Received March 26. 1075)
The complete amino acid sequence of toxin 111 of Nui(i haje (72 residues) has been established mainly by use of a protein sequenator (identification of 70 residues). The two C-terminal residues have been determined by digestion with carboxypeptidases A and B. Addition of succinylated protein o r peptide greatly improved the performance of the sequenator for the Ednian degradation of peptides: on one peptide (39 residues) degradation went to step 34 with a protein program and on two peptides (10 and 13 residues) degradation reached the last amino acid with a peptide program (use of dimethylbenzylamine). Amino acid analysis of tryptic peptides obtained by digestion of the C-terminal cyanogen bromide peptide are in full agreement with the sequence established by automatic degradation. The sequence of toxin 111 of Naja huje is unique and is very similar to that of Nuju nirea a (although there are 9 differences), of Nuju mc4aiioleuca b (1 1 differences) and also to that of Nuju tic@ A (18 differences).
Purification of Nuju liuje venom originating from two difTerent sources (Miami Serpentarium, Miami, Florida. U.S.A., MS-venom; or lnstitut Pasteur, 92, Garches, France, IP-venom) led to isolation of five different neurotoxins [1,2]. MS-venom collected from Egyptian snakes contained two toxins: I and 11. IP-venom, obtained from Ethiopian snakes contained three toxins: 1, 11’ and 111. Toxin I (61 residues) was shown to be a common toxin in these two venoms and identical to toxin a isolated from Nuju haje venom by Botes and Strydom [3]. Toxins I1 and 11’ (61 residues) are very close in their composition. Toxin I11 (72 residues) is the only long toxin present in these venoms and its amino acid composition showed some common characteristics (alanine and phenylalanine, 5 disulfide bridges, presence of methionine) with toxin a of Naju nivea [4]. We now wish to report the determination, mainly by automated sequencing analysis, of the primary structure of toxin 111 isolated from the Ethiopian cobra Naju haje, the N-terminal sequence of which has already been published as an example of the use of automated Edrnan degradation for rapid characterization of newly purified proteins[2]. -
Abbreviations. MS-venom, venom from Miami Serpentarium ; IP-venom, venom from Institut Pasteur; reduced Me-toxin. reduced and methylated toxin; reduced Cm-toxin, reduced and carboxyrnethylated toxin. En;)mes. Trypsin (EC 3.4.21.4); carboxypeptidase A (EC 3.4.12.2); carboxypeptidase B (EC 3.4.12.3). Note. Venom was provided by Institut Pasteur (9ZGarches) from snakes originating from Ethiopia.
MATERIALS A N D METHODS Ma te rial.\
Toxin I11 of Nuja haje was purified as previously described [ 1.21. L- 1-tosylamido-2-phenylet hyl-chloromethyl-ketone-treated trypsin and carboxypeptidase A and B treated with diisopropylphosphofluoridate were obtained from Worthington (Freehold, N.J., [J.S.A.). Sephadex (3-15 (beads, fine grade) was purchased from Pharmacia (Uppsala, Sweden), Biogel P4 from Biorad (Biorad Laboratories, Richmond, California. U.S.A.). Succinic anhydride, iodoacetic acid, dithioerythritol. cyanogen bromide, quadrol and dimethylbenzylamine sequanalgrade reagents were from Pierce Chemical Company (Rockford. Ill., U.S.A.).N-Ethylmorpholine (purum grade) was obtained from Fluka (Buchs, SG Switzerland). Clietnical Motl~icutiorisof Amino Acid Rrsii/iic.\ Reduction arid AIkj1lutiori of Disulj<J Bridge.\. S-Methylation was achieved according to Rochat rt al. [5]. S-Carboxymethylation was performed at pH 8.6 according to Crestfield [6]. except that the 0.25 M Tris buffer was 5 M in guanidine instead of 8 M in urea. In both cases mercaptoethanol was replaced by dithioerythritol. Succinylation of the Amino Groups. The technique of succinylation closely followed the procedure described by Hapner and Willcox [7]. In each case
118
the modified protein was recovered by filtration on Sephadex G-15 equilibrated with 1.0 M acetic acid. Cltwwigc 11). C?twri ogen Bromide
The S-carboxymethylated protein (reduced Cmprotein) was cleaved by cyanogen bromide at 20 "C in anhydrous formic acid for 20 h. according to Inglis and Edman [8]. After the reaction the solution was diluted ten times by water and freeze-dried. The two resulting peptides (residues 1 to 33 and 34 to 72 in the sequence) were separated on a Biogel P4 column (2 x 200 cm) equilibrated in 0.1 M ammonium acetate buffer at pH 7.0. The column effluent was monitored at 230 nm and 280 nni.
Digestion bj3Carbo.vj:,.peptidasesA a i d B The reduced and methylated toxin (0.2 pmol in water) was submitted first to action of carboxypeptidase A. The digestion was performed at 35 ' C i n a pH-stat (pH 8) using an enzyme-to-substrate ratio of 2 : 100 (w/w). The mixture was then heated 15 min at 100°C in order to denature the enzyme. After freeze-drying the substrate was submitted to the action of carboxypeptidase B in a stoppered vial, using N-ethylmorpholine acetate buffer at pH 8.0, 35 "C; the enzyme-to-substrate ratio was 1 : 25 (molar basis). In both cases aliquots of digest were removed at various intervals ( 5 , 10, 20, 30, 60, 120, 180 min), acidified with 1.0 N HCI, dried under vacuum and submitted to amino acid analysis. Digestion by Tqpsin
Reduced and Mcthylated Toxin. The reduced Me-toxin ( 5 pmol) was dissolved in 10 ml of 0.2 M ammonium bicarbonate, pH 8.0, and hydrolyzed by trypsin during 12 h at 37 "C. The enzyme-to-substrate ratio was 5;; (w/w). The digestion was initiated by 3 % trypsin. A second addition of 2 % trypsin was made after 6 h. The reaction was stopped by addition of hydrochloric acid to pH 2.0. After freeze-drying, the tryptic peptides were purified using Biogel P4 columns equilibrated with 0.1 M ammonium acetate buffer at pH 8.6. Cyanogen Bromide Peptide. (Residues 34 to 72 in amino acid sequence.) The peptide 34-72 (3 pmol) obtained by cyanogen bromide cleavage was hydrolyzed by action of trypsin in 10 ml of ammonium bicarbonate buffer. The separation of the tryptic peptides was carried out by the finger-printing procedure: chromatography in butanol- acetic acid - water (5 : 1 : 4 by volume) and high-voltage electrophoresis during 3 h in pyridine - acetic acid - water (1 : 10 : 289 by volume) at pH 6.5, 40 V/cm. The peptides were eluted by 50 o', acetic acid and freeze-dried.
Complete Amino-Acid Analysis of Toxin 111 of "Vtrjo litrjr
Arniiw Acid Anctlvsis
Samples were hydrolyzed by 6.0 N hydrochloric acid in sealed evacuated tubes at 110-C for 20 h in the case of peptides and modified toxins, and 20- 70 h in the case of native protein. The hydrolysates were analyzed in a Spinco amino acid analyzer model 120 C. according to Spackman et al. [9]. Antoinntic
Edmun Degradntion
Automatic Edman degradation was performed using a sequenator: Sequenceur PS 100 (Socosi, 94100, Saint-Maur, France). Two programs have been applied : the protein program using quadrol as described by Edman and Begg [lo] and the peptide program using dimethylbenzylamine [l 3 ] following closely the prescriptions of Jaton [12]. In the last case 4 m g of succinylated sheep apomyoglobin was added to 0.5 pmol of peptide. Identification of the phenylisothiocyanate derivatives of the amino acids was achieved by thin-layer chromatography [13] and by gas chromatography [14] on ii Beckman GC65. The histidine and arginine derivatives were directly identified on paper strips by Pauly and Sakagushi reactions [ 131. Automatic sequencing analyses have been applied on: (a) the reduced and S-methylated toxin (0.5 pmol) using the protein program; (b) the C-terminal cyanogen bromide peptide (0.5 pmol) using also the protein program. (In fact we applied the mixture obtained after succinylation followed by cleavage with cyanogen bromide. Two peptides CBI and CBII were obtained. Only the C-terminal peptide had a free a-amino group.) (c) Peptides obtained after trypsin digestion of reduced Metoxin. Two of these peptides (10 to 13 residues) have been sequenced in the presence of succinylated sheep apomyoglobin; using the peptide program 0.5 pmol of peptide was mixed with 4 mg of protein in dimethylbenzylamine and the mixture injected into the cup. Dimethylbenzylamine was removed by benzene washings and vacuum and the sequenator was then allowed to start the degradation cycles.
RESULTS Automatic sequencing analysis was found to be very efficient in this study as it allowed us to determine 70 residues out of 72. The two last C-terminal residues were obtained by digestion with carboxypeptidase A and B (Fig.1). At first a run on the sequenator with native toxin and reduced Me-toxin permitted the degradation to reach residue 35. Only the identification of methionine at step 33 and of arginine at step 34 remained ambiguous. Edman degradation of the mixture obtained after succinylation of the S-carboxymethylated toxin and cleavage by cyanogen bromide reached 34 steps
C. Kopeyan, F. Miranda. and H. Rochat
119
CBI
----*
10
20
--
T'Z
T'4
30
40
50
T-3
60
70
IRCFITPDVTSQACPDGQNICYTKTWCDNFCGMRGKRVDLGCAATCPTVKPGVDIKCCSTDNCNPFPTRERS c C PA
CPB
Fig. 1. Amino ucid sequetice
Amino-Acid Sequence of Toxin 111 of Nuju haje C’liatlcs KOPEYAN. Fritnqois MIRANDA, and Hervi ROCHAT
Lahoratoirc de Biochimie. Faculte de MCdecine. Secteur Nord et LInitC 38 dc I’lnstiiut National de la Sante ct de la Rzchrrchc Mkdicalc. h l a rsci Ilc.
(Received March 26. 1075)
The complete amino acid sequence of toxin 111 of Nui(i haje (72 residues) has been established mainly by use of a protein sequenator (identification of 70 residues). The two C-terminal residues have been determined by digestion with carboxypeptidases A and B. Addition of succinylated protein o r peptide greatly improved the performance of the sequenator for the Ednian degradation of peptides: on one peptide (39 residues) degradation went to step 34 with a protein program and on two peptides (10 and 13 residues) degradation reached the last amino acid with a peptide program (use of dimethylbenzylamine). Amino acid analysis of tryptic peptides obtained by digestion of the C-terminal cyanogen bromide peptide are in full agreement with the sequence established by automatic degradation. The sequence of toxin 111 of Naja huje is unique and is very similar to that of Nuju nirea a (although there are 9 differences), of Nuju mc4aiioleuca b (1 1 differences) and also to that of Nuju tic@ A (18 differences).
Purification of Nuju liuje venom originating from two difTerent sources (Miami Serpentarium, Miami, Florida. U.S.A., MS-venom; or lnstitut Pasteur, 92, Garches, France, IP-venom) led to isolation of five different neurotoxins [1,2]. MS-venom collected from Egyptian snakes contained two toxins: I and 11. IP-venom, obtained from Ethiopian snakes contained three toxins: 1, 11’ and 111. Toxin I (61 residues) was shown to be a common toxin in these two venoms and identical to toxin a isolated from Nuju haje venom by Botes and Strydom [3]. Toxins I1 and 11’ (61 residues) are very close in their composition. Toxin I11 (72 residues) is the only long toxin present in these venoms and its amino acid composition showed some common characteristics (alanine and phenylalanine, 5 disulfide bridges, presence of methionine) with toxin a of Naju nivea [4]. We now wish to report the determination, mainly by automated sequencing analysis, of the primary structure of toxin 111 isolated from the Ethiopian cobra Naju haje, the N-terminal sequence of which has already been published as an example of the use of automated Edrnan degradation for rapid characterization of newly purified proteins[2]. -
Abbreviations. MS-venom, venom from Miami Serpentarium ; IP-venom, venom from Institut Pasteur; reduced Me-toxin. reduced and methylated toxin; reduced Cm-toxin, reduced and carboxyrnethylated toxin. En;)mes. Trypsin (EC 3.4.21.4); carboxypeptidase A (EC 3.4.12.2); carboxypeptidase B (EC 3.4.12.3). Note. Venom was provided by Institut Pasteur (9ZGarches) from snakes originating from Ethiopia.
MATERIALS A N D METHODS Ma te rial.\
Toxin I11 of Nuja haje was purified as previously described [ 1.21. L- 1-tosylamido-2-phenylet hyl-chloromethyl-ketone-treated trypsin and carboxypeptidase A and B treated with diisopropylphosphofluoridate were obtained from Worthington (Freehold, N.J., [J.S.A.). Sephadex (3-15 (beads, fine grade) was purchased from Pharmacia (Uppsala, Sweden), Biogel P4 from Biorad (Biorad Laboratories, Richmond, California. U.S.A.). Succinic anhydride, iodoacetic acid, dithioerythritol. cyanogen bromide, quadrol and dimethylbenzylamine sequanalgrade reagents were from Pierce Chemical Company (Rockford. Ill., U.S.A.).N-Ethylmorpholine (purum grade) was obtained from Fluka (Buchs, SG Switzerland). Clietnical Motl~icutiorisof Amino Acid Rrsii/iic.\ Reduction arid AIkj1lutiori of Disulj<J Bridge.\. S-Methylation was achieved according to Rochat rt al. [5]. S-Carboxymethylation was performed at pH 8.6 according to Crestfield [6]. except that the 0.25 M Tris buffer was 5 M in guanidine instead of 8 M in urea. In both cases mercaptoethanol was replaced by dithioerythritol. Succinylation of the Amino Groups. The technique of succinylation closely followed the procedure described by Hapner and Willcox [7]. In each case
118
the modified protein was recovered by filtration on Sephadex G-15 equilibrated with 1.0 M acetic acid. Cltwwigc 11). C?twri ogen Bromide
The S-carboxymethylated protein (reduced Cmprotein) was cleaved by cyanogen bromide at 20 "C in anhydrous formic acid for 20 h. according to Inglis and Edman [8]. After the reaction the solution was diluted ten times by water and freeze-dried. The two resulting peptides (residues 1 to 33 and 34 to 72 in the sequence) were separated on a Biogel P4 column (2 x 200 cm) equilibrated in 0.1 M ammonium acetate buffer at pH 7.0. The column effluent was monitored at 230 nm and 280 nni.
Digestion bj3Carbo.vj:,.peptidasesA a i d B The reduced and methylated toxin (0.2 pmol in water) was submitted first to action of carboxypeptidase A. The digestion was performed at 35 ' C i n a pH-stat (pH 8) using an enzyme-to-substrate ratio of 2 : 100 (w/w). The mixture was then heated 15 min at 100°C in order to denature the enzyme. After freeze-drying the substrate was submitted to the action of carboxypeptidase B in a stoppered vial, using N-ethylmorpholine acetate buffer at pH 8.0, 35 "C; the enzyme-to-substrate ratio was 1 : 25 (molar basis). In both cases aliquots of digest were removed at various intervals ( 5 , 10, 20, 30, 60, 120, 180 min), acidified with 1.0 N HCI, dried under vacuum and submitted to amino acid analysis. Digestion by Tqpsin
Reduced and Mcthylated Toxin. The reduced Me-toxin ( 5 pmol) was dissolved in 10 ml of 0.2 M ammonium bicarbonate, pH 8.0, and hydrolyzed by trypsin during 12 h at 37 "C. The enzyme-to-substrate ratio was 5;; (w/w). The digestion was initiated by 3 % trypsin. A second addition of 2 % trypsin was made after 6 h. The reaction was stopped by addition of hydrochloric acid to pH 2.0. After freeze-drying, the tryptic peptides were purified using Biogel P4 columns equilibrated with 0.1 M ammonium acetate buffer at pH 8.6. Cyanogen Bromide Peptide. (Residues 34 to 72 in amino acid sequence.) The peptide 34-72 (3 pmol) obtained by cyanogen bromide cleavage was hydrolyzed by action of trypsin in 10 ml of ammonium bicarbonate buffer. The separation of the tryptic peptides was carried out by the finger-printing procedure: chromatography in butanol- acetic acid - water (5 : 1 : 4 by volume) and high-voltage electrophoresis during 3 h in pyridine - acetic acid - water (1 : 10 : 289 by volume) at pH 6.5, 40 V/cm. The peptides were eluted by 50 o', acetic acid and freeze-dried.
Complete Amino-Acid Analysis of Toxin 111 of "Vtrjo litrjr
Arniiw Acid Anctlvsis
Samples were hydrolyzed by 6.0 N hydrochloric acid in sealed evacuated tubes at 110-C for 20 h in the case of peptides and modified toxins, and 20- 70 h in the case of native protein. The hydrolysates were analyzed in a Spinco amino acid analyzer model 120 C. according to Spackman et al. [9]. Antoinntic
Edmun Degradntion
Automatic Edman degradation was performed using a sequenator: Sequenceur PS 100 (Socosi, 94100, Saint-Maur, France). Two programs have been applied : the protein program using quadrol as described by Edman and Begg [lo] and the peptide program using dimethylbenzylamine [l 3 ] following closely the prescriptions of Jaton [12]. In the last case 4 m g of succinylated sheep apomyoglobin was added to 0.5 pmol of peptide. Identification of the phenylisothiocyanate derivatives of the amino acids was achieved by thin-layer chromatography [13] and by gas chromatography [14] on ii Beckman GC65. The histidine and arginine derivatives were directly identified on paper strips by Pauly and Sakagushi reactions [ 131. Automatic sequencing analyses have been applied on: (a) the reduced and S-methylated toxin (0.5 pmol) using the protein program; (b) the C-terminal cyanogen bromide peptide (0.5 pmol) using also the protein program. (In fact we applied the mixture obtained after succinylation followed by cleavage with cyanogen bromide. Two peptides CBI and CBII were obtained. Only the C-terminal peptide had a free a-amino group.) (c) Peptides obtained after trypsin digestion of reduced Metoxin. Two of these peptides (10 to 13 residues) have been sequenced in the presence of succinylated sheep apomyoglobin; using the peptide program 0.5 pmol of peptide was mixed with 4 mg of protein in dimethylbenzylamine and the mixture injected into the cup. Dimethylbenzylamine was removed by benzene washings and vacuum and the sequenator was then allowed to start the degradation cycles.
RESULTS Automatic sequencing analysis was found to be very efficient in this study as it allowed us to determine 70 residues out of 72. The two last C-terminal residues were obtained by digestion with carboxypeptidase A and B (Fig.1). At first a run on the sequenator with native toxin and reduced Me-toxin permitted the degradation to reach residue 35. Only the identification of methionine at step 33 and of arginine at step 34 remained ambiguous. Edman degradation of the mixture obtained after succinylation of the S-carboxymethylated toxin and cleavage by cyanogen bromide reached 34 steps
C. Kopeyan, F. Miranda. and H. Rochat
119
CBI
----*
10
20
--
T'Z
T'4
30
40
50
T-3
60
70
IRCFITPDVTSQACPDGQNICYTKTWCDNFCGMRGKRVDLGCAATCPTVKPGVDIKCCSTDNCNPFPTRERS c C PA
CPB
Fig. 1. Amino ucid sequetice
