Eur. J. Biochem.

49, 399-405 (1974)

The Amino-Acid Sequence of a Polypeptide

from the Venom of Dendroaspis viridis
Rudolph A. SHIPOLINI and Barbara E. C. BANKS
Departments of Chemistry and Physiology,University College London

(Received June 27/August 13, 1974)

The primary structure of a major polypeptide component from the venom of Dendroaspis viridis
has been determined unambiguously. The molecule (60 amino acids, four disulphide bridges) closely
resembles a relatively non-toxic component of the venom of Dendroaspis angusticeps. The pharma-
cological effects of neither of these are understood. The component from Dendroaspis viridis does
not block neuromuscular transmission in frogs, is without effect on the excitatory or inhibitor actions
of acetylcholine on snail neurons and does not appear to be a lytic factor.

A number of homologous polypeptides, of known
amino acid sequence, having molecular weights in
the range approx. 6800-7800 have been isolated
from the venoms of the Proteroglyphae (for review
see [l], also [2]). The earlier reports were mainly
concerned with components of cobra venoms and
of the venoms of sea-snakes. Attention was focused
only on the more toxic fractions, although chro-
matography had indicated that all the venoms were
very complex mixtures. The most toxic compounds
(LD,o in mice z 0.1 mg/kg) are neurotoxins with a
curare-like action and these all contain 60 - 62 (short)
or 71 - 74 (long) amino acid residues and four or five
disulphide bridges. Of rather lower toxicity (LD,,,
in mice % 2 mg/kg) are the cardiotoxins which may
be associated with lytic or cytotoxic effects. The long
and short neurotoxins and the cardiotoxins form
three immunochemically distinct groups [3].
Recently attention has been turned to the composi-
tion of mamba venoms with regard to both highly
toxic and less toxic components. The venom of the
dominant [4] species of black mamba in East Africa
(Dendroaspis, polylepis) has been shown to contain
both a long and a short neurotoxin [5], which are
closely related to the cobra and sea-snake neurotoxins
both in structure and degree of toxicity. The venom
also contains two other less toxic components, I and K
[6], which have similar molecular weights (60 and
57 amino acid residues respectively) but only three
disulphide bridges. Fractions I and K, with LD,o in
mice of 30-36 mg/kg, are not homologous to the
snake-venom neuro and cardiotoxins but, unusually,
to two mammalian enterosecretory substances, bovine
pancreatic inhibitor and cow colostrum inhibitor.
The mamba polypeptides are considerably less potent
protease inhibitors than are the mammalian peptides.
The venom of Dendroaspis viridis, the West African
green mamba, also contains long and short neuro-
toxins [7] but does not contain polypeptides homo-
logous to fraction I and K, since all the peptides
containing 60-63 amino acids have eight half-
cysteine residues [8].
The venom of the East African green mamba,
Dendroaspis angusticeps, is quite different from other
elapid venoms in that it does not appear to contain
either long or short neurotoxins [9,10]. Although the
crude venom has a toxicity comparable to that of
other mamba venoms (LD5,, in mice z 2 mg/kg),
two major toxins Tal and Ta2 isolated from the
venom show much lower toxicities (LDS0 in mice
zz 20 and > 100 mg/kg respectively). Both these
components are homologous to the neuro and cardio-
toxins but are immunochemically distinct from these
and from each other. The pharmacological effects of
neither are understood.
We wish to report on the amino acid sequence of
a polypeptide (4.9.6.) from the venom of Dendroaspis
viridis which is much less toxic (LD50 > 3.3 mg/kg)
than neurotoxins from the same venom (LD5,, 0.08 -
0.9 mg/kg) and may be comparable in toxicity to
components isolated from the venom of Dendroaspis
angust iceps.
MATERIALS AND METHODS
Toxin 4.9.6 was isolated from the venom of Dendro-
aspis viridis as previously described [9].

Eur. J. Biochem. 49 (1974)


400
Sephadex G-25 superfine, was obtained from Phar-
macia.
Trypsin (treated with diphenylcarbamyl chloride)
was obtained from Sigma (London) Chemical Co.
Ltd.
Chymotrypsin was purchased from BDH and
purified by the method of Maroux [ll].
Thin-layer cellulose chromatography plates were
obtained from Merck.
All reagents were of analytical grade, purchased
from BDH Chemicals Ltd or from Sigma Chemical
Co. Ltd. Reagents and solvents for Edman degrada-
tion were exhaustively purified from reagents of
'sequential' grade preparations.
Column Chromatography
Sephadex G-25 (superfine) was packed into two
columns (1.6 x 90 cm and 1.5 x 60 cm) connected in
series and the resin was equilibrated with 2% (v/v)
aqueous acetic acid. Fractionation was carried out
in the same solvent, at a flow rate of about 12 ml/h
at room temperature. 4-ml fractions were collected
automatically (Radirac collector, LKB Instruments
Ltd.) and the absorptions of each monitored at
254 nm (Uvicord absorptiometer, LKB Instruments
Ltd.).
Modification of Toxin
35 mg of toxin 4.9.6 was reduced and carboxy-
methylated by the method of Crestfield et al. [12]
modified only by the use of a 50-fold excess of dithio-
erythritol as the reducing agent.
The 2-aminoethyl derivative was prepared by
treating the reduced toxin with a 100-fold molar
excess of ethyleneimine, the reagent being added in
four portions at 10-min intervals [13].
Enzymic Digestion
Reduced and carboxymethylated toxin (10 mg/ml)
was incubated with trypsin in 0.2 M N-ethylmorpho-
line-acetic acid buffer, pH 8.2, for 2 h at 37 "C. The
substrate-to-enzyme ratio was 100: 1 (w/w). Enzyme
action was stopped by addition of acetic acid to bring
the pH to 3.5 and the digest was then freeze-dried.
The 2-aminoethyl derivative of the toxin was
treated under similar conditions with chymotrypsin
but at a substrate-to-enzyme ratio of 100:2 (w/w).
No insoluble material appeared.
Fractionation of Pep t ides
The lyophilised digests were dissolved in 1.5 ml
2 % acetic acid and fractionated on a column of Sepha-
dex G-25 (superfine) equilibrated with 2 % acetic acid.
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The Structure of a Snake-Venom Polypeptide
Fractions lying between minima in the elution profile
were pooled.
Further purification was carried out on pooled
fractions from gel filtration by high-voltage electro-
phoresis (60 V/vm, 60- 100 min) on paper (What-
man 3MM, pH 6.5, pyridine-acetic acid- water,
10 : 0.5 : 90, by vol.). Individual fractions were located
by dipping strips, taken from the edges of the paper,
inninhydrin solution (0.2 % in acetone, w/v). Fractions
were eluted from the paper with 5 % acetic acid, 20 h.
The'. eluates were reduced in volume to 300 pl by
rotary evaporation and stored in plastic-capped speci-
men tubes. The purity of each fraction was checked
by chromatography of 3-p1 aliquots on thin-layer
cellulose plates (Merck). The plates were developed
in a butanol- pyridine - acetic acid - water solvent
(90: 60: 18 :72 by vol.) and sprayed with a solution of
ninhydrin in butanol (BDH).
Fractions containing more than one major compo-
nent were further purified by chromatography on
paper (Whatman 3MM) using the same solvent system
as for thin-layer chromatography. Fractions were
located, eluted and concentrated to 300 pl as described
above for the separation by high-voltage electro-
phoresis.
N- Terminal Analysis
The N-terminal amino acids of isolated peptides
were determined, on 3-p1 aliquots, by the dansyl
(5-dimethylaminonaphthalene-1-sulphonyl) technique
[14], the results being taken as a final criterion of
peptide purity. In all cases in which a single N-terminal
amino acid was found, the residual hydrolysate was
subjected to a second dansylation process and the
amino acid composition of the peptide was assessed
qualitatively after chromatography of the dansylated
amino acids on polyamide thin-layer sheets [15].
Additional solvent systems were used to distinguish
between the dansyl derivatives of acidic and hydroxy
amino acids [16] and between the monodansyl deriva-
tives of the basic amino acids [14].
Amino-Acid Analysis
All peptides containing more than five amino acid
residues were subjected to quantitative amino acid
analysis. Approximately 25 pl of peptide solution
was hydrolysed in 1 ml 6 N HCl for 25 h [17] prior
to analysis on a Technicon amino acid analyser
(single-column system).
Amino-Acid Sequence Determination
The N-terminal sequence of the whole toxin was
determined on the S-carboxymethylated derivative
Eur. J. Biochem. 49 (1974)
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R. A. Shipolini and B. E. C. Banks 401

Table 1. The amino-acid composition of peptides isolated from the S-carboxymethyl derivative of toxin 4.9.6 by digestion with
trypsin

Peptide Ammo-acid composition

~ ~~ -

Asp Thr Ser Glu Pro Gly Ala Val S-Cm-Met Ile Leu Tyr Phe Lys His Arg
cys a

T-I 0.8 0.7 0.7 1.0 0.9 1.2 1.1

T-1, 1 1 1 1
T-1, 1 1 1
T-2 0.8 2.7 0.9 3.2 1.1 1.0 0.8 0.9 1.1
T-3 1 1 1 1
T-4 1 1 1 1
T-5 0.9 0.9 0.6 0.6 0.6 1.5 1.1
T-6 0.9 2.0 1.1
T-7 1 1 1 1
T-7, 1 1 1 1 1
T-8 0.8 1.7 0.9 1.1 1.0
T-9 1.8 0.8 0.9 0.7 1.1
Free 1.5

Total 3 5 4 5 3 3 2 2 8 1 5 2 2 2 8 32 3

a S-Cm-Cys, S-carboxymethylcysteine.

by a combination of the methods of Edman and the

dansyl method [14]. The possibility of amidation of
glutamic and aspartic acid residues was examined by
chromatography of the phenylthiohydantoin deriva-
0.15
tives on silica-gel plates (Merck, Silica gel F254 with
fluorescent indicator). 2 0.10

The sequences of peptides obtained by enzymic 9" 0.05

digestion of the modified toxins were determined by
the dansyl-Edman technique [14]. Amidation of acidic
residues was determined from the electrophoretic
mobility of the residual peptides at pH 6.5 [18].
Peptides from the tryptic and chymotryptic digests
are designated T and C respectively and numbered
according to position in the final sequence.
RESULTS
N-Terminal Sequence Determination
The S-carboxymethylated toxin was subjected to
23 cycles of sequential degradation. Clear results
were obtained for 20 steps. The N-terminal sequence
is shown in Fig. 3.
Enzymic Digestion of Toxin 4.9.6
Trypsin. The S-carboxymethylated derivative
(30 mg) was digested with trypsin as described. The
freeze-dried digest, which was not completely soluble
in 1.5 ml 2 % acetic acid, was centrifuged and an ali-
quot of the insoluble residue (T-5) was subjected to
amino acid analysis. 1.5 moles of leucine were found
Q
6
' 9 12 15 18 21 24 27 30 33 36 39 42 4 5
Tube number
Fig. 1. Separation of peptides from a tryptic digest of the
S-carhoxymethyl derivative of 4.9.6 by chromatography on
Sephudex G-25 in 2 % acetic acid
for one mole of toxin derivative. A preliminary separa-
tion of the soluble tryptic peptides was carried out
by gel filtration on Sephadex G-25 SF resin. The elu-
tion profile is shown in Fig. 1. Three fractions, pooled
as indicated, were subjected to preparative high-
voltage electrophoresis on paper (Whatman 3MM)
at pH 6.5. Fraction S-1 gave two major ninhydrin-
positive bands in the acid region, S-2 gave eight and
S-3 gave one. Some of the eluted fractions were shown,
by thin-layer chromatography, to be pure. Other
eluted fractions required further purification by chro-
matography on paper. In all, 11 peptides were isolated
and the amino acid compositions of these summed
to that of the starting material. The results are given
in Table 1.
The complete sequences of all tryptic peptides
were determined by the dansyl-Edman technique.
The results are included in Fig. 3.

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402 The Structure of a Snake-Venom Polypeptide

Table 2. The amino-acid compositions of peptides obtained f r o m the 2-aminoethyl derivative of toxin 4.9.6 by digestion with
chymotrypsin
~~

Peptide Amino-acid composition

~ ~ ~

Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe 2-Aet- Lys His Arg
Cys"

c-1 1 1 1 1
c-2 1.0 3.7 1.7 3.2 1.1 1.0 0.9 0.9 2.1 2.2 1.1
c-2, 0.9 0.9 1.8 1.1 1.1 1.o 1.2 0.9
c-2, 2.7 1.8 1.o 0.8 2.2 1.1
c-3 1 1
c-4 0.9 0.9 0.8 1.5 0.6 1.6 1.2
c-5 1.1 1.2 2.0 0.9 0.9 0.9 1.9 2.3 1.2
C-6 1 1 1 1 1
c-7 1.8 0.8 1.1 3.1 1.2 1.3
Total 3 5 4 5 3 3 2 2 1 5 2 2 2 8 8 2 3

a 2-Aet-Cys, 2-aminoethylcysteine.

\+ 0 .' 32 50 c The amino acid sequences of all the chymotryptic

peptides were determined by the dansyl-Edman techni-
que. The results are included in Fig. 3.
All necessary overlaps were established from the
sequences of the tryptic and chymotryptic peptides.
The total primary sequence of toxin 4.9.6 is given in
Fig. 3.

Tube number
Fig. 2. Separation of peptides from a chymotryptic digest of
the 2-aminoethyl derivative of 4.9.6 by chromatography on
Sephadex G-25 in 2 % acetic acid
By comparison of electrophoretic mobilities it
was shown that the first aspartic acid and the C-ter-
minal glutamic acid residues in peptide T-9 and the
N-terminal glutamic acid residue in peptide T-5 were
not amidated. The second aspartic acid residue in
peptide T-9 was present as asparagine.
Chyrnotrypsin. The 2-aminoethyl derivative of the
toxin (30 mg) was digested with chymotrypsin as
described. The freeze-dried digest was completely
soluble in 2 % acetic acid (1.5 ml) and was fractionated
by gel filtration of Sephadex G-25 SF. The elution
profile is shown in Fig. 2. Three fractions were pooled,
as shown, and each was subjected to high-voltage
preparative electrophoresis on paper at pH 6.5. Frac-
tion S-1 gave three ninhydrin-positive bands, S-2
gave five and S-3 gave two. Eluted fractions were all
heterogeneous when examined by thin-layer chro-
matography. Pure peptides were only obtained after
further chromatography on paper. A total of eight
peptides was isolated, the amino acid compositions
of which summed to that of the whole toxin (Table 2).
DISCUSSION
The presence in mamba venom derived from
Guinea of some 13 polypeptides in the weight-range
of curare-like neurotoxins from other elapid venoms
enables a study of structure - function relations to
be made. It is hoped that features essential for curare-
like action in polypeptides can be identified by com-
parison of neurotoxic and homologous but non-
neurotoxic components. Only the most toxic of the
peptides from the venom of Dendroaspis viridis
(4.7.3, 4.9.3 and 4.11.3) are curare-like [7] but the
remaining components show varying degrees of toxi-
city, with LD5,, values lying in the range 1 - 10 mg/kg.
The polypeptide 4.9.6 is a major component of the
mamba venom (approx. 4.5% dry weight of crude
venom). It has an LD,, greater than 3.3 mg/kg; (the
precise value has nbt been determined because of the
amounts of material needed to give such limited infor-
mation). The primary sequence of this molecule has
been determined unambiguously from the sequences
of peptides isolated from tryptic and chymotryptic
digests of the modified polypeptide.
It was found to be advantageous to carry out the
chymotryptic digest on the 2-aminoethyl derivative
rather than the reduced and carboxymethylated form,
since the former digest contained no insoluble material
and, after freeze-drying, was totally soluble in 2

Eur. J. Biochem. 49 (1974)

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R. A. Shipolini and B. E. C. Banks 403

ao -1
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LD-
- I
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L L L L m
1= c C . c
F- l- el- 2.
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a 1 m m a m r
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Eur. J. Biochem. 49 (1974)

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404 The Structure of a Snake-Venom Polypeptide
5 I0 15
(A: Vet-l I s - C y s - T y r - S e r - H i s-Lys-Thr-Pro-Gln-lsn-Ser-Llc-Thr-l le-
-
( 5 ) Me t I Ie - C y s-Ty r -Ser - t i I s- Ly s-T hr- Pr 0 - 6 I n -Pr 0-S e r -E. 1 E-T hr I IE- -
(C) Thr-Met-Cys-Tyr-Ser-Hi s-Thr-Thr-Thr-Ser-!rg-E.le-l l s-Leu-Thr-
(D; A r g - l Ie - C y s - T y r - l s n - E l s-Eln-Ser-Thr-Thr-Pro-AII -Tyr-Thr-Lys-
Irg
20 25 30
Thr-Cys-E I u-61 u-Lys-Thr-Cys-Tyr-Lys-Phe-Val-Thr-Lys-Leu-Pro-
T hr - Cys- Glu- Elu- Lys- T hr - Cys -T y r-L y s -L y s -S e r-V a ~-~rg -L y s -L e u -P ro -
~sn-Cys-Gly-Glu-dsn-Ser-Cys-Tyr-Rrg-Lys-Ser-lrg-drg-~i~-Pro-Pr~-
S e r - C y s - S 1 y - G I u- F sn -S e r -C y s -T y r - L y s- Ly s- T h r - T r p -S e r - E. s p - H i s - b r g-
61u Tyr Arg
35 40 45
G l y - V a l -I le-Leu-Alz-Lrg-Ely-Cys-Ely-Cys-Pro-Lys-Lys-GI u - l Ie-Phe-
Ala-Val-Val-dla-Gly-drg-Ely-Cys-Cly-Cys-Pro-Ser-Lys-~l u-l'et-
Lys-Het-Va I - L e u 4 Iy - b r g - 6 l y-Cys-8 l y-Cys-Pro-Pro-E I y-Esp--f sp-
G l y - T h r - l Ie - l Ie-GI u-Brg-Gly-Cys-EIy-Cys-Pro-Lys-Val-Lys--Frg-
Prc
50 55 60
R r g - L y s - S e r - l I s-HI s-Cys-Cys-Erg-Ser-Asp-Lys-Cys-lsn-81 u
s-Cys-Cys-lrg-Ser-bssp-Lys-Cys- Asn-EI u
L e u - V a l - d l a - l Ie-Hi
F sn -Leu - E I u - Va I- Ly s -C y s - C y s -T h r -S e r - P r o - R s p - Ly s -C y s - I sn -T y r
GIy-VeI-His-Leu-E1s-Cys-Cys-GIn-Ser-Lsp-Lys-Cys-Psn-8sn
61y I l e TYr
Fig. 4. Amino-acid sequences o j some homologous mamba- F VII) from Dendroaspis angusticeps [9]; (D) 4.1 1.3 (short
venom polypeptides. (A) 4.9.6 from Dendroaspis viridis; (B) neurotoxin) from Dendroaspis viridis [7];and (beneath) toxin a
Ta2 from Dendroaspis angusticeps [lo] ; (C) Tal (originally from Dendroaspispolylepis [51

acetic acid. The specificity of trypsin was normal
except for partial cleavage C-terminal to tyrosine in
position 4 and lysine in position 43, while no cleavage
occurred C-terminal to the lysine in position 57. A
similar result has been reported for closely related
molecules, one isolated from the venom of Dendro-
aspis angusticeps [7] and the other (toxin 4.11.3) from
the Guinea mamba venom [lo], and may be due to
the acidic environment of this lysine residue. Peptide
T-5 was identified both in the supernatant and as the
insoluble residue left when the tryptic digest was
extracted with 2 % acetic acid.
The specificity of chymotrypsin was normal except
that a partial cleavage occurred C-terminal to alanine
in position 13 and a complete cleavage C-terminal to
histidine in position 51.
Toxin 4.9.6 contains no free sulphydryl groups
and is therefore cross-linked by four disulphide
bridges. The molecule is homologous with the short
neurotoxins from other elapid venoms and not with
the factors I and K isolated from the venom of
Dendroaspis polylepis.
The structures of toxin 4.9.6, the two less toxic
components Tal and Ta2 from Dendroaspis angusti-
ceps and of the short neurotoxin 4.11.3 from Dendro-
aspis viridis are given in Fig. 4. It can be seen that
toxin 4.9.6 is very similar to Ta2, there being only
14 amino acid substitutions, mainly between positions
24 and 50. The main difference between these molecules
is in the size of the second and third loops (Fig. 4), the
second being a residue shorter in 4.9.6 than in Ta2,
and the third a residue longer. Neither 4.9.6 nor Ta2
contains the sequence Tyr-X-Y-Z-Trp from residues
23 to 27, so far found to be present in all neurotoxins,
including 4.11.3. The residue at position 25 is lysine
in the four structures given and in all other snake
venom neurotoxins except 4.7.314.9.3 from Dendro-
aspis viridis venom, where the residue is glutamate.
This residue cannot, therefore, be essential for curare-
like action at neuromuscular junctions. The structures
of cardiotoxins have not been included in Fig. 4, since
polypeptides with this ill-defined activity have not
been found in mamba venoms. Toxins 4.9.6 does not
block neuromuscular transmission (R. Miledi, per-

Eur. J. Biochem. 49 (1974)


R. A. Shipolini and B. E. C. Banks
sonal communication) nor is it active against either
the hyperpolarisation or depolarisation responses to
acetylcholine in snail neurons [19]. The compound
does not affect black lipid membranes at a concentra-
tion of 2.5 x g/ml (W. McCormack, personal
communication) nor does it have phospholipase A
activity. It is therefore unlikely to be a ‘lytic’ factor.
It does not show inhibition of trypsin or chymotrypsin
even at a ratio of 50: 1 (G. S. Bailey, personal com-
munication). It is possible that 4.9.6 is the antigen
detected by Botes [lo] in the venom of Dendroaspis
viridis, immunochemically related to Ta2.
Since the pharmacological action of 4.9.6 remains
unknown, it is not profitable to speculate further
about structure- functions relations. The structures
of four other molecules of similar size but different
toxicities are currently being studied.
R. A. Shipolini gratefully acknowledges the award of a
Senior EMBO Fellowship. Mrs M. Kissonerghis is thanked
for her skilled technical assistance. Professors R. Miledi and
C. A. Vernon are thanked for their interest in this work.
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