ARTICLE IN PRESS

Toxicon 49 (2007) 19–29

www.elsevier.com/locate/toxicon

Purification and characterization of Heteroscorpine-1 (HS-1)

toxin from Heterometrus laoticus scorpion venom
Nunthawun Uawonggula, Sompong Thammasiriraka, Arunrat Chaveerachb,
Tarinee Arkaravichienc, Wandee Bunyatratchatad, Wipaporn Ruangjirachuporne,
Pornpimol Jearranaiprepameb, Takeshi Nakamuraf, Michiyuki Matsudaf,
Michimoto Kobayashig, Seisuke Hattorig, Sakda Daduanga,f,
a
Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
b
Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
c
Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
d
Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
e
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
f
Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University,
3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
g
Division of Cellular Genome Proteomics, Institute of Medical Science, University of Tokyo,
Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Received 22 December 2005; received in revised form 1 September 2006; accepted 5 September 2006
Available online 14 September 2006

Abstract

Crude venom from Thai giant scorpion, Heterometrus laoticus, most commonly found in the northeastern area of
Thailand, was evaluated for PD50 (paralytic dose 50) activities from abdominal injection to cricket (Gryllus sp.) and
activities against various kinds of microorganisms. It exhibited good results in disc diffusion assay for Bacillus subtilis,
Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. After purification, toxin showed acceptable
results for PD50 determination in entrapped cricket as well as inhibitory activity against B. subtilis, K. pneumoniae, and
P. aeruginosa with activities over 300 times higher than that of the crude venom. The purified fraction was showed to
contain a single band in SDS-PAGE. MALDI-TOF-MS/MS analysis showed one peak of major protein with 8293 Da.
Partial amino acid sequence show high homology to Scorpine—a polypeptide toxin family with potassium channel
blocking and defensin activity. The novel toxin was named ‘‘Heteroscorpine-1’’ (HS-1) as the first Scorpine from genus
Heterometrus. After full length determination by PCR, HS-1 gene was found to be composed of two exons, separated by
an intron. Deduction revealed 95 amino acid residues with 19 residues as the leading sequence. It showed about 80%
similarity to Panscorpine and Opiscorpine.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Heterometrus laoticus; Heteroscorpine-1 (HS-1); Scorpine; Thai scorpion; Toxin

Corresponding author. Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand.
Tel.:/fax: +66 43 342911.
E-mail address: sakdad@kku.ac.th (S. Daduang).

0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2006.09.003
 ARTICLE IN PRESS
20 N. Uawonggul et al. / Toxicon 49 (2007) 19–29
1. Introduction
Venoms from venomous animals are rich sources
of bioactive compounds. Peptides are compounds
with a variety of actions. Peptides in scorpion
venoms are the most remarkable from their anti-
ion channel blocking activities. They are classified
into four groups according to their effects on sodium
(Na+), potassium (K+), calcium (Ca2+) and chlor-
ide (Cl) channels (Gwee et al., 1996; Legros et al.,
1998; Possani et al., 1999, 2000). Potassium channel
toxins are polypeptides consisting of 31–39 amino
acid residues stabilized by 3 or 4 disulfide bridges
(Possani et al., 1999). Many of them are character-
ized as anti-microbial agents (Kuhn-Nentwig, 2003).
Thus, study of anti-microbial peptides from scorpion
venoms is attractive and predicted as one choice of
novel anti-microbial sources from natural products.
Scorpine is one of the most recently reported as a
family of toxin polypeptides from scorpion venom
having potassium channel block and defensin activity
(Kuhn-Nentwig, 2003). Panscorpine1 was first iso-
lated from African scorpion Pandinus imperator
venom and showed inhibitory effect against Bacillus
subtilis and Klebsiella pneumoniae as well as on the
ookinete and gamete stages of Plasmodium berghei
malaria (Conde et al., 2000). Opiscorpine was the
next Scorpine purified from African Opistophthalmus
carinatus scorpion venom (Zhu and Tytgat, 2004).
It showed anti-fungal activity against Fusarium
oxysporum, the plant pathogenic fungus causing
Fusarium wilt in many plants.
Heterometrus scorpions are commonly found in
South and Southeast Asia. Their venomous potency
is variable and the venom composition has not been
characterized. Surprisingly harmful actions of
H. longimanus were reported from catecholamine
like substances (Gwee et al., 1993). However, toxins
from H. spinifer and H. pulvipes venom were quite
well defined as polypeptides with life threatening
potential from their anti-potassium channel blocking
action (Lebrun, et al., 1997; Srinivasan et al., 2002).
In Thailand, many Heterometrus exist. The Giant or
Black or Asian forest scorpion, Heterometrus laoti-
cus, is the most commonly found in the northeastern
area. It is about 10-cm long and has a deep black
body (Uawonggul et al., 2006). Venom composition
is still unknown but envenomation rarely causes
1
To minimize confusion between name of family and name of
member, Scorpine from P. imperator (Conde et al, 2000) is called
‘‘Panscorpine’’ (Zhu and Tytgat, 2004).
a serious case. In this study, a polypeptide from
H. laoticus venom was purified and characterized for
its activities against microorganisms. Scanning elec-
tron microscopy was used to clarify the inhibitory
effects on bacteria. The purified polypeptide was
determined for gene and amino acid sequences. It has
been named ‘‘Heteroscorpine-1’’ or ‘‘HS-1’’ with
high homology to members of Scorpine family and
many K+ channel blockers.
2. Materials and methods
2.1. Scorpion and scorpion venom
H. laoticus scorpions were from suburban areas
around Khon Kaen City, Thailand. Venom milking
was performed by fixing them in a confining device
and shocking with electrical pulse of 100 V and 1-msec
duration at 10 Hz frequency using a Grass stimulator
model S88 (modified from Gopalakrishnakone et al.,
1995). Crude venom was kept at 4 1C for not longer
than two weeks.
2.2. Anti-microorganism assay and scanning electron
microscopy
Anti-microbial activity was tested using the disc
diffusion method. A Mueller–Hinton agar plate at
35 1C was used for bacteria whereas a potato dextrose
agar plate at 30 1C was used for fungi. Diameters of
inhibition zones were measured. For bacteria, cells
from the inhibition zone were fixed by 2.5%
glutaraldehyde overnight before coating with gold
and observing under scanning electron microscope
LEO 1450VP (LEO Electron Microscopy, UK).
2.3. Toxin purification and biological assay
Crude venom (50 mg protein) was purified by
1.6  100-cm Sephadex G50 (Fluka, Switzerland)
column with ammonium acetate buffer, pH 4.7 as
the main buffer. Fractions were biologically assayed
for paralytic dose 50 (PD50) and inhibitory activities
on microorganisms. PD50 was determined from
abdominal injection to cricket (Gryllus sp.; modified
from Ji et al. (1996)). Those with relatively high
activity were further subjected to CM Toyopearl
650 M cationic column (Tosoh Corporation, Japan)
according to the method reported by Thammasirirak
et al. (2006), but the buffer was 20 mM ammonium
acetate buffer, pH 4.7, and elution was by NaCl
gradient (0–0.5 M). Purity was further checked by
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N. Uawonggul et al. / Toxicon 49 (2007) 19–29
Tris-Tricine SDS-PAGE following the Schagger and
von Jagow protocol (1987).
2.4. Edman degradation for N terminus
determination
The classic manual standard method was performed.
A sample in 50% pyridine and 4-N,N-dimethylami-
noazobenzene-4-isothiocyanate (DABITC) solution
was incubated with phenylisothiocyanate (PITC) and
washed with heptane:ethyl acetate (2:1). The aqueous
phase was collected, completely dried and then
incubated with 100% trifluoroacetic acid (TFA). After
drying again, water and butyl acetate were added and
separated by spinning down. The organic phase was
dried, then 50% TFA added to obtain phenylthiohy-
dantoin (PTH)–amino acid and dried. The dried
PTH–amino acid was redissolved in ethanol and
spotted on polyamide sheet using DABITC and
diethylamine in 50% pyridine as a marker. After
two-dimensional TLC using acetic acid:water (1:2) as
the first and toluene:n-hexane:acetic acid (2:1:1) as the
second mobile phase, color was developed by HCl
vapor.
2.5. Amino acid sequence determination
After separation by electrophoresis on a Tris-
tricine SDS-PAGE, proteins were transferred onto
a polyvinylidene difluoride (PVDF) membrane
(Problot, Applied Biosystems), reduced, S-carbox-
ymethylated, and digested in situ with Achromo-
bacter protease I (lysylendopeptidase-C; Iwamatsu,
1992). Matrix-assisted laser desorption ionization-
time of flight (MALDI-TOF) tandem mass spectro-
metry (MS/MS) analysis of selected peptides for
amino acid sequence determination was carried out
using a mass spectrometer (4700 Proteomics Analy-
zer, Applied Biosystems, USA).
2.6. Two dimensional polyacylamide gel
electrophoresis (2D-PAGE)
Sample (100 mg) was separated in the first dimen-
sion with 7 cm immobilized pH gradient strip pH
3–10 (Amersham Biosciences, Sweden). The
strip was rehydrated for 12 h and focussed
for 9250 total Vh using EttanTM IPGphor (Amer-
sham Bioscience). Then, it was washed in equilib-
ration buffer with 2% DTT following with the
buffer containing 5% iodoacetamide. The second
dimension was resolved by 16.5% Tris-tricine
21
SDS-PAGE. Protein was stained by Coomassie
brilliant blue R 250 staining and again by silver
staining.
2.7. Primer design and polymerase chain reaction
(PCR)
Degenerate and nondegenerate primers were
designed from amino acid sequences obtained from
MS/MS analysis and from conserved regions of
nucleotide sequences of the Scorpine members and
K+ channel blocker (Conde et al., 2000; Diego-
Garcia et al., 2005; Legros et al., 1998; Zhu et al.,
1999; Zhu and Tytgat, 2004). Primer sequences and
lengths of the amplified bands are shown in Table 1.
Genomic DNA was extracted from frozen scorpion
tissues using a standard protocol. The PCR reaction
comprised 2 ng genomic DNA template, 0.8 mM
dNTPs, 1 mM forward and reverse primer, 7.5 mM
MgCl2 and 2.5 units Taq polymerase (Expand High
FidelityPLUSPCR system kit, Roche, Germany).
PCR reactions were performed at 94 1C, 5 min as
initial denaturing temperature, followed by 30
cycles of 30 s of denaturation at 94 1C, 30 s–1 min
of annealing at 45–55 1C and 30 s of elongation at
72 1C. Final elongations were at 72 1C for 7 min.
2.8. Cloning and sequencing
PCR products were amplified before sequencing.
Gel elution was by Qiaex gel extraction kit (Qiagen,
USA), and pDrive cloning was by PCR cloning kit
(Qiagen, USA). After bacterial transformation and
cell proliferation, plasmid was extracted using Mini-
preps DNA purification kit (Promega, USA) and
further purified by Wizards Minipreps DNA pur-
ification resin (Promega, USA). Plasmid concentra-
tion was measured by Nanodrop (Nanodrop
Technologies, USA). Nucleotide was sequenced using
a 373oxl DNA analyzer (Applied Biosystems, USA).
2.9. Bioinformatics
Homology and similarity analyses were performed
by Clustal W software available from /http://www.ebi.
ac.uk/clustalwS. Similarity searches were performed
using FASTA 3 program from /http://fasta.
genome.jpS /http://www.ebi.ac.uk/fastaS. Relation-
ships among sequences were predicted by phylogenic
tree, constructed using the MEGA 3.1 /http://
www.megasoftware.netS. Theoretical mass determina-
tion and pI was by /http://br.expasy.org/cgi-bin/
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22 N. Uawonggul et al. / Toxicon 49 (2007) 19–29

Table 1
Primer design and PCR product sizes

Forward primers Reverse primers Product size (bp)

HSF2: 50 -ATG GG(A/C) AAT TGC GA(A/G) HSR2: 50 -CC(A/G) CA(C/T) TT(A/G) CAT TT(A/ 77
AC(G/T) CA(C/T)-30 degenerate from the amino G) GT(A/T) CCG TG-30 from HGTKCKCG
acid sequence MGNCETHC
HSF7: 50 -GGA CT(C/G) (A/G/C)T(A/G) GC(C/G) HSR3: 50 -GCA GAA TCC CTT TTC ACC TGA 1286
ATT GCC TC(C/T) TGC-30 CG-30 designed from known amino acid sequence of
HS-1 residues 75–82, TSGEKGFC
HSF11: 50 -ACG AGT CTT TTA CGC CTC GGT HSR6: 50 -(G/T)(C/T)C CC(C/T) (C/T)TT (C/T)GG 329
TTC C-30 CT(G/T) CAA TTA-30 designed from Opiscorpine3
and Panscorpine at the stop codon toward 30 ends
HSF4: 50 - (A/C)(A/C)A AAA CAA GAT GAA HSR10: 50 - AGT TTT CGC CTA TCG AAC TCA 249
CA(A/G) CAA GC -30 CC -30
HSF33: 50 - CTG TCG AGT TGC GGA ACG CTA HSR15: 50 -TGC CGA AAC TCT CGG AAA GGC 225
G -30 TC -30
HSF33 HSR10: 50 - AGT TTT CGC CTA TCG AAC TCA 346
CC-30
HSF12: 50 - TGA GGG TCT AAT ATA AGC CAG HSR12: 50 - TCC CGG AGA GCG GGA CAA CG - 735
TCG –30 30

Table 2
The inhibitory activity against microorganisms after H. laoticus Thai scorpion crude venom (crude venom) or purified toxin from peak no.
89 of CM Toyopearl cationic column chromatography (CM89) treatments using the disc diffusion method

Species Types of microorganisms Crude venom CM89

Average SD Average SD

Acidovorax avenae subsp. citrulli Bacteria,gram 0.00 0.00 — —

Bacillus subtilis Bacteria,+gram 5.20 0.45 1.50 0.71
Citrobacter freundii Bacteria,gram 0.00 0.00 — —
Escherichia coli Bacteria,gram 0.00 0.00 — —
Klebsiella pneumoniae Bacteria,gram 10.57 0.79 2.50 0.71
Pseudomonas aeruginosa Bacteria,gram 8.67 0.58 1.50 0.71
Ralstonia solanacearum Bacteria,gram 0.00 0.00 — —
Staphylococcus aureus Bacteria,+gram 2.40 0.89 0.00 0.00
Salmonella group B Bacteria,gram 0.00 0.00 — —
Fusarium oxysporum f. sp. lycopersici race 1 Fungus 0.00 0.00 — —
Fusarium oxysporum f. sp. lycopersici race 2 Fungus 0.00 0.00 — —
Fusarium oxysporum f. sp. lycopersici race 3 Fungus 0.00 0.00 — —
Sclerotium rolfsii Fungus 0.00 0.00 — —

Inhibition zone diameters are in mm.

peptide-mass.pl?P14790S and /http://br.expasy.org/
cgi-bin/pi_toolS, respectively.
3. Results
3.1. Purification of an anti-bacterial peptide from H.
laoticus venom and its activity
PD50 of crude venom in cricket was 0.67 ml. It
inhibited growth of many bacteria in disc diffusion
assay (Table 2). Then it was subjected to peptide
purification. Sephadex G-50 gel filtration column
chromatography revealed many protein peaks
(Fig. 1(A)). The pool fraction (fraction nos. 27–35)
with high biological activity was further subjected to
CM-Toyopearl cationic exchange column (Fig. 1(B)).
Protein in fraction no. 89 (CM89), eluted at around
0.35–0.4 M NaCl, showed the highest activity with
one band from Tris-tricine SDS-PAGE analysis
(Lane CM89, Fig. 1(C)). Native-PAGE running at
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N. Uawonggul et al. / Toxicon 49 (2007) 19–29 23

Fig. 1. Purification of the anti-microorganism peptide from Heterometrus laoticus scorpion crude venom. Crude venom was loaded onto
Sephadex G50 gel filtration chromatography (A). Pool fractions (gray shaded) with high anti-microorganism activity were further loaded
onto CM Toyopearl 650 M cationic column chromatography (B). NaCl gradient, 0–0.5 M, was used from fraction 49–100. Fraction no. 89
(asterisk) showed the highest activity. Purity was checked by 16.5% Tris-tricine SDS-PAGE analysis (C) lane V, crude venom, lane CM89,
fraction no. 89 from CM Toyopearl cationic column. N terminus was determined using Edman degradation (D). Molecular mass was
checked by MALDI-TOF mass spectroscopy (E). pI of proteins in CM89 was checked by 2D-PAGE (F).
 ARTICLE IN PRESS
24 N. Uawonggul et al. / Toxicon 49 (2007) 19–29

pH 4.4 showed a single band (data not shown).

CM peak no.89

0.00413 mmole
CM89 apparently showed only one spot of PTH-
glycine as an amino acid at N-terminus from the first

1.5 mmc
cycle of Edman degradation analysis (Fig. 1(D)),

23.44
Pseudomonas aeruginosa

356
showed one major peak with 8293.496 Da on mass
determination by mass spectroscopy (Fig. 1(E)) and
one spot on 2D-PAGE (Fig. 1(F)) with pI about 9.

Crude venom
CM89 showed inhibitory action to B. subtilis, K.

6.25 mmole
6.4 mmc
pneumoniae and Pseudomonas aeruginosa (Table 2)

100.00
with more than 300 times higher activity than crude

1
venom (Table 3).

CM peak no.89

363 mm/mmole
0.00413 mmole
3.2. Effects of the peptide on bacteria under scanning
electron microscope

2.5 mmc

23.65
B. subtilis, K. pneumoniae and P. aeruginosa, treated

358
Klebsiella pneumoniae
with crude venom or CM89, were observed under
scanning electron microscope to demonstrate effects

1.02 mm/mmole
Crude venom
on bacteria (Fig. 2). Untreated bacterial cells had

6.25 mmole
10.57 mmc
normal and smooth cell surfaces (Figs. 2(A)–(C)1)

100.00
whereas the crude venom (Figs. 2(A)–(C)2) and
CM89 (Figs. 2(A)–(C)3) treated cells showed apparent Purification of HS-1 from H. laoticus scorpion venom, its PD50 activity and bacterial inhibitory activities

1
changes in their morphology. They showed rough-

CM peak no. 89
ening and blebbing of the bacterial surfaces.

605 mm/mmole
0.00413 mmole
3.3. Partial amino acid sequence determination and
1.5 mmc

28.85
homology alignments

437
CM89 was transferred to a PVDF membrane.
Bacillus subtilis

After digestion with lysylendopeptidase-C in situ, 1.69 mm/mmole

Crude venom

peptides were subjected to tandem mass spectro-

6.25 mmole
5.2 mmc

metry. Amino acid sequences of two peptides of 807 100.00

PD50, volume of solutions that makes 50% of animals paralyzed.

and 2502 Da were successfully determined as
1

GFCHGTK and CVAN(L/I)DTMGNCETH, re-

spectively (Fig. 3). Amino acid sequence similarity
CM peak no. 89

363 mm/mmole
0.00413 mmole

searched by FASTA revealed a very high homology

0.0125 ml1b

to two members of the Scorpine family, Panscorpine

Diameter of clear zone from disc diffusion assay.

from P. imperator (Conde et al., 2000) and

0.83

Opiscorpine from O. carinatus (Zhu and Tytgat,

13

2004), with about 65% and 63% identity, res-

pectively. Thus, the polypeptide purified from
PD50 in cricketa

Crude venom

H. laoticus was named ‘‘Heteroscorpine-1’’ or

6.25 mmole

0.832 mm/
1.49 ml1b

‘‘HS-1’’ in abbreviation as the first Scorpine purified

100.00
mmole

from Heterometrus venom.

1

3.4. Gene and complete amino acid sequence

determination
0.238 mmole1 ml1
3.03 mmole1 ml1
Specific activity
Total activity
Total protein

The genomic DNA encompassing the HS-1

1/PD50.
Yield (%)

coding region was amplified by PCR and sequenced.

Table 3

PCR reaction started from HSF2 and HSR2 as the

Fold

b
a

first pairs of primer. They revealed amino acid

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N. Uawonggul et al. / Toxicon 49 (2007) 19–29 25

Fig. 2. Scanning electron microscopy of Bacillus subtilis (A), Klebsiella pneumoniae (B) and Pseudomonas aeruginosa (C). Those without
any treatments (1), treated with Heterometrus laoticus scorpion crude venom (2), or purified fraction no. 89 from CM Toyopearl (3) by disc
diffusion method, were prepared for examination under scanning electron microscope. Bars indicate sizes in nm.

Fig. 3. Determination of amino acid sequences of two proteolytic peptides of 807 and 2502 Da by MALDI TOF MS/MS. Tandem mass
spectra of the two peptides with their sequences (GFCHGTK and CVAN(L/I)DTMGNCETH) are shown. Complete y ion series within
the presented mass range were observed in the spectra indicating confident identifications.

residues 75–79. HSF7 and HSR3 were used to
determine amino acid residues 20–74. For determi-
nation of amino acid residues 24–95, HSF11 and
HSR6 were used as primers. To determine residues
6–19, HSF4 and HSR10 were used. Final portion of
residues 1–5 was determined by HSF33 and HSR15
and confirmed by different PCR reactions using
HSF33 and HSR10. Intron was determined by
HSF7 and HSR3 and again, by HSF12 and HSR12.
Sequence determination by many reactions of PCR
revealed 1440 nucleotides (Fig. 4).
After deducing and aligning with the known
Scorpine, HS-1 was seen to consist of 95 amino acid
residues with 19 residues of a leading sequence
at the N-terminus (Zhu and Tytgat, 2004). A total
of 6 cysteine residues were observed predicting
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26 N. Uawonggul et al. / Toxicon 49 (2007) 19–29

3 disulfide bridge formations within the molecule.
One intron, with length of 1073 nucleotides, was
flanked by two exons. Alignment of HS-1 amino
acid sequence with Panscorpine and Opiscorpine
showed about 82% and 78% similarity, respectively
and about 40% similarity to many potassium
channel blockers (Fig. 5(A)) with close evolution
to those Scorpines in phylogenetic tree analysis
(Fig. 5(B)). This strongly confirmed that HS-1 is a
member of the Scorpine family with potassium
channel blocking and defensin activity.
PCR amplification using genomic DNA as a
template revealed an intron of HS-1 gene located
between two exons within Asn codon of the peptide
coding region (Fig. 4) similar to that found in the
Opiscorpine3 (Opi-3) gene (Zhu and Tytgat, 2004).
It starts with GT and ends with AG as commonly
found in eukaryotes. Comparing intron of HS-1
with that of Opi-3, the whole sequence showed 28%
similarity (Table 4). However, once missing parts
were ignored, the sequence near the 50 and 30 splice
sites exhibited very high homology (Table 4). This
indicates that the sequences near those sites are very
important for biological functions.
4. Discussion
Due to the resistance of pathogenic microorgan-
isms to drugs today, novel antimicrobial agents
from various sources, especially from nature, are
under wide investigation in laboratories around the
world. Scorpion venom is one of the most interest-
ing, since it is a rich source of active molecules.
Their long existence for more than 400 million years
leads to the prediction of highly efficient defense
mechanisms against pathogenic microorganisms. In
this study, we have tried to purify and characterize
the activity of toxin in Thai local scorpion venom.
At the beginning, the crude venom was tested for
paralysis action in cricket and inhibitory action
against various kinds of microorganisms. It showed
acceptable results to various kinds of microorgan-
isms. Finally, the novel toxin was purified and its
activity was confirmed again. The purity of the
protein was unquestionable since not only a single
band was seen on SDS-PAGE analysis, but a single
spot on Edman sequencing analysis and a major
peak from MS also emphasized its high purity.
The purified peptide showed remarkable inhibition
to 3 bacterial species by many hundred folds of
activity compared to the crude venom. After
full-length determination, the amino acid sequence
revealed about 80% similarity to Scorpine family
members. Thus, it is thought to be a member of
the Scorpine family with potassium channel block-
ing and defensin activity and named ‘‘Heteroscor-
pine-1’’ or ‘‘HS-1’’.
We started our study from PD50 determination in
cricket, since toxins from venom of all members of
Family Scorpiones are potassium ion channel

Fig. 4. Heteroscorpine-1 (HS-1) gene and its amino acid sequences. Exons are nucleotides in capital letters. Intron is in small letters.
Untranslated region is capital letters in box. Primer positions are indicated by dotted arrows. Deduced amino acid residues are shown in
capital letters. Residues underlined are the partial amino acid sequences determined by MS/MS analysis. The double underline denotes the
leading sequence. Grey-shaded sequence is Cys pattern of invertebrate defensins, CX416CX2HCX69GX1CX49CX1C, predicted by Froy
and Gurevitz (2003).
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N. Uawonggul et al. / Toxicon 49 (2007) 19–29 27

Fig. 5. Sequence alignment (A) and evolutionary relationship shown by phylogenetic analysis based on primary structures (B) of members
of Scorpine family and K+ channel blocker. Heteroscorpine-1(HS-1) is in this study. Opiscorpine3 is from Opistophthalmus carinatus
(Zhu and Tytgat, 2004). Panscorpine is from Pandinus imperator (Conde et al., 2000). Tityus Scorpine is from Tityus costatus Karsch
(Diego-Garcia et al., 2005). TsTXKb from Tityus serrulatus (Legros et al., 1998), BmTXKb2 from Buthus matensii (Zhu et al., 1999) and
AaTXKb from Androctonus australis (Legros et al., 1998). Identical residues are double asterisked. Conserved replacements are asterisked.
Leading sequences are underlined.

Table 4
HS-1 and Opi-3 Intron alignments

HS-1 intron Opi-3 intron (Zhu and Tytgat, 2004) % similarity

Whole sequence, 1–1073 Whole sequence, 1–1160 28

near 50 splice site 1–424, Omit 83–106, 311–330 near 50 splice site 1–382 78
near 30 splice site 897–1073 30 splice site, 916–1160 38
near 30 splice site, 897–1073 30 splice site 916–1160, Omit 949–955, 976–1006 and 1049–1077 80
near 30 splice site, 989–1073 30 splice site, 1078–1160 82

blockers. The assay is a classical method as the test
for ion channel blocker as reported by Ji et al.
(1996). After comparing gene and amino acid
sequences to the known ones, HS-1 was shown to
be a potassium channel blocker. Such a primitive
technique is recommended here as the preliminary
method for activity assay for any kind of polypep-
tide from scorpion venom predicted as an ion
channel blocker, before the more advanced techni-
ques are used.
HS-1 inhibited growth of three bacterial species
without gram specificity similar to Panscorpine.
This suggests that the action of both does not
mainly attack the bacterial cell wall. However, it
showed no inhibitory effects to fungus F. oxysporum
f. sp. lycopersici, unlike Opiscorpine. This difference
is not obvious and needs further investigation.
Clarification of the mechanism of anti-bacterial
action was investigated by electron microscopy. The
scanning electron microscopy exhibited obvious
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28 N. Uawonggul et al. / Toxicon 49 (2007) 19–29
blebbing of cell surfaces of bacteria susceptible to
HS-1 without any protrusion. There were no pores
observable, indicating that the mechanisms of
inhibition did not involve the penetration of
peptides into the cell wall and/or cell membrane
and pore formation. Potassium ion channel block-
ing activity of HS-1 may play a role in blebbing.
However, the mechanism has to be clarified in
depth.
The amino acid sequence deduced from oligonu-
cleotide without leading sequence (76 amino acids)
has theoretical monoisotopic molecular mass and pI
of 8293.07 Da and 8.79, respectively, whereas the
mass practically determined by mass spectrometry
was 8293.496 Da and pI from 2D-PAGE analysis
was about 9. These correlate remarkably between
the nucleotide sequence of the gene and the putative
encoded protein purified from the venom. After
alignment, HS-1 showed about 80% similarity to
Panscorpine and Opiscorpine. Moreover, HS-1 and
Opiscorpine obviously showed 3 disulfide bridges
and completely followed the typical Cys pattern of
invertebrate defensins (CX416CX2HCX69GX1
CX49CX1C) estimated by Froy and Gurevitz
(2003) (Fig. 4). Thus it can be confidently reported
that HS-1 is a member of the Scorpine family and
has a very high tendency to be an antimicrobial
agent.
Analysis of an intron in the HS-1 gene compared
to that of the Opi-3 gene is very interesting.
Surprisingly, many parts of the introns are missing,
whereas the rest has very high similarity. This
implies a complication of evolution among members
in Family Scorpionidae. Rate of point mutation
looks very low, but deletions or insertions of gene
elements are outstanding. However, high similarities
between both introns exhibit a close relationship in
evolution of the two species, O. carinatus in Africa
and H. laoticus in Southeast Asia. Zhu and Tytgat
(2004) tried to discuss introns of members of
Scorpine family in detail. Although many points
of an intron of HS-1 are similar to that of Opi-3,
several points are different. For example, the
putative U2AF splicing factor binding region of
HS-1 has nearly 100% similarity to Opi-3 gene.
However, repetitive elements of GTATT found
upstream of the branch site in Opi-3 gene are
missing in HS-1 intron indicating that this element
may not be important for biological functions. To
characterize intron of the Scorpine family, large
amounts of data from many genes of Scorpine
members are necessary.
Although the purpose of this study is to find out
the novel chemicals or agents with potent inhibitory
activity against microorganisms, the more impor-
tant objective is an attempt to study venom and
toxin of Thai local endangered venomous species.
Nowadays many venomous insects, fish and arthro-
pods locally found in the tropics are disappearing
without any record of the biological function of
their venom, whereas the rest are close to extinction
from the direct elimination of those dangerous
creatures including devastation of their habitat and
chemicals used in modern agricultural processes.
Urgent studies are necessary.
Acknowledgments
This work was partially supported by the Japan
Society for the Promotion of Science (JSPS)
through a fellowship to SD for part of this research
in Japan between August 1st, 2004 and March 31st,
2005. We deeply appreciate Mrs. Manami Sone and
Professor Naoki Mochizuki of the Department of
Structural Analysis, The National Cardiovascular
Center, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565,
Japan for nucleotide sequencing work.
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