Indian Journal of Biochemistry & Biophysics

Vol. 43, April 2006, pp 88-93

Induction, purification and characterization of an antibacterial peptide

scolopendrin I from the venom of centipede Scolopendra subspinipes mutilans
Ren Wenhua1, Zhang Shuangquan1*, Song Daxiang2, Zhou Kaiya2 and Yang Guang2
1
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University,
Nanjing 210097, P. R. China
2
Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University,
Nanjing 210097, P. R. China
Received 15 June 2005; revised 17 November 2005
The crude venom of the centipede Scolopendra subspinipes mutilans, injected with Escherichia coli K12D31 for 3-4
days showed broad-spectrum antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. It showed
good antibacterial activity against E. coli K12D31 at different temperatures, pH, and ionic strengths. The crude venom was
heated at 100°C for 30 min, centrifuged at 10,000 rpm for 30 min at 4°C and the supernatants were obtained, from which an
antibacterial fraction having a molecular mass of 3000-5000 Da, was further separated by ultrafiltration. A homogeneous
antibacterial peptide named scolopendrin I, having a molecular mass of 4,498 Da, was isolated using cation-exchange
chromatography and two steps of reverse-phase high performance liquid chromatography (RP-HPLC). Scolopendrin I did
not show any hemolytic and agglutination activities at the concentration below 30 µM.
Keywords: Centipede, Scolopendra subspinipes mutilans, antibacterial peptide, scolopendrin I, venom, E. coli K12D31, induction,
hemolytic activity, agglutination activity

The centipede Scolopendra subspinipes mutilans is
widely used in traditional medicine for the treatment
of neural, respiratory and cardiovascular diseases in
many Asian countries1. However, only a few reports
are available about purification and characterization
of proteins/peptides from the centipede. A high-
molecular mass acidic and heat-labile cardiotoxic
protein (toxin S) was isolated from the venom of S. s.
dehaani2. Saxiphilin, a transferrin was purified from
the centipede Ethmostigmus rubripes could detect the
paralytic shellfish toxins3. Also, a novel serine
protease scolonase, composed of 277 amino acids was
characterized from S. s. mutilans4.
In this study, an antibacterial peptide tentatively
named scolopendrin I was purified from the crude
venom of S. s. mutilans when injected with E. coli
K12D31. The molecular mass, antibacterial, hemolytic
and agglutination activities of scolopendrin I were
preliminarily investigated.
Materials and Methods
Adult centipedes (Scolopendra subspinipes
mutilans) each weighing around 3 g were procured
__________
*To whom correspondence should be addressed
Tel.: 86-25-83598216; E-mail: spidervenom@163.com
Abbreviations: poly IC, polyinosinic-polycytidylic acid; TFA,
trifluoroacetic acid; HPLC, high performance liquid chromatography;
RP-HPLC reverse-phase HPLC, PBS, phosphate-buffered saline,
MIC, minimal inhibitory concentration
from Chuzhou, Anhui Province, P. R. China. Gram-
positive strains Bacillus subtilis ACCC 11062,
Staphylococcus aureus CFCC 1117 and Streptococcus
pyogenes CVCC 594; Gram-negative strains
Escherichia coli ACCC 12069, Pseudomonas
aeruginosa CMCC (B) 10101, Shigella dysenteriae
CMCC (B) 51832 and Proteus mirabilis CMCC (B)
49005; and fungal strains Saccharomyces cerevisiae
ACCC 2032, Aspergillus niger ACCC 30005 and
Mucor bacilliformis AS 3.3420, used in the study
were provided by the China General Microbiological
Culture Collection Center, Beijing, China.
Induction of antibacterial substances, extraction of crude
venom and assay of antibacterial activity
Antibacterial molecules were induced as
described5. Briefly, each centipede was injected with
approx. 106 viable, log-phase E. coli K12D31 150 µL,
physiological saline (130 mM NaCl, 5 mM KCl, 1
mM CaCl2) 50 µL and poly IC 50 µL. After 0.5, 1,
1.5, 2, 3, 4, 5, 6, 7 and 8 day, the crude venom from
these ten groups of centipede (5 animals in each
group) was collected by an electrical milking
procedure6 and centrifuged at 10,000 rpm for 30 min
at 4°C. The supernatants were lyophilized and stored
at -70°C in ultracold freezer for further assay.
Antibacterial activity of crude venom was
determined by measuring zones of growth inhibition
in thin LB-agar plates with E. coli K12D31
 WENHUA et al: ANTI BACTERIAL PEPTIDE FROM VENOM OF CENTIPEDE
(welldiffusion assay)7. Each well of 1 mm in diameter
was loaded with 5 µL venom and then incubated at
37°C for 12 hr. The minimum inhibitory
concentration (MIC) of crude venom against
microorganisms was tested as described8. Unlike
bacteria, fungi were inoculated in potato-dextrose at
25°C. The optical density at 620 nm (OD620) was
measured.
Effect of different temperatures, pH, and ionic strengths on
antibacterial activity of crude venom
Effect of temperature
The lyophilized powder of crude venom from
centipede induced by E. coli for 4 days was dissolved
in PBS (0.02 M, pH 5.0) (final conc. 1 µg/µL) and
then incubated at ten different temperatures: -20, 4,
15, 25, 37, 50, 70, 90, 100 and 121°C for 30 min.
Effect of pH
The lyophilized powder of crude venom from
centipede induced by E. coli for 4 days was dissolved
in disodium hydrogen phosphate-citric acid buffer
(final conc. 1 µg/µL) at pH 2.2, 3.0, 4.0, 5.0, 6.0, 7.0
and 8.0, for 48 hr.
Effect of ion strength
The lyophilized powder of crude venom from
centipede induced by E. coli for 4 days was dissolved
in pH 6.0 and pH 8.0 disodium hydrogen phosphate-
citric acid buffer (0.2, 0.02 and 0.002 M) (final conc.
1 µg/µL), for 48 hr.
Antibacterial activity of all the above crude venom
samples i.e., at different temperatures, pH, and ionic
strengths, was tested by 96-well microtiter plate. Each
well contained 40 µL LB medium culture, with
approx. 1 × 105 E. coli K12 D31/mL and 5 µL of the
crude venom samples. Microbial growth was
monitored by optical density (OD) at 492 nm, after
incubation for 18 hr at 37°C. The controls were
performed using bacteria alone, in the presence of the
same buffer.
Purification of scolopendrin I
The crude venom, which showed activity against
E. coli K12 D31, was dissolved in ultrapure water
(Arium® 611 water ultrapurifier, Sartorius, Germany),
heated at 100°C for 30 min in water bath and
centrifuged at 10,000 rpm for 30 min at 4°C. The
supernatants after concentration and ultrafiltration
(using Vivaspin 0.5 ml concentrator Vivascience,
Sartorius AG, Germany) were divided into three parts
with bases on the molecular mass of <3,000 Da,
3,000-5,000 Da and >5,000 Da, respectively and their
antibacterial activity was assayed7. The ultrafiltrate of
89
3,000-5,000 Da was separated by cation-exchange
chromatography on TSK-gel SP-5PW column (Toso,
Japan), initially equilibrated with 0.05 M ammonium
acetate buffer, pH 5.1. The column was eluted with a
linear gradient of 0-80% of 1 M ammonium acetate
buffer, pH 5.1 over 30 min, at a flow rate of 0.5
ml/min. The fraction that showed antibacterial activity
was collected and lyophilized.
The lyophilized powder was diluted with 1% TFA
and purified by RP-HPLC using a Hypersil BDS C18
column (4.6 × 150 mm, 5 µm, Dalian Elite Analytical
Instruments Co. Ltd., DEAIC), equilibrated with 1%
TFA. The column was eluted with a linear 0-50%
gradient of acetonitrile in acidified water over 30 min,
at a flow rate of 0.7 ml/min and the active fraction
was collected and lyophilized. The active fraction was
further purified by a RP-HPLC using a Kromasil KR-
60 5CN column (4.6 ×150 mm, EKA Chemicals AB,
Sweden), equilibrated with 1% TFA and eluted with a
linear 0-5% gradient of acetonitrile in acidified water,
at a flow rate of 0.7 ml/min.
All chromatography steps were carried out at room
temperature on a HPLC detection system (Hewlett
Packard 1050). The column eluent was monitored for
absorbance at 280 nm. The fractions with one
individual peak were hand-collected and lyophilized
under vacuum. Only one peak was available in the
final purification step (Fig. 7), which was collected,
lyophilized and tentatively named scolopendrin I.
At all the purification steps, antibacterial activity of
the products was determined by measuring growth
inhibition zones (in mm) on thin agarose plates,
inoculated with E. coli K12 D31. Luria agarose
(pH 6.4) was mixed with 60 µL of 10-4 dilution
(approx. 2 × 105 cells) of log-growth phase of E. coli
K12D31. The lyophilized powder of the product
obtained at each step was dissolved in sterile saline
solution with a concentration of 100 µg/mL. The 10
µL solution was filled in wells and the plates were
incubated at 37°C for 24 hr. Synthetic cecropin B
(Sigma) and sterile saline solution were used as
positive and negative controls, respectively. Total and
specific antibacterial activities of the products from
each step were calculated. One unit of antibacterial
factor activity was defined to be equivalent to the
activity of 0.1 µg of synthetic cecropin B9.
Tricine-SDS-PAGE
Tricine-SDS-PAGE of scolopendrin I was carried
out as described earlier10. Gel was cut vertically down
the middle after electrophoresis. One half was stained
90 INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, APRIL 2006
with Coomassie blue R250 and the other half was
overlaid with media containing viable cells of E. coli
K12D31 to display the antibacterial activity zone.
Molecular mass
Molecular mass of scolopendrin I was determined
using MALDI-TOF-MS with a Bruker ReflexTM
(Bremen, Germany) by the National Center of
Biomedical Analysis, the Chinese Academy of
Military Medical Sciences, Beijing, China.
Hemolytic and agglutination activities of scolopendrin I
Scolopendrin I dissolved in PBS buffer (50 mM
sodium phosphate buffer, 150 mM NaCl, pH 7.2)
(final conc. was 2.5, 5, 10, 15, 20, 25 and 30 µM) was
mixed with the same volume of mouse erythrocytes in
the same buffer (final 1%, v/v). The negative control
(0% hemolysis) consisted of erythrocytes in PBS
buffer, whereas the positive control (100% hemolysis)
consisted of the erythrocytes, mixed with honeybee
venom melittin, dissolved in PBS buffer. All samples,
including dissolved scolopendrin I and the controls,
were incubated with gentle shaking (50 rpm) at 37°C
for 1 hr, placed on ice and centrifuged at 3,000 rpm
for 10 min at 4°C to obtain the supernatants. The
hemolytic activity of scolopendrin I was determined
by measuring the absorbance of the supernatant at 546
nm. The agglutination activity was assayed visually.
A negative control was set as above, but the positive
control used was phytohaemagglutinin, not melittin.
Results and Discussion
Antimicrobial activity and physicochemical properties of
crude venom
Being a soil animal, S. subspinipes is inevitably
exposed to microorganisms, such as bacteria and fungi,
Fig. 1―Antibacterial activity of crude venom of Scolopendra
subspinipes mutilans, injected with E. coli K12D31, as indicated by
the diameter of inhibition zone [Values are averaged from
duplicated experiments]
etc. Thus, it is anticipated to produce some antibacterial
substances for predation and self-protection. After
S. subspinipes was injected with E. coli K12D31 and
poly IC, its venom showed antibacterial activity.
Although no antibacterial activity was detected in the
crude venom at 0.5 day, after 1 day, diameter of the
inhibition zone increased to 5.2 cm and thereafter, the
activity increased gradually to peak after 3-4 days.
Thus, centipedes injected for 4 days were used for the
isolation of antibacterial peptide. The activity almost
remained stable from the 5th to 8th day (Fig. 1).
The crude venom exhibited antimicrobial activity
against Gram-positive bacteria B. subtilis, S. aureus and
S. pyogenes, Gram-negative bacteria E. coli,
P. aeruginosa and S. dysenteriae and the fungi
S. cerevisiae, A. niger and M. bacilliformis (Table 1).
Thus, the activity was relatively broad spectrum, though,
no obvious activity was found against P. mirabilis.
Compared with the controls, the venom sample (from
centipede induced by E. coli for 4 days) treated at
different temperatures, pH and ionic strengths showed
Table 1―Antimicrobial activity of crude venom of Scolopendra
subspinipes mutilans
Minimum inhibitory
conc. (µg/mL)
Bacillus subtilis ACCC 11062 4.1
Staphylococcus aureus CFCC 1117 5.6
Streptococcus pyogenes CVCC 594 7.2
Escherichia coli ACCC 12069 3.3
Pseudomonas aeruginosa CMCC (B) 10101 7.2
Shigella dysenteriae CMCC (B) 51832 4.1
Proteus mirabilis CMCC (B) 49005 NA
Saccharomyces cerevisiae ACCC 2032 14.4
Aspergillus niger ACCC 30005 5.6
Mucor bacilliformis AS 3.3420 14.4
NA, no activity
Fig. 2―Effect of temperature on crude venom of S. s. mutilans
[OD values at 492 nm of E. coli K12D31 incubated with crude
venom at ten different temperatures for 30 min]
 WENHUA et al: ANTI BACTERIAL PEPTIDE FROM VENOM OF CENTIPEDE 91

lower OD values, indicating the antibacterial activity incubated at 100°C for 30 min, the fraction 3,000-
had thermal (Fig. 2), pH (Fig. 3) and ionic strength 5,000 Da showed highest antibacterial activity,
(Fig. 4) stability. The thermal stability, insensitivity to compared to other fractions. Therefore, heat-treatment
changes of pH and ionic strength of crude venom was chosen as the first step for the purification of
showed a great similarity to insect antibacterial scolopendrin I, and then, ultrafiltrate of 3,000-5,000
peptides11. Da was collected and fractionated on TSK-gel SP-
5PW column. The yield and the antibacterial activity
Purification of scolopendrin I
From the crude venom, without being heated up to against E. coli K12 D31 of the products from different
purification steps are given in Table 2.
100°C for 30 min, five fractions, based on the
molecular mass i.e., <3,000 Da, 3,000-5,000 Da,
5,000-10,000 Da, 10,000-30,000 Da and >30,000 Da
were separated by ultrafiltration. All the fractions
showed antibacterial activity, suggesting that
centipede could produce peptides/proteins with
various molecular masses after induction. When the
crude venom was heated up to 100°C for 30 min, total
amount of protein decreased significantly from
1364.11 mg to 298.32 mg, while 44.88% antibacterial
activity (i.e. purification yield9) was retained. The
specific antibacterial activity, however, increased Fig. 4―Effect of ionic strength on the crude venom of S. s.
mutilans [OD values at 492 nm of E. coli K12 D31 incubated with
significantly after the heat-treatment (Table 2). Thus, crude venom at pH 6.0 and pH 8.0 disodium hydrogen phosphate-
heat-treatment was efficient to remove unwanted citric acid buffer for 48 hr]
proteins at the beginning of the purification
procedure. Furthermore, when the crude venom was

Fig. 5―Cation-exchange chromatography of the ultrafiltrate of

Fig. 3―Effect of pH on the crude venom of S. s. mutilans [OD
values at 492 nm of E. coli K12D31 incubated with the crude
venom at different pH for 48 hr]
3,000-5,000 Da from crude venom on a TSK-GEL SP-5PW
column [The height of bars is proportional to antibacterial activity
of the fractions against E. coli. The solid line represents
absorbance of fractions at 280 nm and broken line represents a
gradient of 1 M ammonium acetate]

Table 2―Antibactericidal activity against E. coli K12 D31 and yield of products from each purification step for scolopendrin I
[Synthetic cecropin B was used as positive control and sterile saline solution as the negative control. One unit of antibacterial factor
activity was defined to be equivalent to the activity of 0.1 µg of synthetic cecropin B]
Total protein (mg) Total activity (KU) Specific activity (KU/mg) Purification yield (%)
Crude venom 1364.11 82.42 0.060 100
Heated at 100°C for 30 min 298.32 36.99 0.124 44.88
Ultrafiltrate (3000-5000 Da) 35.65 7.028 0.197 8.53
Cation-exchange 1.21 5.603 4.631 6.79
(using HPLC P-5PW)
RP-HPLC (using BDS C 18) 0.78 2.711 3.475 3.29
RP-HPLC (using KR-60 5CN) 0.77 2.695 3.500 3.27
92 INDIAN J. BIOCHEM. BIOPHYS., VOL. 43, APRIL 2006

Nucleic acid, which carries negative charge, was
removed from the sample by the cation-exchange
chromatography. Three peaks A, B and C, all having
antibacterial activity, were obtained. As the activity of
fraction C was most significant (Fig. 5), it was
collected, purified and separated by RP-HPLC on
Hypersil BDS C18 column, as shown in Fig. 6. The
highest peak inhibited the growth of E. coli. K12D31,
and was further purified by RP-HPLC using Kromasil
KR-60 5CN (Fig. 7), pooled and lyophilized. The
purified fraction was tentatively named scolopendrin
I. Its molecular mass as determined by MALDI-TOF
mass spectrometry was 4,498 Da (Fig. 8).
Tricine-SDS-PAGE showed only one band with
molecular mass in the range of 4.0-6.0 kDa, suggesting
scolopendrin I is a small polypeptide (Fig. 9A). It
exhibited an antibacterial zone in the same position

Fig. 6―RP-HPLC of fraction C pooled from cation-exchange Fig.7―RP-HPLC of fraction from C18 RP-HPLC on a Kromasil
chromatography on a Hypersil BDS C18 column [The solid line KR-60 5CN column [The solid line represents fractions
represents absorbance of fractions at 280 nm and the broken line absorbance at 280 nm and the broken line represents a linear
represents a linear gradient of acetonitrile in constant 1% TFA] gradient of acetonitrile in constant 1% TFA]

Fig. 8―MALDI-TOF mass spectra of scolopendrin I

 WENHUA et al: ANTI BACTERIAL PEPTIDE FROM VENOM OF CENTIPEDE
Fig. 9―Tricine-SDS-PAGE of scolopendrin I [Lane M, molecular
mass markers; lane A, scolopendrin I; and lane B, zone of growth
inhibition, corresponding to the position of peptide band. The gel
was overlaid with viable cells of E.coli K12D31, imbedded in LB
agar. Incubation was at 37°C for 24 hr]
(Fig. 9B). The above results suggest that scolopendrin
I is a homogeneous antibacterial peptide. The
solubility of crude venom in ultrapure water heat-
treatment at 100°C for 30 min, suggest water
solubility and high thermal stability of scolopendrin I.
Also, purification of this fraction by cation-exchange
chromatography suggests its alkaline nature.
However, other properties, such as antimicrobial
spectrum, amino acid composition, primary structure,
etc. are required be studied further.
Scolopendrin I did not show hemolytic activity at
the concentration below 30 µM (Fig. 10). The negative
control also did not show hemolytic activity. In
contrast, the positive control melittin showed 100%
hemolysis at a concentration of about 5 µM. On the
other hand, hadrurin, an antibacterial peptide from the
scorpion showed 75% hemolysis activity at a
concentration around 10 µM12. Another antibacterial
peptide tachystatin C, from the horseshoe crab showed
hemolysis below 25 µM13. Scolopendrin also showed
no agglutination activity against mouse erythrocyte at
the concentration below 30 µM on macroscopic
observation; in contrast, positive control showed haem-
agglutination activity.
Oligo-3-aminopyridine, a potent antibacterial agent
against S. aureus has minimum inhibitory
concentration (MIC) of 25 µg/ml14. Although the MIC
of scolopendrin I was not tested, the MIC of crude
venom of Scolopendra subspinipes against S. aureus
93
Fig. 10―Hemolytic activity of scolopendrin I [The samples (2.5
to 30 µM) were mixed with mouse erythrocytes and incubated for
1 hr at 37°C. Hemolytic activity was observed by monitoring the
increase in the absorbance at 546 nm. Closed squares represent for
scolopendrin I. Open and closed circles represent negative and
positive control, respectively. Values are averaged from repeated
experiments]
was found to be 5.6 µg/ml, indicating the presence of
some more potent antibacterial agents in the venom.
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