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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 from Chuzhou, Anhui Province, P. R. China. Gram-
widely used in traditional medicine for the treatment positive strains Bacillus subtilis ACCC 11062,
of neural, respiratory and cardiovascular diseases in Staphylococcus aureus CFCC 1117 and Streptococcus
many Asian countries1. However, only a few reports pyogenes CVCC 594; Gram-negative strains
are available about purification and characterization Escherichia coli ACCC 12069, Pseudomonas
of proteins/peptides from the centipede. A high- aeruginosa CMCC (B) 10101, Shigella dysenteriae
molecular mass acidic and heat-labile cardiotoxic CMCC (B) 51832 and Proteus mirabilis CMCC (B)
protein (toxin S) was isolated from the venom of S. s. 49005; and fungal strains Saccharomyces cerevisiae
dehaani2. Saxiphilin, a transferrin was purified from ACCC 2032, Aspergillus niger ACCC 30005 and
the centipede Ethmostigmus rubripes could detect the Mucor bacilliformis AS 3.3420, used in the study
paralytic shellfish toxins3. Also, a novel serine were provided by the China General Microbiological
protease scolonase, composed of 277 amino acids was Culture Collection Center, Beijing, China.
characterized from S. s. mutilans4.
In this study, an antibacterial peptide tentatively Induction of antibacterial substances, extraction of crude
venom and assay of antibacterial activity
named scolopendrin I was purified from the crude
Antibacterial molecules were induced as
venom of S. s. mutilans when injected with E. coli
described5. Briefly, each centipede was injected with
K12D31. The molecular mass, antibacterial, hemolytic
approx. 106 viable, log-phase E. coli K12D31 150 µL,
and agglutination activities of scolopendrin I were
physiological saline (130 mM NaCl, 5 mM KCl, 1
preliminarily investigated.
mM CaCl2) 50 µL and poly IC 50 µL. After 0.5, 1,
Materials and Methods 1.5, 2, 3, 4, 5, 6, 7 and 8 day, the crude venom from
Adult centipedes (Scolopendra subspinipes these ten groups of centipede (5 animals in each
mutilans) each weighing around 3 g were procured group) was collected by an electrical milking
__________ procedure6 and centrifuged at 10,000 rpm for 30 min
*To whom correspondence should be addressed at 4°C. The supernatants were lyophilized and stored
Tel.: 86-25-83598216; E-mail: spidervenom@163.com at -70°C in ultracold freezer for further assay.
Abbreviations: poly IC, polyinosinic-polycytidylic acid; TFA,
trifluoroacetic acid; HPLC, high performance liquid chromatography;
Antibacterial activity of crude venom was
RP-HPLC reverse-phase HPLC, PBS, phosphate-buffered saline, determined by measuring zones of growth inhibition
MIC, minimal inhibitory concentration in thin LB-agar plates with E. coli K12D31
WENHUA et al: ANTI BACTERIAL PEPTIDE FROM VENOM OF CENTIPEDE 89

(welldiffusion assay)7. Each well of 1 mm in diameter 3,000-5,000 Da was separated by cation-exchange


was loaded with 5 µL venom and then incubated at chromatography on TSK-gel SP-5PW column (Toso,
37°C for 12 hr. The minimum inhibitory Japan), initially equilibrated with 0.05 M ammonium
concentration (MIC) of crude venom against acetate buffer, pH 5.1. The column was eluted with a
microorganisms was tested as described8. Unlike linear gradient of 0-80% of 1 M ammonium acetate
bacteria, fungi were inoculated in potato-dextrose at buffer, pH 5.1 over 30 min, at a flow rate of 0.5
25°C. The optical density at 620 nm (OD620) was ml/min. The fraction that showed antibacterial activity
measured. was collected and lyophilized.
Effect of different temperatures, pH, and ionic strengths on
The lyophilized powder was diluted with 1% TFA
antibacterial activity of crude venom and purified by RP-HPLC using a Hypersil BDS C18
Effect of temperature column (4.6 × 150 mm, 5 µm, Dalian Elite Analytical
The lyophilized powder of crude venom from Instruments Co. Ltd., DEAIC), equilibrated with 1%
centipede induced by E. coli for 4 days was dissolved TFA. The column was eluted with a linear 0-50%
in PBS (0.02 M, pH 5.0) (final conc. 1 µg/µL) and gradient of acetonitrile in acidified water over 30 min,
then incubated at ten different temperatures: -20, 4, at a flow rate of 0.7 ml/min and the active fraction
15, 25, 37, 50, 70, 90, 100 and 121°C for 30 min. was collected and lyophilized. The active fraction was
Effect of pH further purified by a RP-HPLC using a Kromasil KR-
The lyophilized powder of crude venom from 60 5CN column (4.6 ×150 mm, EKA Chemicals AB,
centipede induced by E. coli for 4 days was dissolved Sweden), equilibrated with 1% TFA and eluted with a
in disodium hydrogen phosphate-citric acid buffer linear 0-5% gradient of acetonitrile in acidified water,
(final conc. 1 µg/µL) at pH 2.2, 3.0, 4.0, 5.0, 6.0, 7.0 at a flow rate of 0.7 ml/min.
and 8.0, for 48 hr. All chromatography steps were carried out at room
temperature on a HPLC detection system (Hewlett
Effect of ion strength
The lyophilized powder of crude venom from Packard 1050). The column eluent was monitored for
centipede induced by E. coli for 4 days was dissolved absorbance at 280 nm. The fractions with one
in pH 6.0 and pH 8.0 disodium hydrogen phosphate- individual peak were hand-collected and lyophilized
citric acid buffer (0.2, 0.02 and 0.002 M) (final conc. under vacuum. Only one peak was available in the
1 µg/µL), for 48 hr. final purification step (Fig. 7), which was collected,
Antibacterial activity of all the above crude venom lyophilized and tentatively named scolopendrin I.
samples i.e., at different temperatures, pH, and ionic At all the purification steps, antibacterial activity of
strengths, was tested by 96-well microtiter plate. Each the products was determined by measuring growth
well contained 40 µL LB medium culture, with inhibition zones (in mm) on thin agarose plates,
approx. 1 × 105 E. coli K12 D31/mL and 5 µL of the inoculated with E. coli K12 D31. Luria agarose
crude venom samples. Microbial growth was (pH 6.4) was mixed with 60 µL of 10-4 dilution
monitored by optical density (OD) at 492 nm, after (approx. 2 × 105 cells) of log-growth phase of E. coli
incubation for 18 hr at 37°C. The controls were K12D31. The lyophilized powder of the product
performed using bacteria alone, in the presence of the obtained at each step was dissolved in sterile saline
same buffer. solution with a concentration of 100 µg/mL. The 10
µL solution was filled in wells and the plates were
Purification of scolopendrin I
incubated at 37°C for 24 hr. Synthetic cecropin B
The crude venom, which showed activity against
(Sigma) and sterile saline solution were used as
E. coli K12 D31, was dissolved in ultrapure water
positive and negative controls, respectively. Total and
(Arium® 611 water ultrapurifier, Sartorius, Germany),
specific antibacterial activities of the products from
heated at 100°C for 30 min in water bath and
each step were calculated. One unit of antibacterial
centrifuged at 10,000 rpm for 30 min at 4°C. The
factor activity was defined to be equivalent to the
supernatants after concentration and ultrafiltration
activity of 0.1 µg of synthetic cecropin B9.
(using Vivaspin 0.5 ml concentrator Vivascience,
Sartorius AG, Germany) were divided into three parts Tricine-SDS-PAGE
with bases on the molecular mass of <3,000 Da, Tricine-SDS-PAGE of scolopendrin I was carried
3,000-5,000 Da and >5,000 Da, respectively and their out as described earlier10. Gel was cut vertically down
antibacterial activity was assayed7. The ultrafiltrate of 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 etc. Thus, it is anticipated to produce some antibacterial
overlaid with media containing viable cells of E. coli substances for predation and self-protection. After
K12D31 to display the antibacterial activity zone. S. subspinipes was injected with E. coli K12D31 and
poly IC, its venom showed antibacterial activity.
Molecular mass
Although no antibacterial activity was detected in the
Molecular mass of scolopendrin I was determined
crude venom at 0.5 day, after 1 day, diameter of the
using MALDI-TOF-MS with a Bruker ReflexTM
inhibition zone increased to 5.2 cm and thereafter, the
(Bremen, Germany) by the National Center of
activity increased gradually to peak after 3-4 days.
Biomedical Analysis, the Chinese Academy of
Thus, centipedes injected for 4 days were used for the
Military Medical Sciences, Beijing, China.
isolation of antibacterial peptide. The activity almost
Hemolytic and agglutination activities of scolopendrin I remained stable from the 5th to 8th day (Fig. 1).
Scolopendrin I dissolved in PBS buffer (50 mM The crude venom exhibited antimicrobial activity
sodium phosphate buffer, 150 mM NaCl, pH 7.2) against Gram-positive bacteria B. subtilis, S. aureus and
(final conc. was 2.5, 5, 10, 15, 20, 25 and 30 µM) was S. pyogenes, Gram-negative bacteria E. coli,
mixed with the same volume of mouse erythrocytes in P. aeruginosa and S. dysenteriae and the fungi
the same buffer (final 1%, v/v). The negative control S. cerevisiae, A. niger and M. bacilliformis (Table 1).
(0% hemolysis) consisted of erythrocytes in PBS Thus, the activity was relatively broad spectrum, though,
buffer, whereas the positive control (100% hemolysis) no obvious activity was found against P. mirabilis.
consisted of the erythrocytes, mixed with honeybee Compared with the controls, the venom sample (from
venom melittin, dissolved in PBS buffer. All samples, centipede induced by E. coli for 4 days) treated at
including dissolved scolopendrin I and the controls, different temperatures, pH and ionic strengths showed
were incubated with gentle shaking (50 rpm) at 37°C
for 1 hr, placed on ice and centrifuged at 3,000 rpm Table 1―Antimicrobial activity of crude venom of Scolopendra
for 10 min at 4°C to obtain the supernatants. The subspinipes mutilans
hemolytic activity of scolopendrin I was determined Minimum inhibitory
by measuring the absorbance of the supernatant at 546 conc. (µg/mL)
nm. The agglutination activity was assayed visually. Bacillus subtilis ACCC 11062 4.1
A negative control was set as above, but the positive Staphylococcus aureus CFCC 1117 5.6
control used was phytohaemagglutinin, not melittin. Streptococcus pyogenes CVCC 594 7.2
Escherichia coli ACCC 12069 3.3
Results and Discussion
Antimicrobial activity and physicochemical properties of Pseudomonas aeruginosa CMCC (B) 10101 7.2
crude venom Shigella dysenteriae CMCC (B) 51832 4.1
Being a soil animal, S. subspinipes is inevitably Proteus mirabilis CMCC (B) 49005 NA
exposed to microorganisms, such as bacteria and fungi, Saccharomyces cerevisiae ACCC 2032 14.4
Aspergillus niger ACCC 30005 5.6
Mucor bacilliformis AS 3.3420 14.4
NA, no activity

Fig. 1―Antibacterial activity of crude venom of Scolopendra


subspinipes mutilans, injected with E. coli K12D31, as indicated by Fig. 2―Effect of temperature on crude venom of S. s. mutilans
the diameter of inhibition zone [Values are averaged from [OD values at 492 nm of E. coli K12D31 incubated with crude
duplicated experiments] 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


3,000-5,000 Da from crude venom on a TSK-GEL SP-5PW
column [The height of bars is proportional to antibacterial activity
Fig. 3―Effect of pH on the crude venom of S. s. mutilans [OD of the fractions against E. coli. The solid line represents
values at 492 nm of E. coli K12D31 incubated with the crude absorbance of fractions at 280 nm and broken line represents a
venom at different pH for 48 hr] 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 and was further purified by RP-HPLC using Kromasil
removed from the sample by the cation-exchange KR-60 5CN (Fig. 7), pooled and lyophilized. The
chromatography. Three peaks A, B and C, all having purified fraction was tentatively named scolopendrin
antibacterial activity, were obtained. As the activity of I. Its molecular mass as determined by MALDI-TOF
fraction C was most significant (Fig. 5), it was mass spectrometry was 4,498 Da (Fig. 8).
collected, purified and separated by RP-HPLC on Tricine-SDS-PAGE showed only one band with
Hypersil BDS C18 column, as shown in Fig. 6. The molecular mass in the range of 4.0-6.0 kDa, suggesting
highest peak inhibited the growth of E. coli. K12D31, 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 93

Fig. 9―Tricine-SDS-PAGE of scolopendrin I [Lane M, molecular Fig. 10―Hemolytic activity of scolopendrin I [The samples (2.5
mass markers; lane A, scolopendrin I; and lane B, zone of growth to 30 µM) were mixed with mouse erythrocytes and incubated for
inhibition, corresponding to the position of peptide band. The gel 1 hr at 37°C. Hemolytic activity was observed by monitoring the
was overlaid with viable cells of E.coli K12D31, imbedded in LB increase in the absorbance at 546 nm. Closed squares represent for
agar. Incubation was at 37°C for 24 hr] scolopendrin I. Open and closed circles represent negative and
positive control, respectively. Values are averaged from repeated
(Fig. 9B). The above results suggest that scolopendrin experiments]
I is a homogeneous antibacterial peptide. The
solubility of crude venom in ultrapure water heat- was found to be 5.6 µg/ml, indicating the presence of
treatment at 100°C for 30 min, suggest water some more potent antibacterial agents in the venom.
solubility and high thermal stability of scolopendrin I.
Also, purification of this fraction by cation-exchange References
chromatography suggests its alkaline nature. 1 Pempberton, R.W (1999) J Ethnopharmacol 65, 207-216
However, other properties, such as antimicrobial 2 Gomes A, Datta A, Sarangi B, Kar P K& Lahiri S C (1983)
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the concentration below 30 µM (Fig. 10). The negative 5 Qu X M, Steiner H, Engstrom A, Bennich H & Boman HG
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