1119
Purification and characterization of hemocyte
Phenoloxidases in
CHILO SUPPRESSALIS
Walker
(lePidoPtera: crambidae)
Seyyedeh Kimia Mirhaghparast1, Arash Zibaee1,*, Hassan Hoda2 and Mahmoud Fazeli-Dinan3
1Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran. 2Biological Control Department, Iranian Institute of Plant Protection, Amol, Iran.
3Department of Medical Entomology and Vector Control, Health Sciences Research Center, Faculty of Health,
Mazandaran University of Medical Sciences, Sari, Iran
*corresponding author: arash.zibaee@gmx.com, arash.zibaee@guilan.ac.ir
abstract: In the current study, two phenoloxidases (POs) from the larvae of Chilo suppressalis Walker were extracted and purified by column chromatography using Sepharyl G-100 and DEAE-Cellulose fast flow column. Two proteins possessing PO activity, named as POI and POII, were extracted by purification, 5.08- and 5.62-fold, respectively, with 8.94% and 7.31% recoveries, respectively. Also, the specific activities of POI and POII were 0.478 and 0.529 U/mg protein, respectively. Finally, the molecular weights of POI and POII were calculated as 94.6 and 95.7 kDa, respectively. Kinetic parameters of the purified phenoloxidases by Lineweaver-Burk analysis were Vmax of 2.27 and 1.11 U/mg protein and Km of 15.51 and 17.31 mM for POI and POII, respectively. Mg2+ and Cu2+ significantly increased the PO activities. Ca2+ decreased the activity of POI and
showed no statistical effects on POII activity. EDTA and DTC significantly inhibited the activities of the purified enzymes, while triethylenetetramine hexaacetic acid (TTHA) and RGTA showed no significant effects on enzymatic activities.
key words: Chilo suppressalis; phenoloxidase; purification; characterization received: January 7, 2015; revised: February 3, 2015; accepted: February 3, 2015
introduction
Insect phenoloxidases contain a laccase-type enzyme (EC 1.10.3.2) that oxidizes o- or p-diphenols to qui-nones involved in sclerotization of the cuticle (Ara-kane et al., 2005; Dittmer et al., 2004) and a tyrosine-like enzyme that hydroxylates tyrosine (EC 1.14.18.1) and oxidizes o-diphenols to quinones (EC 1.10.3.1) involved in immune reactions (Gorman et al., 2007; Beckage, 2008). Phenoloxidases (POs) are available as inactive zymogens, prophenoloxidases (ProPOs). These inactive forms are polypeptides of approxi-mately more than 80kDa molecular weight that con-tain two atoms of copper per protein molecule and exhibit structural homology with the hemocyanins of arthropods and hexamerin storage proteins of insects (Ashida and Brey, 1997). Phenoloxidases (POs) have critical roles in immune responses, e.g. the final stages
of nodule formation and encapsulation to completely disable invading objectives (González-Santoyo and Córdoba-Aguilar, 2012). Since POs are available as zymogens, a protease cascade must change propheno-loxidase by the action of the hemolymph serine prote-ases (Kim et al., 2002). The proteprote-ases attack the Arg-Phe bond of POs and remove a N-terminal 50 amino acid fragment (Fujimoto et al., 1995; Kim et al., 2002). In the immune process, phenoloxidase catalyzes the conversion of phenols to quinones then it polymer-izes quinones to melanin for deposition in nodules or capsules around invading objects (Klowden, 2007).
Insect phenoloxidases are interesting since of their important engagement of molecular and physio-logical processes in insect immunity and the potential to be used for population control of agricultural pests.
is a serious pest of rice in the Middle East, south-ern Asia, north Africa and some European countries that utilize the inner parts of rice stems (Zibaee et al., 2009). Chemical spraying can be inefficient since no chemicals reach the larvae. Nowadays, researches have been concentrated on the microbial control of larvae by various entomopathogenic fungi, mainly
Beauveria bassiana sensu latto and Metarhizium anisopliae s.l.(Zibaee and Ramzi, 2014; Zibaee and Malagoli, 2014).Our previous study revealed that different concentrations of hexaflumuron and pyri-proxyfen, two insect growth regulators, inhibited the PO activity of C. suppressalis (Mirhaghparast and Zi-baee, 2013). It is mandatory to understand fully the nature and biochemical properties of insect POs to target them for pest control tactics. Hence, the ob-jectives of the current study were (i) the extraction and purification of PO from the hemocytes of C. sup-pressalis larvae, and (ii) determination of enzymatic activity using ions and chelating agents.
materials and methods
insect rearing
Egg patches of C. suppressalis were collected from rice fields and placed in containers containing rice seed-lings. Insects were reared using a modified method of Zibaee et al.(2009) at 28±1°C, 80% relative hu-midity (RH) under conditions of 16 h light:8 h dark. Laboratory conditions were checked, the containers were cleaned, and fresh stems were provided for lar-vae every day.
collection of hemolymph
Hemolymph of C. suppressalis was collected from the first abdominal proleg of larvae by a 50-μL sterile glass capillary tube (Sigma-Aldrich Co.). The hemo-lymph was immediately diluted by an anticoagulant solution (0.01 M ethylenediaminetetraacetic acid, 0.1 M glucose, 0.062 M NaCl, and 0.026 M citric acid, pH 4.6), as described by Azambuja et al. (1991).
Po preparation and assay
The collected samples were centrifuged at 28500×g
for 5 min; the supernatant was discarded and the pel-let was washed gently by universal buffer (20 mM, pH 7, containing succinate, glycine and 2-morpho-lino ethanesulfonic acid (Frugoni, 1957; Leonard et al., 1985). Cells were homogenized in 200 μL of the buffer, centrifuged at 28500×g for 15 min, and the he-mocyte lysate supernatant (HLS) was used to measure PO activity. Samples were pre-incubated with the buf-fer at 30°C before 50 μL of 10 mM aqueous solution of substrate L-dihydroxyphenylalanine (L-DOPA) was added to the reaction medium. The mixture was incu-bated for 5 min at 30°C and PO activity was measured at 492 nm. One unit of PO activity represents the amount of enzyme required to produce an increase in OD490 of 0.01 per min (Dularay and Lackie, 1985). Purification of Po
rate of 60 mL/h. Fractions showing PO activity were pooled and stored at −20°C for subsequent analysis. Polyacrylamide gel electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) using a 4% (w/v) stacking gel and a 10% (w/v) separating gel as described by Laemmli (1970), was used to determine the purity and molecular mass of the purified PO. Molecular mass of the enzyme was estimated by using standard markers, β-galactosidase (116 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), lactate de-hydrogenase (35.5 kDa), restriction endonuclease Bsp 981 (25 kDa), β-lactoglobulin (18.4 kDa) and lyso-zyme (14.4 kDa). After SDS-PAGE, proteins on the polyacrylamide gel were stained with 0.2% Coomassie brilliant blue R-250.
kinetic parameters of Po
Kinetic parameters of the purified POs were carried out according to the method described by Eisenthal and Cornish-Bowden (1974) using L-DOPA. Concen-trations of the substrates were 2, 4, 6, 8 and 10 mM. Reaction mixture contained 50 μL of universal buf-fer, 30 μL of the substrate (mentioned concentrations) and 20 μL of the purified PO. Incubation time was continued for 5 min and absorbance was read at 492 nm. The obtained data were inserted in Sigma-Plot software (version 6) to calculate Vmax and Km values. effect of ions and chelating agents
on the purified Po
The effects of mono- and divalent cations on the pu-rified PO of C. suppressalis were determined by us-ing NaCl, KCl, CaCl2, CuCl2 and MgCl2. The reac-tion mixture was 50 μL of universal buffer, 30 μL of L-DOPA as substrate, 30 μL of the cations in 5 mM of concentration and 20 μL of the purified PO. In-cubation time was continued for 5 min and absor-bance was read at 492 nm. The effects of enzymatic inhibitors on PO activity were determined using 5 mM of ethylenediaminetetraacetic acid (EDTA),
di-ethyldithiocarbamate (DTC), ethylene glycol-bis(2-aminoethylether)-N,N,Nʹ,Nʹ-tetraacetic acid (EGTA) and triethylenetetramine-N,N,N’,N”,N”’,N”’-hexaace-tic acid (TTHA). Reaction mixture was 50 μL of uni-versal buffer, 30 μL of L-DOPA as substrate, 30 μL of the inhibitors and 20 μL of the purified POs. Incuba-tion time was continued for 5 min and absorbance was read at 492 nm.
Protein assay
Protein concentrations were determined according to the method described by Lowry et al. (1951). statistical analysis
The experiments were designed based on a complete-ly randomized scheme. All data were compared by one-way analysis of variance followed by Tukey’s post hoc test at p ≤ 0.05.
results and discussion
pu-rified in several insect species (Anderson et al., 1989; Kopacek et al., 1995; Asano an Ashida, 2001; Feng et al., 2008; Xue et al., 2006; Wang et al., 2010; Ajamhas-sani et al., 2012; Zibaee et al., 2011; Delkash-Roudsari et al., 2015). The majority of studies detected one isoform of PO in their models (Lockey and; Ourth,
1992; Lee and Anstee, 1995; Asada and Sezaki, 1999; Zufelato et al., 2004; Zibaee et al., 2011; Delkash-Roudsari et al., 2015), while others purified two or three isoforms (Fujimoto et al., 1993; Kopácek et al., 1995; Yasuhara et al., 1995; Müller et al., 1999), which is in accordance with our results (two isoforms in C.
table 1. Purification of phenoloxidase in C. suppressalis.
Purification steps unit activity (u)
Protein (mg/dl)
specific activity
(u/mg protein) recovery (%) Purification fold
Crude 0.123 1.30 0.094 100
-Ammonium sulfate 30 0.078 0.68 0.114 63.41 1.21
Ammonium sulfate 70 0.075 0.62 0.120 60.97 1.27
Sepharyl G-100 0.045 0.34 0.132 36.58 1.40
POI 0.011 0.023 0.478 8.94 5.08
POII 0.009 0.017 0.529 7.31 5.62
*All steps were carried out at 4˚C.
suppressalis). Cui et al. (2000) suggested that multiple PO isoforms have evolved by gene duplications and divergence. Variations in environmental conditions and exposure to various stresses such as pathogens possibly contributed to their maintaining and diver-sity in insect populations.
It has now been observed in insects that the POs have a putative thiol-ester site and two distinct copper binding regions, CuA and CuB, including 6 histidine residues that are considered to be ligands for the two copper atoms (Beckage, 2008). In our study, Mg2+ and
Cu2+ increased POI and POII activities as compared
to the control (Table 2). Also, general and specific chelating agents were used to prove the presence of metal ions in active sites of the purified POs. Results demonstrated the inhibitory effects of EDTA (general chelating agent) and DTC (copper chelating agent) against purified POs of C. suppressalis (Table 3). Ther observation of the higher activity of the POs in the presence of Cu2+ indicates that the enzyme is a
metal-loprotein, and thus small changes at the metal center can amplify its biological activity. The involvement of metal ions in media containing purified PO causes an increase in the β-sheet content and increased activity of the enzyme (Feng et al., 2008). Similar results have been reported by other authors as well (Andersson et al., 1989; Ashida et al., 1983; Dunphy, 1991; Feng et al., 2008; Ajamhassani et al., 2011; Zibaee et al., 2011a). Finally, metal ions affect enzymatic activity
via several possible mechanisms: (i) they can activate electrophiles and nucleophiles by taking and releas-ing electrons; (ii) they prevent the reactions of nu-cleophiles; (iii) ions increase the efficiency of enzyme activity by keeping the enzyme and substrate in close proximity through the coordination bonds,and (iv)
fig. 2. Double reciprocal plot to show kinetic parameters of the purified phenoloxidases from C. suppressalis. (1/Vmax=intercept on the 1/V0 ordinate, −1/Km=intercept on the negative side of the 1/[S] abscissa).
table 2. Effects of mono- and divalent cations on the purified POs of C. suppressalis.
cations concentrations (mm)
relative activity of Poi
(%)±se
relative activity of Poii
(%)±se
Control 0 100 100
Na+ 5 91±6.32 101±6.98
K+ 5 102±8.17 10±14.78
Ca2+ 5 74±2.41* 96±23.74
Mg2+ 5 131±11.26* 128±31.25*
Cu2+ 5 158±22.48* 179±14.03*
*Statistical differences are indicated by asterisks (Tukey’s test, p ≤ 0.05). table 3. Effects of mono- and divalent cations on the purified POs of C. suppressalis.
cations concentra-tions (mm)
relative activity of Poi
(%)±se
relative activity of Poii
(%)±se
Control 0 100 100
EDTA 5 55±3.47* 47±5.18*
EGTA 5 100±9.25 103±18.25
DTC 5 48.67±5.19* 28±1.84*
TTHA 5 98±13.15 100±22.58
ions put active groups in the enzyme and substrate structure in an effective position and increase the ef-ficiency of the enzyme-substrate complex as well as the stability of the enzyme (Zibaee et al., 2011b).
In insects, POs are important in not only immune responses but also as a potential target for pest con-trol. Namely, it may be possible to control population outbreaks of pests by inhibiting the enzyme or dis-turbing its structure. This may provide a useful basis for developing novel insecticides that could replace synthetic ones causing critical environmental threats such as pest resistance, accumulation of toxic residues in the food chain, and pest resurgence. Although we have purified the enzyme in C. suppressalis, further researches are necessary to reach a stable and effi-cient pest control via PO inhibition. Purification of POs will be important for studying the enzyme inhi-bition or activation in the presence of natural toxins produced by entomopathogens and synthetic pesti-cides used in agricultural practice. For example, in our previous study (Mirhaghparast and Zibaee, 2013), we determined significant inhibition of the enzyme by the insect growth regulators (IGR) hexaflumuron and pyriproxyfen. Generally, IGR affect molting, metamorphosis, reproduction and other physiologi-cal processes in insects (Tunaz and Uygun, 2004), and we showed that hexaflumuron and pyriproxyfen may attack immune responses against invading objects, leading to a decrease in the survival of C. suppressalis
in the environment.
authors’ contributions: The authors have contributed equally to the research paper.
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