Purification and characterization of hemocyte Phenoloxidases in Chilo suppressalis Walker ( lePidoPtera : crambidae )

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, 5.08and 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 quinones involved in sclerotization of the cuticle (Arakane et al., 2005;Dittmer et al., 2004) and a tyrosinelike 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 approximately more than 80kDa molecular weight that contain 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 prophenoloxidase by the action of the hemolymph serine proteases (Kim et al., 2002).The proteases 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 polymerizes 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 physiological processes in insect immunity and the potential to be used for population control of agricultural pests.Chilo suppressalis Walker (Lepidoptera: Crambidae) is a serious pest of rice in the Middle East, southern 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 pyriproxyfen, two insect growth regulators, inhibited the PO activity of C. suppressalis (Mirhaghparast and Zibaee, 2013).It is mandatory to understand fully the nature and biochemical properties of insect POs to target them for pest control tactics.Hence, the objectives of the current study were (i) the extraction and purification of PO from the hemocytes of C. suppressalis 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 seedlings.Insects were reared using a modified method of Zibaee et al. (2009) at 28±1°C, 80% relative humidity (RH) under conditions of 16 h light:8 h dark.Laboratory conditions were checked, the containers were cleaned, and fresh stems were provided for larvae 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 hemolymph 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 pellet was washed gently by universal buffer (20 mM, pH 7, containing succinate, glycine and 2-morpholino 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 hemocyte lysate supernatant (HLS) was used to measure PO activity.Samples were pre-incubated with the buffer 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 incubated 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 OD 490 of 0.01 per min (Dularay and Lackie, 1985).

Purification of Po
The method of Pang et al. (2005) was used to purify the PO of C. suppressalis in a three-step procedure.Samples were initially subjected to ammonium sulfate precipitation by using fractions of 30% and 70% concentrations.Two different ammonium sulfate precipitations were collected by centrifugation at 28500×g, and the pellets obtained in each treatment were suspended in a minimal volume of universal buffer (pH 7).The precipitated samples were transferred to a dialyzing tube and placed in 200 mL of the buffer for 20 h.The final ammonium sulfate precipitation was subjected to gel filtration containing a Sepharyl G-100 column (2 cm × 100 cm) equilibrated with universal buffer pH 7 containing 0.05% (v/v) Triton X-100.Fractions (1.5 mL) were collected at a flow rate of 20 mL/h with the same buffer.Fractions showing the highest PO activity of Sepharyl G-100 column were applied to a DEAE-Cellulose fast flow column (3 cm × 30 cm) equilibrated with universal buffer pH 7.0.After washing the column with the same buffer, bound proteins were eluted with a linear gradient of sodium chloride (0-0.5 M) in the equilibrating buffer.Fractions (1.5 mL each) were collected at a flow PURIFICATION OF PHENOLOXIDASE IN C. SUPPRESSAlIS rate of 60 mL/h.Fractions showing PO activity were pooled and stored at −20°C for subsequent analysis.

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.Concentrations of the substrates were 2, 4, 6, 8 and 10 mM.Reaction mixture contained 50 μL of universal buffer, 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 V max and K m values.

effect of ions and chelating agents on the purified Po
The effects of mono-and divalent cations on the purified PO of C. suppressalis were determined by using NaCl, KCl, CaCl 2 , CuCl 2 and MgCl 2 .The reaction 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.Incubation time was continued for 5 min and absorbance 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(2aminoethylether)-N,N,Nʹ,Nʹ-tetraacetic acid (EGTA) and triethylenetetramine-N,N,N' ,N",N"' ,N"'-hexaacetic acid (TTHA).Reaction mixture was 50 μL of universal buffer, 30 μL of L-DOPA as substrate, 30 μL of the inhibitors and 20 μL of the purified POs.Incubation 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 completely 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
In the current study, two phenoloxidases were purified from hemocytes of C. suppressalis, namely POI and POII.Fractions 23-28 out of 43 taken fractions showed the highest phenoloxidase activities in the Sepharyl G-100 column (Fig. 1a).These fractions were loaded into DEAE-cellulose fast flow column for further purification.Two peaks containing fractions 19-21 and 25-27 were observed in the column and their purities were checked by SDS-PAGE (Figs. 1b, 1c).The purification parameters revealed specific activities of 0.478 and 0.529 U/mg protein, 8.94% and 7.31% recoveries and 5.08-and 5.62-fold purification for POI and POII, respectively (Table 1).Molecular weights of the POs were calculated at 94.6 and 95.7 kDa, respectively (Fig. 1c).Finally, the kinetic parameters of the POs using Lineweaver-Burk analysis revealed a Vmax of 1.11 and 2.27 U/mg protein for POI and POII while the Km obtained 15.51 and 17.31 mM, respectively (Fig. 2).Since POs are one of the main components of insect immunity, they may have potential as targets of pest control.POs have been 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;Ajamhassani 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.  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 diversity 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, Mg 2+ and Cu 2+ 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 Cu 2+ indicates that the enzyme is a metalloprotein, 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 releasing electrons; (ii) they prevent the reactions of nucleophiles; (iii) ions increase the efficiency of enzyme activity by keeping the enzyme and substrate in close proximity through the coordination bonds,and (iv)  ions put active groups in the enzyme and substrate structure in an effective position and increase the efficiency 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 control.Namely, it may be possible to control population outbreaks of pests by inhibiting the enzyme or disturbing 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 efficient pest control via PO inhibition.Purification of POs will be important for studying the enzyme inhibition or activation in the presence of natural toxins produced by entomopathogens and synthetic pesticides 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 physiological 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.
*All steps were carried out at 4˚C.

table 2 .
Effects of mono-and divalent cations on the purified POs of C. suppressalis.

table 3 .
Effects of mono-and divalent cations on the purified POs of C. suppressalis.