Purification and characterization of a phenoloxidase in the hemocytes of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae): effects of insect growth regulators and endogenous inhibitors

Abstract

A phenoloxidase was extracted and purified from hemocytes of Ephestia kuehniella by using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose fast flow chromatographies. At the final stage of purification, a protein was purified by molecular mass of 78.5 kDa, specific activity of 1.17 U/mg protein, recovery of 20.48% and purification fold of 16.71. The purified PO showed the highest activity at pH 4–5 and temperatures of 35–40 °C. Na⁺, K⁺, Mn⁺, Zn²⁺ and Mg²⁺ decreased activity of the purified PO but Ca²⁺ and Cu²⁺ increased the enzymatic activity. EDTA (General chelating agent), DTC (Copper chelating agent) and EGTA (Calcium chelating agent) significantly decreased PO activity but TTHA (Magnesium chelating agent) showed no statistically significant effects. Kinetic parameters of the purified enzyme showed the highest V_max when L-DOPA was used as substrate but no significant differences were observed in case of K_m for used L-DOPA, pyrocatechol and hydroquinone. In vitro inhibition of the purified PO by using two insect growth regulators, Hexaflumuron and Pyriproxyfen, revealed IC₅₀ of 96.41 and 38.59 µg/ml for these compounds, respectively. Kinetic studies using different concentrations of L-DOPA and IC₅₀ concentrations of the two IGRs revealed the increase of K_m value versus control and competitive inhibition. Finally, column chromatography of hemolymph revealed peak III showing endogenous inhibitors of phenoloxidase by molecular weight of 27.3 that showed competitive inhibition on the PO.

• Biochemical property
• Ephestia kuehniella
• endogenous Inhibitor
• insect growth regulator
• phenoloxidase

Introduction

Immune systems of insects utilize phagocytosis, nodule formation and encapsulation as cellular responses and antimicrobial peptides as humeral responses against non-self-agents1. In all mentioned responses, phenoloxidases (POs) have crucial roles in final stages to disable invading agents completely. In fact, invading agents are recognized by pattern recognition proteins (PGRPs) leading to serine protease cascade in hemolymph. The protease changes prophenoloxidase (Inactive zymogen) to phenoloxidase2. On the other hand, the enzyme is activated by hemolymph serine proteases at Arg-Phe bond by the removal of a N-terminal fragment including 50 amino acid residues3^,4. Activated phenoloxidase catalyzes conversion of phenols to quinones. Finally, melanin is synthesized from polymerization of quinone to be deposited in formed nodule or capsule around invading agent1.

It has now been known that there are two types of phenoloxidases in insects. The first is laccase-type enzymes (EC 1.10.3.2) that oxidize o- or p-diphenols to quinones5^,6. The second is tyrosine-like enzymes that hydroxylate tyrosine (EC 1.14.18.1) and oxidize o-diphenols to quinones (EC 1.10.3.1)6. The first type involves sclerotization and tanning of cuticle but the second type refers to PO involved in immune responses of insects. Insect prophenoloxidases (Inactive form of PO) structurally are homologous with hemocyanins of arthropods and hexamerin proteins of insects. The enzyme is a polypeptide of approximately higher than 50 kDa molecular weight containing 0.14–0.22% of copper, two atoms of copper per protein molecule6.

Study on insect phenoloxidases is important due to not only determining the molecular and physiological impacts of POs on insect immunity but also targeting the enzyme to decrease population of agricultural pests. In fact, any disruptions in PO activities may lead to death of host or increase efficiency of microbial agents in biocontrol programs. Although the process might be of interest, a few studies have been carried out to purify and screen PO inhibitors in insects. The processes need careful attention since the enzyme is unstable and rapid loss of enzymatic activity may occur during purification process. So far, proPO has been purified and characterized from some insect species including Hemiptera, Lepidopteran, Dipteran, Blattodea and Orthoptera7. Hence, objectives of the current study are (i) extraction and purification of PO from hemocytes of E. kuehniella larvae, (ii) determination of enzymatic parameters by using different substrates, pH, temperature and chelating agents, (iii) determining the effect of two insect growth regulators and (iv) extraction of an endogenous inhibitor from hemolynph of the larvae.

Materials and methods

Insect rearing

E. kuehniella was reared on artificial diet containing wheat bleached flour (43 g), yeast, Saccharomyces cerevisiae (6 g) and glycerine (20 ml) in plastic containers (17 × 9 × 5 cm) at 25 ± 1 °C, 70% of humidity and 16L:8D conditions8.

Collection of hemolymph

Hemolymph of E. kuehniella was collected from the first abdominal proleg of larvae (20 µl of hemolymph per larva) by a 50 μl sterile glass capillary tube (Sigma-Aldrich Co., London, UK). The hemolymph was immediately diluted with an anticoagulant solution (0.01 M ethylenediamine tetraacetic acid, 0.1 M glucose, 0.062 M NaCl, and 0.026 M citric acid, pH 4.6)9.

PO preparation and assay

The collected samples were centrifuged at 13 000 rpm for 5 min; the supernatant was discarded and the pellet was washed gently twice with a universal buffer (20 mM, pH 7, containing succinate, glycine and 2-orpholino ethansulforic acid; Frugoni10. Cells were homogenized in 500 μl of the buffer, centrifuged at 13 000 rpm for 15 min, and the hemocyte lysate supernatant (HLS) was used to assess PO.

Samples were pre-incubated with buffer at 30 °C for 30 min before the addition of 50 μl of 10 mM aqueous solution of substrate L-dihydroxyphenylalanin (L-DOPA). 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 OD490 of 0.01 per min11.

Purification of PO

The method of Pang et al.12 was used to purify PO of E. kuehniella in a three-step procedure as follows:

Ammonium sulfate treatments

Samples were first subjected to ammonium sulfate precipitation by using fractions of 30% and 70% concentrations. The two different ammonium sulfate treatments were collected by centrifugation at 5000 rpm and the pellets obtained in each treatment were suspended in a minimal volume of universal buffer (pH 7). Precipitated samples were transferred to dialyze tube, put in 200 ml of the buffer for 20 h.

Sepharyl G-100 gel filtration

The ammonium sulfate fractions were subjected to gel filtration containing Sepharyl G-100 column (2 cm × 100 cm) equilibrated with universal buffer pH 7 containing 0.05% (v/v) Triton X-100. Fractions of 1.5 ml were collected at a flow rate of 20 ml/h with the same buffer.

DEAE-Cellulose fast flow separation

Fractions showing the highest PO activity of sepharyl G-100 column were applied to a DEAE-Cellulose fast flow column (3 × 30 cm) equilibrated with universal buffer pH 6.0. 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 rate of 60 ml/h. Fractions with PO activity were pooled and stored at −20 °C for further analysis.

Polyacrylamide gel electrophoresis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) was used to determine the purity and molecular mass of the enzyme using a 4% (w/v) stacking gel and a 10% (w/v) separating gel. The molecular mass of the enzyme was estimated by using standards: β-galactosidase (116 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), lactate dehydrogenase (35.5 kDa), restriction endonuclease Bsp 981 (25 kDa), β-lactoglobulin (18.4 kDa) and lysozyme (14.4 kDa). After SDS-PAGE, proteins on the polyacrylamide gel were stained with 0.2% Coomassie brilliant blue R-250.

Effect of pH and temperature on the purified PO

The effects of temperature and pH on the purified PO of E. kuehniella were examined by using 10 mM solution of L-dihydroxyphenylalanine (L-DOPA) as substrate and various ranges of pH and temperature. Optimal pH was determined using universal buffer at a range of 3–10. The effect of temperature on PO activity was determined by incubating the reaction mixture at 20, 25, 30, 35, 40, 45, 50, 60 and 70 °C for 5 min. Reaction mixture was 50 μl of buffer, 30 μl of L-DOPA and 20 μl of the enzyme. pH of reaction and temperature were changed as described earlier. Incubation time was continued for 5 min and absorbance was read at 492 nm.

Kinetic parameters of PO

Kinetic parameters of the purified PO were determined using different substrates L-DOPA, catechol and hydroquinone. Concentrations of the substrates were 2, 4, 6, 8 and 10 mM. Reaction mixture was 50 μl of universal buffer, 30 μl of substrates (mentioned concentrations) and 20 μl of the purified PO. Incubation time was continued for 5 min and absorbance was read at 492 nm. Obtained data were inserted in Sigma-Plot software (version 6, San Jose, CA) to calculate V_max and K_m values.

Effect of ions and chelating agents on the purified PO

The effects of mono- and di-valent cations (concentrations of 0.5, 3 and 5 mM) on the purified PO of E. kuehniella were determined by using Na⁺, K⁺, Ca²⁺, Cu²⁺, Mg²⁺, Zn²⁺ and Mn⁺. Reaction mixture was 50 μl of universal buffer, 30 μl of substrates (mentioned concentrations), 30 μl of the cations in given concentration and 20 μl of the purified PO. Incubation time was continued for 5 min and absorbance was read at 492 nm. The effect of enzyme inhibitors on PO activity was studied using different concentrations (2, 4, 6, 8 and 10 mM) of ethylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DTC), N,N,N′,N′-tetraacetic acid (EGTA) and triethylenetetramine hexaacetic acid (TTHA). Reaction mixture was 50 μl of universal buffer, 30 μl of substrates (mentioned concentrations), 30 μl of the cations in given concentration and 20 μl of the purified PO. Incubation time was continued for 5 min and absorbance was read at 492 nm.

In vitro effects of hexaflumuron and pyriproxuyfen on the purified PO

Different concentrations (30 μl) of hexaflumuron and pyriproxyfen were prepared (5–100 µg/ml) and pre-incubated with 30 μl of L-DOPA (10 mM) in 50 μl of universal buffer for 5 min. Then, 20 μl of the purified PO was added and reaction was continued for additional 5 min prior to read absorbance at at 492 nm. OD values was changed to relative activity (%), inserted in POLO-PC software to calculate IC values (Inhibitory concentration).

Effects of hexaflumuron and pyriproxyfen on kinetic parameters of the Purified PO

IC₅₀ concentrations (30 μl) of hexaflumuron and pyriproxyfen were preincubated with 50 μl of universal buffer and different concentrations of L-DOPA (30 μl, 2–10 mM) for 5 min. Then, 20 μl of the purified PO was added and reaction was continued for additional 5 min prior to read absorbance at 492 nm. Data were inserted to Sigma Plot software (Version 6) to calculate kinetic parameters and type of enzymatic inhibition.

Extraction of PO endogenous inhibitor from hemolymph of E. kuehniella

The method described by Sugumaran and Nellaiappan13 was adopted by a slight modification to extract and purify endogenous inhibitor of PO from the hemolymph of E. kuehniella. The collected hemolymph was centrifuged at 13 000 rpm for 30 min at 4 °C. Obtained supernatant was mixed with phosphate buffer (20 mM, pH 7) in 1:1 ration. In addition, it was incubated for 30 min at 5 °C. The mixture was subjected to 0 to 60% ammonium sulfate fractionation. The precipitate was collected by centrifugation at 5000 rpm for 30 min, dissolved in minimum amount of phosphate buffer (6 ml) and dialyzed against 300 µl of buffer for 20 h. The protein precipitated inside the dialysis bag was collected by centrifugation at 5000 rpm for 30 min, dissolved in 5 ml of a buffer containing 100 mM sodium acetate, pH 6.0 containing 0.5 M NaCl, 1 mM CaCl₂, 1 mM MgCl₂ and 1 mM MnCl₂ and chromatographed on Sepharyl G-100 column pre-equilibrated with the same buffer. Fractionation was carried out taking 37 fractions. Five peaks were obtained after measuring protein in each fraction. PO activity was determined by using 50 μl of universal buffer, 30 μl of L-DOPA, 30 μl of sample from each peak and 20 μl of the purified Enzyme. Incubation was continued for 5 min prior to read absorbance at 492 nm. SDS-PAGE was carried out showing molecular mass and purity of the peak caused PO inhibition.

Protein assay

Protein concentrations were assayed according to the method described by Lowry et al.14. The method recruits reaction of Cu²⁺, produced by the oxidation of peptide bonds with Folin–Ciocalteu reagent. In the assay, 20 µl of sample was added to 100 µl of reagent, and incubation was made for 30 min prior to read absorbance at 545 nm (Recommended by Ziest Chem. Co., Tehran, Iran).

Statistical analysis

The experiments were designed based on a complete randomized scheme. All data were compared by one-way analysis followed by Tukey test at p ≤ 0.05).

Results

Purification of PO from E. kuehniella

The enzyme was purified by a three-step procedure from hemocytes of E. kuehniella (Table 1). Precipitated samples from 70% ammonium sulfate were subjected to Sepharyl G-100 column leading to collection of 73 fractions. Fractions 25–42 showed the highest PO activity, and were pooled and subjected to DEAE-cellulose fast flow column (Supplementary 1). In ion-exchange chromatography, 30 fractions were collected that fractions 11–17 showed the highest enzymatic activity. These fractions were pooled and loaded into SDS-PAGE to find purity and molecular mass of the purified protein (Supplementary 1). SDS-PAGE revealed a single band of purified protein in comparison with crude and sepharose G-100 samples by molecular mass of 78.5 kDa (Supplementary 2). At the end of purification process, a molecule was purified by 78.5 kDa of molecular weight, specific activity of 1.17 U/mg protein, recovery of 20.48% and purification fold of 16.71 (Table 1).

Table 1. Purification of PO from the hemolymph of E. kuehniella.

Purification steps	U	Protein (mg)	U/mg protein	Recovery (%)	Purification fold
Crude sample	0.41 ± 0.054	2.21	0.070 ± 0.021	100	1
Amonium sulfate 70%	0.163 ± 0.032	1.35	0.120 ± 0.017	39.75	1.71
Sepharyl G-100	0.099 ± 0.047	0.70	0.142 ± 0.025	24.14	2.02
SEAE-Cellulose fast flow	0.084 ± 0.070	0.072	1.17 ± 0.097	20.48	16.71

Effect of pH and temperature on the purified PO

Although the highest activity of the purified PO was obtained at pH 5, no statistical differences were observed between pHs 4 and 5 (Figure 1). The PO activity decreased from pH 6 and reached zero at pH 10 (Figure 1). Analysis of PO activity versus temperature revealed that peak activity was found at 35 and 40 °C although the enzymatic activity sharply increased from 25–40 °C and no activity was observed at 70 °C (Figure 1).

Figure 1. Optimal pH and temperature of the purified PO from the hemolymph of E. kuehniella. Statistical differences have been shown by different letters (Tukey test, p ≤ 0.05).

Kinetic parameters of the purified PO

Kinetic parameters of the purified PO are shown in Figure 2 and Table 2 by using L-DOPA, pyrocatechol and hydroquinone. Linweaver–Burk analysis revealed that K_m of the enzyme had no statistical differences among the used substrates but the highest and the lowest V_max values were observed in case of L-DOPA and Pyrocatechol (Table 2).

Figure 2. Double reciprocal plot to show the kinetic parameters of the purified PO from E. kuehniella by using L-DOPA, pyrocatechol and hydroquinone as substrates. (1/Vmax = intercept on the 1/V0 ordinate, −1/Km = intercept on the negative side of the 1/[S] abscissa).

Table 2. Kinetic parameters of the purified PO from E. kuehniella.

Substrate	Vmax* (U/mg protein)	Km* (mM)	R^2
L-DOPA	1.92 ± 0.12a	8.32 ± 2.36a	0.97
Pyrocatechol	0.420 ± 0.34c	10.27 ± 4.55a	0.80
Hydroquinone	1.19 ± 0.25b	9.77 ± 3.13a	0.96

*Statistical differences have been shown by different letters (Tukey test, p ≤ 0.05).

Effects of ions and chelating agents on the purified PO

Different concentrations of cations induced significant effects on activity of the purified PO from E. kuehniella (Table 3). Different concentrations of Na⁺, K⁺, Mn⁺, Zn²⁺ and Mg²⁺ significantly decreased the enzymatic activity in a dose-dependent manner that the highest negative effect was observed in case of Mn⁺ (Table 3). Ca²⁺ and Cu²⁺ significantly increased the activity of the purified PO although the highest increase was obtained by using 5 mM concentration of Cu²⁺ (Table 3). EDTA (General chelating agent), EGTA (Calcium chelating agent) and DTC (Copper chelating agent) significantly decreased the activity of the purified PO that DTC had the highest inhibitory effect (Table 4). No statistical differences were observed in case of TTHA (Magnesium chelating agent; Table 4).

Table 3. Effects of mono- and divalent cations on the purified PO activities of E. kkuehniella.

Cations	Concentrations (mM)	Relative activity of PO (%) ± SE*
Na^+	Control (0)	100 ± 5.72b
Na^+	0.5	58 ± 6.78a
Na^+	3	78 ± 4.12a
Na^+	5	54 ± 14.54a
K^+	Control (0)	100 ± 12.98a
K^+	0.5	92.98 ± 5.70ab
K^+	3	76.97 ± 5.62c
K^+	5	78.94 ± 4.31bc
Mn^+	Control (0)	100 ± 6.25a
Mn^+	0.5	30.17 ± 15.17b
Mn^+	3	10.94 ± 1.93b
Mn^+	5	0 ± 0c
Zn^2+	Control (0)	100 ± 4.36a
Zn^2+	0.5	91.08 ± 7.36ab
Zn^2+	3	79.06 ± 2.01ab
Zn^2+	5	63.06 ± 26.57b
Mg^2+	Control (0)	100 ± 27.30a
Mg^2+	0.5	72.04 ± 6.81ab
Mg^2+	3	33.33 ± 19.24c
Mg^2+	5	52.59 ± 8.21b
Ca^2+	Control (0)	100 ± 21.85b
Ca^2+	0.5	322 ± 17.20a
Ca^2+	3	284 ± 8.14a
Ca^2+	5	296 ± 21.37a
Cu^2+	Control (0)	100 ± 12.28c
Cu^2+	0.5	224 ± 16.75b
Cu^2+	3	303 ± 8.11ab
Cu^2+	5	317 ± 15.64a

*Statistical differences have been shown by different letters (Tukey test, p ≤ 0.05).

Table 4. Effects of chelating agents on the purified PO activities of E. kkuehniella.

Compounds	Concentrations (mM)	Relative activity of PO (%) ± SE*
EDTA	Control (0)	100 ± 6.49a
	2	71.91 ± 7.00ab
	4	53.55 ± 7.01b
	6	57.30 ± 9.77b
	8	63.25 ± 3.64b
	10	25.09 ± 5.09c
DTC	Control (0)	100 ± 22.55a
	2	30.27 ± 15.16b
	4	9.07 ± 3.62c
	6	10.92 ± 6.64c
	8	8.96 ± 0.60c
	10	3.27 ± 0.94c
EGTA	Control (0)	100 ± 27.35a
	2	28.98 ± 3.62b
	4	45.65 ± 5.47b
	6	77.53 ± 6.91ab
	8	39.85 ± 20.17b
	10	69.56 ± 6.52ab
TTHA	Control (0)	100 ± 14.06b
	2	102.15 ± 5.68a
	4	105.5 ± 24.35a
	6	117.84 ± 20.25a
	8	112.2 ± 30.65a
	10	110 ± 15.83a

*Statistical differences have been shown by different letters (Tukey test, p ≤ 0.05).

Effects of hexaflumuron and pyriproxyfen on the purified PO

Table 5 show dose-response line regressions and IC₅₀ values of hexaflumuron and pyriproxyfen on the purified PO of E. kuehniella (Supplementary 3). IC₅₀ of 96.41 and 38.59 µ/ml of hexaflumuron and pyriproxyfen against the purified PO were found respectively (Table 5). Additionally, effects of these two IGRs on kinetic parameters of the enzyme revealed higher K_m value of the treated enzyme versus control (Supplementary 4; Table 6). Meanwhile, no effects were found on V_max value of treated and control samples (Table 6).

Table 5. Inhibition parameters of hexaflumuron and pyriproxyfen on the purified PO of E. kuehniella.

IGR	IC50 (µg/ml)	Slope ± SE	CL (upper and lower limits)	X^2	df
Hexaflumuron	96.41*	1.51 ± 0.107	60.73–161.53	12.73	4
Pyriproxyfen	38.59	1.59 ± 0.136	15.51–86.83	29.12	3

*Asterisk shows statistical differences between two used IGRs by t-test (p ≤ 0.05).

Table 6. Kinetic parameters of the purified PO from E. kuehniella in control and treated conditions of hexaflumuron and pyriproxyfen.

Treatments	Vmax (U/mg protein)	Km (mM)	R^2
Control*	1.92 ± 0.12a	8.32 ± 2.36a	0.97
Hexaflumuron treated	2 ± 0.54a	10.92 ± 1.35b	0.94
Pyriproxyfen treated	1.78 ± 0.39a	15.66 ± 0.79c	0.96

*Control means without any IGR. Used substrate for the experiment was L-DOPA. Statistical differences have been shown by different letters (Tukey test, p ≤ 0.05).

Extraction of PO endogenous inhibitor from hemolymph of E. kuehniella

Fractionation of samples for extraction of PO endogenous inhibitor revealed five peaks on Sepharyl G-100 column. Fractions regarding each peak were carefully collected and their effects were assayed on the purified PO by using L-DOPA as substrates. It was revealed that Peak III significantly decreased the enzymatic activity to lower than 50% in comparison with other fractions (Supplementary 5a, b). SDS-PAGE of the sample in peak III revealed a single band 27.3 kDa of molecular weight (Supplementary 5c). Meanwhile, kinetic studies on the enzyme inhibited by purified inhibitor revealed competitive inhibitor since K_m value of the treatment and control were 14.94 mM and 6.87 mM. No statistical differences were found in case of V_max (Supplementary 5d).

Discussion

Phenoloxidases are one of the main components of insect immunity. Numerous attempts have been made to purify the enzyme, find its molecular mass and tissue allocation2^,15. The enzyme have now been purified and characterized in some insect arthropods. In the current study, one isoform of PO was purified from hemocytes of E. kuehniella by molecular mass of 78.5 kDa. Majority of the relevant studies have purified a PO by one isoform but two or three isoforms have been purified in some studies2^,3. Meanwhile, PO was measured to be 78.5 kDa of molecular mass that corresponds with reports on POs of other insects2. Liu et al.16 believed that different biochemical properties of POs like pH, response to xenobiotic and isoforms could be attributed to species and its survival conditions that appeared in in vitro studies. PO is one of the sensitive enzymes in insects. Several studies reported that the enzymes were affected whenever an insect was treated by chemicals or even host plants. Also, number of isoforms depends on species and its immune capability.

Temperature and pH are the two key factors in the biochemical media that crucially affect enzymatic reactions. Any changes of these factors could alter enzymatic activity leading to affect on physiological process. Although purified PO from hemocytes of E. kuehniella had optimal pH of 5 and temperature of 40 °C but no statistical differences have been observed by pH of 4 and 35 °C. Majority of studies have been reported acidic or neutral pHs for activities of POs but alkaline PO have been reported in Helicoverpa virescens Fabricius (Lepidoptera: Noctuidae) and H. cunea17^,18. These differences could be correlated with pH of hemolymph that indicate type of species and survival conditions16^,19. Enzymes have temperature-dependent activities that are correlated with enzymatic stability and temperature of biochemical media. Hence, biological reactions occur faster with increasing temperature upto the point of enzyme denaturation, above which the enzyme activity and the rate of reaction decrease sharply.

It was found that the purified PO from hemocytes of E. kuehniella could oxidize L-DOPA, pyrocatechol and hydroquinone although the higher V_max was ordinary observed in case of L-DOPA, hydroquinone and pyrocatechol. Although the lower K_m was observed in case of L-DOPA but there was no statistical differences among used substrates. The results of substrate specificity showed that the purified PO from E. kuehniella was probably a kind of tyrosinase not a laccase or a cathecol oxidase. Type of PO isoforms and evolutionary processes may affect the amount of K_m and of course the efficiency of the enzyme to act on the substrate20. Meanwhile, binding affinity of PO could be affected by the nature of the active site of PO. More precisely, differences in substrate-protein contact points or differences in the size of the substrate-binding pocket can affect PO binding affinity in different insects20.

Metal ions are other factors that affect enzymatic activity via several possible mechanisms, e.g. they can activate electrophile and nucleophile binding and releasing electrons20. Although different cations significantly affected activity of the purified PO from hemocytes of E. kuehniella but the highest effects were observed by using Cu²⁺ and its specific chelating agent (DTC). Meanwhile, similar effects of Cu²⁺ and its specific chelating agent (EGTA) on increasing of the purified PO activity could be attributed to possible substitution between the metals during enzyme activation20. It was determined that proPO are polypeptides which contain 0.14–0.22% copper, indicating two atoms of copper per protein molecule8. Increased activity of the PO from hemocytes of E. kuehniella demonstrate that the enzyme is a metalloprotein that and small changes at the metal center can amplify its biological role. Moreover, metal ions (Mainly di-valent cations) increase PO activity by changing its protein folding16^,20. In fact, binding of metal ions significantly affect secondary structure of some peptides depending on their nature20. It has now been determined that secondary structures of the purified PO have the lowin β-turn (14.33%) and the α-helix content (37.28%)20. Involvements of the metal ions to media containing purified PO cause augmentation of the β-sheet and increase activity of the enzyme20.

Majority of the studies dealing with effect of hormones and IGRs indicate changes of PO activity in vivo. Since, hormones and IGRs affect number of circulating hemocytes interpretation of their direct effects on PO activity could not be reliable. Hence, the current study determined direct effects of hexaflumuron and pyriproxyfen on the purified PO activity by determing IC₅₀ values and kinetic parameters of the enzyme. IC₅₀ calculation revealed that pyriproxyfen is more effective than hexaflumuron because of the lower IC₅₀. Moreover, Linewever–Burk analysis revealed increase of K_m value when the enzyme was incubated with two IGRs showing competitive inhibition. In case, inhibitor binds to enzyme so that substrate and inhibitor compete to engage active site21. In such a way, increasing of substrate concentration could lead to natural activity of enzyme21. In a similar study, Mirhaghparast and Zibaee22 showed that different concentrations of hexaflumuron and pyriproxyfen resulted IC₅₀ values of 0.047 and 0.11 μg/ml on the purified PO of C. suppressalis. Yan-Yan et al.23 reported that concentrations of hexaflumuron up to 153 mg/L increased PO activity of S. littura then the enzymatic activity decreased along with higher used concentrations.

In the present study, a low molecular weight inhibitor was extracted and purified from hemolymph of E. kuehniella by measuring PO activity in various fractions. The inhibitor showed competitive inhibition by increasing of K_m value versus control. PO inhibitors of insects have been extracted and purified from cuticle. Tsukamoto et al.24 purified three low molecular weight inhibitors from pupal extracts of house flies. These inhibitors showed competitive inhibition of endogenous phenoloxidase activity. The endogenous inhibitor of PO regulates activity of the enzyme during infection, wounding and/or metamorphosis and prevents undesired melanization and sclerotization reactions somehow showing protecting role of intrinsic organs. In case, more detailed studies are required to sequence the protein and find its the structure–function relationship with the enzyme.

Conclusions

Purification and characterization of insect’s POs are mandatory since those are the unique enzymes that play critical roles in melanization and sclerotization of cuticle, wound healing and immune reactions. Moreover, several studies have shown that these enzymes could be a target to control insect pests. Currently, several compounds like IGRs, cantharidin, 4-Hexylresorcinol, Kojic acid and Quercetin that are safe for non-target organisms and environment. These researches will lead to a basic tool for the development of new insecticides in integration with microbial agents. As a future project, protein and genomic sequence of the enzyme must be elucidated and its expression must be determined in intact and immune challenge conditions.

Acknowledgements

This study was supported by research deputy in university of Guilan.

Declaration of interest

The authors report no declarations of interest.