Purification and characterization of prophenoloxidase from Galleria mellonella L.

Abstract

Prophenoloxidase (PPO) was purified from Galleria mellonella L. A 67-fold purification of the proenzyme with 352% yield was achieved by using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. The purified enzyme was migrated as a single band on SDS-polyacrylamide gel electrophoresis. K_m and V_max values were 0.017 M and 1430.45 EU for catechol. Inhibition of PPO was investigated with inhibitors such as p-aminobenzoic acid, etyleneglycol, and ascorbic acid. Among them, ascorbic acid showed the strongest inhibitory activity with IC₅₀ value of 2.94 μM. The current paper represents new strategies for the biological control of the Galleria mellonella L. insect.

Keywords::

• Galleria mellonella
• prophenol oxidase
• affinity chromatography
• inhibition

Introduction

The immunity of insects consists of humoral and cellular defense actions. Humoral defense refers to antibacterial proteins and other immune-related molecules generated by the fat body and the hemocytes that are secreted into plasma to sequestrate and clear the foreign bodies from the hemocoel (Theopold et al. 2004). Cellular immunity includes phagocytosis, nodule formation, and encapsulation (Uçkan et Al. 2011. Several factors, including asphyxiation, the local production of cytotoxic quinones or semiquinones via the PPO activation cascade during melanization, free radicals and antibacterial peptides have been suggested to function as killing agents towards internal parasites and other foreign entities that enter the insect's haemocoel (Nappi et al. 1995, 2000, Gillespie et al. 1997, Lavine and Strand 2002, Jiravanichpaisal et al. 2006). Phenoloxidase is one of the most important enzymes that are involved in the innate immune system of insects (Cerenius and Söderhäll 2004). Phenoloxidase was also associated with other physiologically important biochemical processes such as sclerotization of the insect cuticle and wound healing (Ashida and Brey 1995).

Phenoloxidase (EC 1.14.18.1) is a copper-containing enzyme, widely distributed in nature, which is responsible for melanization in animals and browning in plants (Gowda and Paul 2002, Shelby and Popham 2006). PPO also catalyzes the ortho-hydroxylation of monophenols and the oxidation of o-diphenols to o-quinones (Gowda and Paul 2002). Phenoloxidase exists in insect hemolymph or hemocytes as a proenzyme in its inactive form called prophenoloxidase. PPO is activated by a serine proteinase (prophenoloxidase activating enzyme) cascade triggered by microbial carbohydrates such as β-1,3-glucan, peptidoglycan, and lipopolysaccharides in insects (Söderhäll and Cerenius 1998).

It is possible that inhibition of PPO could lead to abrogation of insect defense mechanisms and this theory is logical to regard PPO as a target of new insecticides with novel modes of action in pest control (Du et al. 2010). If the enzyme activity is inhibited or disturbed, pests may lose normal defense ability, which would be a significant method to control pests by using the PPO inhibitors (Wang et al. 2005, Xue et al. 2007, Xiao et al. 2008). The greater wax moth G. mellonella is an important Lepidopteran pest in beehives because it feeds on pollen and generally destroys the combs and frequently is used as a model animal for the investigation of insect immunity and host-parasitoid interactions (Uçkan et al. 2001, Er et al. 2011, Ergin et al. 2006).

To elucidate the molecular and physiological mechanisms of PPO, a detailed study of the enzyme is necessary (Beckage 2008). However, more attention has been paid to the investigation of PPO due to the instability and rapid loss of enzymatic activity during the purification process (Feng et al. 2008). PPO has been purified from a number of different insects, including the lepidopterans Bombyx mori (Ashida 1971), Manduca sexta (Aso et al. 1985, Hall et al. 1995, Jiang et al. 1997), Hyalophora cecropia (Andersson et al. 1989), Galleria mellonella (Kopácek et al. 1995), and Ostrinia furnacalis (Feng et al. 2008). The current study demonstrates the purification and characterization of PPO from G. mellonella in terms of substrate specificities, optimum pH and temperature, heat inactivation, and degrees of inhibition by general PPO inhibitors in addition to its molecular weight.

Material and Methods

Insects

Laboratory colonies of G. mellonella were established from individuals that were collected from the honeycombs maintained by beekeepers around Bal kesir, Turkey. Insects were reared at 25 ± 1°C, 60 ± 5% RH, and with a photoperiod of 12 : 12 h, L : D. The G. mellonella colony was maintained by feeding the insects with honeycomb to maintain similarity to their natural media in beehives. (Uçkan et al. 2004, 2010).

Collection of hemolymph

Last instar larvae of approximately the same sizes were used for the experiments. Larvae were precooled at 4°C, then bled by piercing the first hind leg with a sterile 19-gauge needle. Hemolymph was collected with a glass microcapillary tube (Sigma Chemical Co., St. Louis, MO) and pooled into an Eppendorf tube kept on ice.

Extraction and purification procedure

The extraction procedure was adopted from Wesche- Ebeling &Montgomery (1990). 1.7 ml hemolymph was taken in 25 ml of 0.5 M phosphate buffer (pH 7.3) containing 5% poly(ethylene glycol) and 10 mM ascorbic acid. After filtration of the homogenate through muslin, the filtrate was centrifuged at 15.000 g for 30 min, and the supernatant was collected. A crude protein precipitate was made by adding (NH₄)₂SO₄ to 80% saturation and the precipitate was collected by centrifugation at 15.000 g for 20 min, redissolved in 5 mM phosphate buffer (pH 6.30), and dialyzed against the same buffer (Wesche-Ebeling and Montgomery 1990). The homogenate was purified with affinity chromotography. The affinity gel used was synthesized according to the method of Arslan and Erzengin (2004). Briefly, the enzyme solution was applied to the affinity column (1 3 10 cm), equilibrated with 0.05 M phosphate buffer (pH 5.0). The affinity gel was washed with the same buffer. PPO was eluted with the solution of 0.05 M phosphate buffer (pH 7.0) containing 1 M NaCl.

Electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using the method of Laemmli (1970). Samples were applied to 12% polyacrylamide gels. The slab gels of 1.5 mm thickness were run at a constant current of 180 mV. Gels were stained for protein using a standard Coomassie Blue method.

Spectrophotometric assays

Kinetic assays were carried out by measuring the increase in absorbance at 420 nm and at 25^oC for catechol with a Biotek automated recording spectrophotometer. The reaction was carried out in a light path quartz cuvette. The sample cuvette contained 130 μL of substrates in various concentrations prepared in the homogenization buffer (pH 6.8) and 40 μL of the enzyme. For each measurement, the volume of solution in the quartz cuvette was kept constant at 1 mL. The reference cuvette contained all of the components except the substrate, with a final volume of 1 mL (Arslan et al. 1997).

Determination of protein content

The protein content was determined according to the Bradford method using bovine serum albumin as standard (Bradford 1976).

Enzyme kinetics and substrate specificity

PPO activity was assayed using catechol as substrate. The rate of the reaction was measured in terms of the increase in absorbance at the absorption maxima of the corresponding quinone products for catechol substrate. Enzyme activity was calculated from the linear portion of the curve. One unit of PPO activity was defined as the amount of enzyme that causes an increase in absorbance of 0.001 unit/min for 1 ml of enzyme at 25°C (Arslan et Al. 2004). Catechol substrate, Michaelis-Menten constant (K_m), and maximum velocity (V_max) were determined according to the method of Lineweaver-Burk.

Inhibition of PPO activity

IC₅₀ values of different inhibitors p-aminobenzoicacid, etyleneglycol, and ascorbic acid were determined on PPO. In order to determine IC₅₀ values, 0.1 M catechol was used as substrate. At first the activity of enzyme was assay without inhibitor. This measure was accepting 100% activity for graph and than enzyme activity assay with different inhibitor concentration. In order to determine IC₅₀ values were drawn by using a % activity-inhibitor concentration graph. IC₅₀ values were determined from these graphs.

Results and Discussion

Extraction and purification of PPO

In this study, Galleria mellonella L. PPO was purified with affinity chromatography. The purification procedures are summarized in Table I. As seen in Table I, PPO was purified up to 67.2-fold. Different purification protocols have been used for PPO enzymes from different sources (Jiang et al. 1998). In order to purify PPO from different sources, we used Triton X-100, ammonium sulfate precipitation, dialysis, affinity chromatography, sephadex G-200, and Phenyl sepharose hydrophobic chromatography (Arslan et al. 2004, Jiang 1999, Weemes et al. 1998). However, pear PPO was purified generally in two or more steps asTriton X-100, ammonium sulfate precipitation, dialysis and acetone precipitation (Siddiq and Cash 2000, Weemes et al. 1998, Carbonaro and Mattera 2001).

Table I. Purification of polyphenol oxidase from Galleria Mellonella L.

Purification Step	Volume (mL)	Activity (U/mLdak)	Total activity	Total Protein (mg)	Spesific activity (U/mg protein)	Overal Yield (%)	Purification fold
Extract	25	25	625	25.225	24.777	-	-
Ammonium sulphate precipitation	30	550	13750	14.97	918.504	2200	37.1
Affinity chromatography	2	1100	2200	1.322	1664.145	352	67.2

The molecular weight of PPO was estimated on SDS-PAGE with a single band of approximately 66.2 kDa (Figure 1). The molecular mass of PPO from other species has been reported as follows: Sunn pest Eurygaster integriceps Puton (Hemiptera: Scutelleridae), 22 kDA (Aras et al. 2011), Hyalophora cecropia, 76 kDA (Andersson et al. 1989).

Figure 1. SDS-PAGE of the purified phenoloxidase from Galleria Mellonella L.

Substrate specificity and enzyme kinetics

PPO activity in purified enzyme was examined with regard to its diphenolase activities. The substrate specificity of the enzyme was investigated using one chemical name of catechol as substrate. The Lineweaver-Burk plot analysis of this enzyme preparation showed K_m value of 0.017 M for catechol and V_max value was 1430.45 (U/mL.min.) for catechol (Figure 2, Table II). Km of 2.25, 1.35, 1.30, and 0.17 mM were reported for Heliothis virescens Fabricius (Lepidoptera: Noctuidae) (Lockey and Ourth 1992), Spodoptera littoralis Fabricius (Lepidoptera: Noctuidae) (Lee and Anstee 1995), Drosophila melanogaster L. (Diptera: Drosophilidae) (Asada and Sezaki 1999), and Apis mellifera L. (Hymenoptera: Apididae) (Zufelato et al. 2004), respectively. The Km of Galleria mellonella L. PO is higher than that of other insects, indicating the lower affinity of the enzyme to catechol.

Figure 2. Lineweaver-Burk graph of PPO.

Table II. Kinetic values of Galleria Mellonella L.

KM (M)	Vmax (U/mLdak)	Vmax / KM
0.017	1430.45	84144

The V_max/K_m ratio is called the “catalytic power,” and it is a better parameter to find the most effective substrate (Dogan et al. 2000). The ratio V_max/K_m is 84144. The large ranges in the apparent K_m values of PPO reported may be due to different reasons: different assay methods used for different varieties, different origins of the same variety, and different values of pH of extraction (Rocha et al. 1998).

Inhibition of PPO

Inhibition of PPO by ascorbic acid, p-aminobenzoic acid, and ethyleneglycol has been investigated. It was found that the presence of all chemicals causes the inhibition of PPO. Figure 3 shows the effect of ascorbic acid, p-aminobenzoic acid, and ethyleneglycol inhibitor on PPO using catechol as substrate. The enzymatic browning by a specific inhibitor may involve a single mechanism or may be the result of the interplay of two or more mechanisms of inhibitor action. Inhibition of PPO was investigated with inhibitors such as ascorbic acid, p-aminobenzoicacid, and etyleneglycol using catechol as substrate. Among them, ascorbic acid showed the strongest inhibitory activity with IC₅₀ value of 2.94 μM. Ethylen glycol was a poor effective inhibitor for PPO (IC₅₀ 25.44 μM) (Table III).

Figure 3. The effect of ascorbic acid, p-aminobenzoic acid, and ethyleneglycol inhibitor on PPO.

Table III. The values IC₅₀ of some compounds on Galleria Mellonella L. PPO.

Compounds	IC50 (μM)
Ascorbic acid	2.94
p-amino benzoic acid	10.76
Ethylen glycol	25.44

All in all, the present article reports for the first time the isolation and qualitative characterization of the PO enzyme in the Galleria mellonella L. pest. In consideration of the role played by PO in the insect immunity, this information could be profitable not only to comparative biochemists but also to researchers that see in the immune system a possible target for biological control of insect pests.

Declaration of interest

The authors report no conlicts of interest. The authors alone are responsible for the content and writing of the paper.