Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds

Plant Physiology and Biochemistry 70 (2013) 61e68

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Research article

Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds Ana C.B. Cruz a, Fábio S. Massena a, Ludovico Migliolo a, e, Leonardo L.P. Macedo e, Norberto K.V. Monteiro a, Adeliana S. Oliveira a, Francisco P. Macedo b, Adriana F. Uchoa b, Maria F. Grossi de Sá d, Ilka M. Vasconcelos c, Andre M. Murad d, Octavio L. Franco e, *, Elizeu A. Santos a a

Laboratório de Química e Função de Proteínas Bioativas, Departamento de Bioquímica, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil b Laboratório de Moscas das Frutas, Departamento de Biologia Celular e Genética, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil c Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, CE, Brazil d Embrapa e Recursos Geneticos e Biotecnologia, Brasilia, DF, Brazil e Centro de Analises Proteômicas e Bioquímica, Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, 70790160 Brasília, DF, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 January 2013 Accepted 29 April 2013 Available online 18 May 2013

The present study aims to provide new in vitro and in vivo biochemical information about a novel Kunitz trypsin inhibitor purified from Piptadenia moniliformis seeds. The purification process was performed using TCA precipitation, TrypsineSepharose and reversed-phase C18 HPLC chromatography. The inhibitor, named PmTKI, showed an apparent molecular mass of around 19 kDa, visualized by SDS-PAGE, which was confirmed by mass spectrometry MALDI-ToF demonstrating a monoisotopic mass of 19.296 Da. The inhibitor was in vitro active against trypsin, chymotrypsin and papain. Moreover, kinetic enzymatic studies were performed aiming to understand the inhibition mode of PmTKI, which competitively inhibits the target enzyme, presenting Ki values of 1.5
108 and 3.0
101 M against trypsin and chymotrypsin, respectively. Also, the inhibitory activity was assayed at different pH ranges, temperatures and reduction environments (DTT). The inhibitor was stable in all conditions maintaining an 80% residual activity. N-terminal sequence was obtained by Edman degradation and the primary sequence presented identity with members of Kunitz-type inhibitors from the same subfamily. Finally after biochemical characterization the inhibitory effect was evaluated in vitro on insect digestive enzymes from different orders, PmTKI demonstrated remarkable activity against enzymes from Anthonomus grandis (90%), Plodia interpuncptella (60%), and Ceratitis capitata (70%). Furthermore, in vivo bioinsecticidal assays of C. capitata larvae were also performed and the concentration of PmTKI (w/w) in an artificial diet required to LD50 and ED50 larvae were 0.37 and 0.3% respectively. In summary, data reported here shown the biotechnological potential of PmTKI for insect pest control. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Bioinsecticidal Kunitz trypsin inhibitor Insect pests Piptadenia moniliformis

1. Introduction

Abbreviations: ED50, effective dose to inhibit 50% of an enzymatic pool; HPLC, high-performance liquid chromatography; TCA, trichloroacetic acid; Ki, inhibition constant; DTT, dithiothreitol; LD50, lethal dose to kill 50% of an insect population; MALDI-ToF, matrix assisted laser desorption ionization time of flight mass spectrometry; TFA, trifluoroacetic acid. * Corresponding author. Tel.: þ55 61 34487167; fax: þ55 61 33474797. E-mail address: [email protected] (O.L. Franco). 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.04.023

Proteinaceous inhibitors are proteins widely distributed in nature possessing the ability to interact with enzymes of different origins, suppressing partial or totally the catalytic activity of these enzymes. In plants, they have been found in storage tissues, such as leaves, fruits, tubers and seeds. A variety of physiological functions have been attributed for these proteins, especially its role in the plant-defense mechanisms against insect pest attack [1,2]. Serine protease inhibitors of the Kunitz superfamily have gained particular attention due to the potent activity against insect larval midgut serine

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proteases. The use of proteinaceous inhibitors as candidates for control strategies of insects is hopeful, since insect digestive proteases are promising targets in the control of various insects. Kunitz inhibitors were capable of inhibiting the proteolytic activity of serine proteases from lepidopterans, such as the black cutworm (Agrotis ipsilon Hufnagel), the corn earworm (Heliothis zea), the tobacco budworm (Heliothis virenscens Fab.), the western spruce budworm (Choristeneura occidentalis) [3], and coleopterans such as the cotton boll weevil (Anthonomus grandis) [4], the cowpea weevil (Callosobruchus maculatus) [5e8] and the bean weevil (Zabrotes subfasciatus) [8,9] and dipterans such as Ceratitis capitata [6,8e10]. Increased mortality, several deformations, decreased weight gain, and reduction of the fecundity and oviposition were common effects observed when Kunitz inhibitors were added to the artificial diets of insects [5e7,11]. The fruit fly (Diptera: Tephritidae) is an insect pest that attacks a great variety of fruit-tree species that are economically important in Brazil such as mango, caja, caja-mirim, siriguela, guava, guabiroba, jabuticaba, rose apple, cherry, carambola, orange, tangerine, abiu and sapodilla trees. The species C. capitata (Wiedemann), commonly known as the Mediterranean fruit fly, is considered the main pest of fruticulture in the world, attacking an extensive host range including more than 350 plant species. Millions of dollars are annually lost and many programs were created to control and to eradicate this pest [12]. Great quantities of insecticides are often used, causing damage to the environment and consumer health. Solutions for this situation are based on the development of novel, target-specific compounds reducing the impact on the environment and increasing effectiveness in the combat against this insect [13,14]. Compounds such as lectins, vicilins, proteases and amylases inhibitors can be considered possible alternatives to the use of pesticides to control insect pests [5,6,15]. In this view, the tropical Fabaceae tree of the Mimosoideae subfamily, Piptadenia moniliformis (Benth.), known popularly as Catanduva and angico-de-bezerro, typically found in the caatinga northeastern part of Brazil, had a Kunitz-type trypsin inhibitor purified and biochemically characterized. The inhibitory activity was tested: in vitro assays of insect pests from different orders and in vivo bioassays models during the larval development of C. capitata (fruit fly). 2. Results 2.1. Purification of P. moniliformis trypsin inhibitor The PmTKI purity degree was observed by SDS-PAGE and MALDI-ToF mass spectrometry analyses (Fig. 1). Both methodologies showed a major molecular mass of 19.296 Da. This average molecular mass also showed, by MS analysis, a double charge (M þ Hþ2) of 9.648 Da, indicating the absence of contaminants and higher purity degree Moreover, this mass was unmodified in the presence or absence of reducing agent as observed by SDS-PAGE gel. Moreover, purification procedures resulted in a purification of a 690-fold with a 0.15% yield (Table 1). 2.2. N-terminal amino acid sequence analysis The alignment of the N-terminal amino acid sequence of the PmTKI with other inhibitors had strong homology with Kunitz protease inhibitor of the Mimosoideae subfamily of the Leguminosae (Table 2). The alignment homology of PmTKI inhibitor with Kunitz protease inhibitors from Acacia confusa [16], Enterolobium contortisiliquum [17], Prosopis juliflora [18], Adenanthera pavonina [19] and Leucaena leucocephala [20] were 86, 86, 80, 76 and 70%, respectively. This strong homology indicates that PmTKI is a member of the Kunitz inhibitor family.

Fig. 1. Elution profile on RP-HPLC analytical column C4. The fractions obtained from TrypsineSepharose column were separated by RP-HPLC column at a flow rate of 1 mL min1. The PmTKI purity degree (indicated by the arrow) was analyzed by SDSPAGE stained with Coomassie blue (inset square); (1) protein molecular weight markers; (2) purified PmTKI; (3) PmTKI treated with DTT. (B) Mass spectrometry analysis of PmTKI indicating molecular mass of 19.296 Da.

2.3. Stability of PmTKI Studies of temperature effects over PmTKI showed that the inhibitory activity was stable at 60  C, losing only 20% of activity at 100  C (Fig. 2A). Pre-incubation of the inhibitor in the pH range (2.0e12.0) did not affect trypsin inhibition activity (Fig. 2B). In addition, the activity also was assayed after pre-incubation with several concentrations of DTT (1, 10 and 100 mM). PmTKI was not affected by reducing agent and lost only 30% of activity after 2 h, demonstrating the stability of this inhibitor does not depend exclusively on the disulfide bound (Fig. 2C). 2.4. Specificity of PmTKI to serine and cysteine proteinases and kinetics studies PmTKI inhibited chymotrypsin (43%), another serine proteinases of the same class of trypsin which was completely inhibited (100% Table 1 Total yield of PmTKI purification steps. Steps

Total IUa

Total protein (mg)

Specific activity (IU mg1)

Purification (times)

Recovery (%)

CE TCA precipitation TrypsineSepharose PmTKI

16,284 21,775 473.2 169.0

207.0 146.9 2.24 0.3

78.7 148.2 211.3 563.3

1.0 1.4 92.4 690

100 71 1.1 0.15

a One Inhibition Unity (IU) was defined as the inhibitor amount that decreased the absorbance at 410 nm by 0.01 O.D. under the trypsin inhibition assay.

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Table 2 N-terminal alignment of PmTKI with others Kunitz-type inhibitors. Kunitz-type plant proteinase inhibitors: AcTKI, Acacia confusa; EcTKI, Enterolobium contortisiliquum; PjTKI, Prosopis juliflora; ApTKI, Adenanthera pavonina and LiTKI, Leucaena leucocephala. Name

Primary sequences

Identity (%)

PmTKI AcTKI EcTKI PjTKI

KELLDADGDLLRNGGTYYILPVFRGKGGGL KELLDADGDILRNGGAYYILPALRGKGGGL KELLDSDGDILRNGGTYYILPALRGKGGGL QELLDVDGEILRNGGSYYILPAFRGKGGGL

87 87 80

ApTKI

RELLDVDGNFLRNGGSYYIVPAFRGKGGGL

77

LiTKI

QVLVDLDGDPLYNGMSYYILPVARGKGGGL

70

Reference This report Hung et al., 1992 Batista et al., 1996 Negreiros et al., 1991 Richardson et al., 1986 Oliva et al., 2000

of inhibition). Cysteine proteinases were also tested and a weak inhibition to papain (23%) was observed (Table 3). In order to determine the PmTKI inhibition mechanism against trypsin and chymotrypsin, the inhibition kinetic data were analyzed by Dixon and LineweavereBurk plots (Fig. 3). The analysis suggested a competitive-mode kinetic inhibition for trypsin obtained with the LineweavereBurk diagram (Fig. 3B). In the Dixon plot, a family of intersecting lines was obtained by plotting 1.velocity1 according inhibitor concentration at each substrate concentration. The Ki values are determined by lines intersection that converges above the x-axis. Dixon plot analysis revealed inhibition constants of 1.5
108 and 3.0
101 M for trypsin and chymotrypsin inhibitions, respectively. PmTKI showed to have higher affinity against trypsin in comparison to chymotrypsin. 2.5. PmTKI inhibitory assay against protease extracts from insect pests PmTKI was assayed against different insect gut proteases from Coleopteran, Lepidopteran and Dipteran pests (Table 4). Among the different gut proteases tested, PmTKI showed high in vitro inhibition against A. grandis (boll weevil), C. capitata (fruit fly) and Plodia interpunctella (Indian Meal Moth) with 90, 70.5 and 60%, respectively. On the other hand C. maculatus (cowpea weevil), Z. subfasciatus (bean weevil) and Telchin licus (sugarcane Giant borer) proteases were weakly inhibited by PmTKI with 9.5, 6.0 and 24.0%, respectively. 2.6. Insect bioassays Based on in vitro enzymatic studies, standard feeding trials were carried out to assess the potential bioinsecticidal effects of PmTKI against C. capitata pest, which was used as a model. Fig. 4A and B shows the influence of PmTKI on the weight and number of survivors of C. capitata during larval developmental when larvae were fed a diet containing different concentrations of PmTKI. The inhibitor added in the artificial diet of these pests was effective against C. capitata and produced 50% mortality to this fruit fly at the level of 0.37% (LD50, lethal dose). It also affected the mass of larvae in 50.0% with an ED50 (effective dose) at the level of 0.3%. 2.7. Discussion The Kunitz-type inhibitors have been exhaustively studied because of their capacity to protect plants against insect pest digestive enzymes. These properties confer the potential for wide application using over-expression technology, for example, using economically viable cultivars. These inhibitors are characterized by

Fig. 2. Stability of PmTKI (A) pH stability of the inhibitor after incubation in the indicated pH for 30 min at 37  C; (B) temperature stability after incubation for 30 min at the indicated temperature; (C) reducing environment after incubation for 15, 30, 60 and 120 min using DTT agent. The residual trypsin inhibitory activity was measured by using BApNA as substrate in 50 mM TriseHCl buffer, pH 7.5. Each mean represents three replicates (SE).

a molecular mass in the range of 16e32 kDa. In this study, a trypsin inhibitor from P. moniliformis (PmTKI) of 19.2 kDa was purified, which was consistent with the molecular mass of other Kunitz trypsin inhibitors [5,6,8e10,21,22]. An alignment of the N-terminal Table 3 Inhibitory activity of PmTKI toward serine and cysteine proteinases. Enzyme

Source

PmTKI inhibition %  SE

Trypsin Chymotrypsin Elastase Bromelain Papain

Bovine pancreas Bovine pancreas Porcine pancreas Pineapple stem Papaya latex

99  43  3 nd 23 

nd: not detectable; SE: standard error.

0.4 3.0 1.5 1.0

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Fig. 4. Effects of PmTKI (%, w/w) on C. capitata larvae development in artificial diet system: (A) % larva mass decrease; (B) % mortality larvae. Each mean represent six replicates (SE).

Fig. 3. Kinetic analysis of PmTKI over trypsin and chymotrypsin activities. Data were illustrated and Ki values were determined by Dixon plots. (A) Inhibition of trypsin activity using different BApNA (0.5 and 1.0 mM) concentrations (B) LineweavereBurk diagram for trypsin with three inhibitor concentrations. The symbols represented different inhibitor concentrations being circles 3.2
108 M, triangles 1.6
108 M, squares 0.8
108 M and diamonds being enzyme concentration of 0.3 mg mL1.

Table 4 Inhibitory activity of PmTKI toward digestive insect proteinases. Enzyme Coleopteran A. grandis (Cotton boll weevil) Z. subfasciatus (Mexican bean weevil) C. maculatus (Cowpea weevil) Lepidopteran P. interpunctella (Meal moth) T. licus (Giant borer) Dipteran C. capitata (Fruit fly) SE corresponds to standard error.

% inhibition  SE 90.0  5.2 6.0  3.3 9.5  2.8 60.0  2.7 24.0  2.0 70.5  0.4

amino acid sequence of PmTKI with the other inhibitors revealed a higher identity with Kunitz inhibitors of the Mimosoideae subfamily. The alignment of the inhibitors showed an asparagine residue in the position 13 (Asn13) that is strictly conserved between members of the Kunitz inhibitor family. Asparagine residue was confirmed to be important for the inhibitory activity of these proteins [23]. PmTKI presented 30 amino acid residues and higher identity degrees were observed suggesting that PmTKI is a new member of the Kunitz-type inhibitors family. PmTKI showed a high degree of thermal, pH and reducing environment stability. Furthermore PmTKI was not sensitive to reducing agent as DTT suggesting a composition of a single polypeptide chain as previously observed for other Kunitz-type inhibitors [8e10,21,22]. Some inhibitors of this subfamily are formed by a single polypeptide chain, as found for inhibitors purified from Pithecellobium dumosum [8e10] and Dimorphandra mollis [24] seeds. PmTKI data was similar showing a single polypeptide chain, as confirmed by SDS-PAGE in reducing conditions. It is possible that PmTKI possess intramolecular disulfide bridges in its structure, which is responsible for the inhibitory stability of these proteins against physical and chemical agents. However, the complete amino acid sequence of PmTKI is required to confirm the presence of intramolecular disulfide bridges. Moreover, the study of temperature effect on PmTKI showed a small decrease in the inhibitory activity when pre-incubated for 30 min at temperatures between 60 and 100  C. Pre-incubation of the inhibitor in the pH range (2e12) for 30 min did not affect trypsin inhibitory activity. The high degree of thermal and pH stability observed for these inhibitors has also been observed

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for Kunitz-type inhibitors from seeds of P. dumosum [8e10] and Putranjiva roxburghii [25]. Specificity studies of PmTKI have shown a greater inhibition of trypsin proteolytic activity than toward chymotrypsin but it did not affect the activity from pig pancreas elastase. The activity of two cysteine proteases has also been analyzed. PmTKI was not effective against pineapple bromelain, however, it was able to inhibit the papaya plant papain activity in about 23%, reinforcing the idea that these inhibitors can act on other enzyme classes as observed for Crotalaria pallida [6], A. pavonina [7] and P. dumosum [8e10], all representatives of the subfamily Mimosoideae [26]. Inhibition kinetics showed that PmTKI is a competitive inhibitor against trypsin and chymotrypsin, similar to inhibitors from P. dumosum [8e10], P. roxburghii [25], Brassica juncea [27] and Archidendron ellipticum [28]. The Ki value of PmTKI was 1.5
108 and 3.0
101 M against trypsin and chymotrypsin, respectively, confirming its stronger affinity toward trypsin than to chymotrypsin in agreement with the reports of others inhibitors [6,9,21]. In order to verify the activity of the purified PmTKI, digestive proteinases from Coleopteran, Lepidopteran and Dipteran pests were extracted and assayed against BApNA. PmTKI showed the highest inhibition against trypsin-like enzymes from A. grandis, (90%) and P. interpunctella (60%) and from C. capitata (70.5%). A number of Kunitz related inhibitors from plants were evaluated in vitro for the potential to decrease the activity of digestive enzymes from Lepidopteran, Coleopteran and Dipteran larvae. Among these, CpaTI (C. pallida trypsin inhibitor) was active against serine proteinases from the larvae of A. grandis with inhibitory activity of 52% [6]. Kunitz inhibitors purified from Tamarindus indica [5], D. mollis [24], A. pavonina [7], C. pallida [6], Prosopis juliflora [29] and P. dumosum [8] inhibited in the range of 70e87% inhibition the trypsin-like digestive enzymes of C. maculatus, a Coleptera: Bruchidae. In contrast, PmTKI was more effective for trypsin-like enzymes of P. interpunctella (Lepidoptera: Pyralidae) than PdKI [8], PdKI-2 [9]. In addition, trypsin-like digestive enzymes from C. capitata (Diptera, fruit fly) were in vitro inhibited (53, 65 and 70%, respectively) by CpaTI [6], PdKI [8] and PdKI-2 [9], similar to PmTKI. The determination of the in vitro specificity of an inhibitor against insect digestive enzymes is a good tool to analyze its effects on the development of insects in an artificial diet system containing inhibitors. The bioinsecticidal effects of PmTKI, at doses from 0.05 to 1% (w/ w), were tested in a feeding-trial model against C. capitata larvae, an important and economically damaging pest in tropical and subtropical regions. The results showed strong deleterious effects of PmTKI on the mass and survival of C. capitata during larval developmental, when larvae were fed with a diet containing various concentrations of the inhibitor. The larval mass was decreased by 50% (ED50) and mortality was 50% (LD50), at a level of 0.3% and 0.37% (w/w), respectively. This partial effect of PmTKI on the mortality and the larvae mass was probably due to the fact that the larvae digestive system is based on both chymotrypsin- and trypsin-like serine proteinases [30] with either a high specificity or resistance of PmTKI to trypsin-like enzymes. Bioassays against dipteral insect pests are rarely reported in the literature. For example, when SKTI (Soybean Kunitz Trypsin Inhibitor) was added at level of 1.15% in artificial diets and offered to Lucilia cuprina, a strong reduction in the weight of larvae was observed at around 80%. This susceptibility can be explained because the major proteinases from these larvae are trypsin-like proteinases [31]. Otherwise, TTI and CapTI were inactive against C. capitata larvae at high concentrations with only 3e4% (w/w) of the inhibitor being observed to have deleterious effects [5,6]. The use of proteinase inhibitors that target gut enzymes shows high promises for insect pest control.

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3. Material and methods 3.1. Material Enzymes: Papain, Bromelain, Bovine chymotrypsin, Bovine Trypsin and Porcine Elastase; Substrates: BApNA, BTpNA and Azocasein were purchased from Sigma Chemical Co. (St. Louis, MO). The following abbreviations are used: BApNA, N-benzoyl-DLarginyl-p-nitroanilide; BTpNA, N-benzoyl-L-tyrosyl-p-nitroanilide; and SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. All were purchased from Sigma (St. Louis, MO, USA). 3.2. Purification of P. moniliformis trypsin inhibitor Catanduva (P. moniliformis) seeds were obtained from ICMBio/ MMA (Floresta Nacional de Nísia Floresta, Instituto Chico Mendes de Conservação da Biodiversidade) seed bank, Rio Grande do Norte, Brazil. Finely ground Catanduva seed meal was extracted (1:10, w/ v) with 50 mM TriseHCl buffer, pH 7.5 for 30 min at 20  C. After centrifugation for 30 min at 8000
g at 4  C, the supernatant was named crude extract (CE) and precipitated with trifluoroacetic acid (TCA) at final concentrations of 1, 2, 3 and 4%. The fractions obtained were named F1, F2, F3 and F4 and were then dialyzed against distilled water, freeze-dried and submitted to anti-tryptic assays. The fraction denominated F1 showed high inhibitory activity against bovine trypsin and was utilized for the next purification steps. This fraction was applied to a TrypsineSepharose 4B column (4.0 cm
2.5 cm column), equilibrated with 50 mM TriseHCl buffer, pH 7.5. Fractions of 1.5 mL were manually collected at a flow rate of 30 mL h1. The retained peak was eluted with a 5 mM HCl solution, dialyzed and the lyophilized was submitted to analytical RP-HPLC C4 Vydac (5 mm, 250 mm
10 mm) chromatography with a gradient of solvent B (60% acetonitrile/0.1% TFA/H2O) using the following mobile phase conditions: H2O:ACN:TFA (95:05:0.1, v:v:v) for 5 min, then a linear gradient to H2O:ACN:TFA (05:95:0.1, v:v:v) in 60 min at a flow rate of 2.5 mL min1. The purified peak, named PmTKI, was subjected to further analysis. The experiments were conduced at room temperature and monitored at 216 and 280 nm. Fractions were manually collected and lyophilized. The protein quantification was measured by Bradford (1976) procedure with bovine serum albumin (BSA) as protein standard. 3.3. Preparation of the insect gut proteinases P. interpunctella, C. maculatus and Z. subfasciatus were supplied by Laboratório de Química e Função de Proteínas from the Department of Biochemistry, UFRN, Brazil. A. grandis and T. licus were obtained from Centro Nacional de Recursos Genéticos e Biotecnologia (CENARGEN/EMBRAPA), Brasília, Brazil. C. capitata were obtained from Laboratório de Mosca das Frutas from the Departament of Cellular Biology and Genetics, UFRN, Brazil. Larvae and adult insect proteases were obtained after dissection and extraction of the guts [32]. The guts were surgically removed from the animal and placed into an iso-osmotic saline (150 mM NaCl) solution. Gut tissue was stirred and centrifuged at 10,000
g at 4  C, for 10 min. The supernatants were then recovered and used for in vitro assays. Additionally, all insect gut homogenates were prepared in TriseHCl 50 mM at different pHs. The better activity presented for C. maculatus, Z. subfasciatus, A. grandis, T. licus, Alabama argilacea and C. capitata was pH 8.5 and for P. interpunctella pH 9.5 (data not shown). 3.4. Antitrypsin activity The trypsin inhibitory assay was performed by using BApNA as a substrate [2]. Ten microliters of trypsin (0.3 mg mL1 in 2.5 mM

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HCl) solution was incubated for 15 min at 37  C with 60 mL of inhibitor solution (0.5 mg mL1) and 120 mL of 50 mM TriseHCl buffer, pH 7.5. The molar ratio used was of 1:1 for all assays here realized with PmTKI versus trypsin. Reactions were started with an addition of 500 mL of 1.25 mM BApNA solution, prepared in 1% DMSO (v:v) and 50 mM TriseHCl buffer, pH 7.5. After 15 min at 37  C, the reaction was stopped by an addition of 150 mL of 30% acetic acid glacial solution. The assay colorimetric was measured by absorbance at 405 nm. One unit of inhibitory activity was defined as being the amount of inhibitor that decreased absorbance by 0.01 at 405 nm. All assays were made in triplicate and the results of each series were expressed as the average value  standard deviation (SD). In addition, the ability of PmTKI to inhibit the serine (porcine elastase) and cysteine (papain and bromelain) proteinases were assayed using 1% azocasein solution as a substrate, as described by Xavier-Filho et al. [33]. Prior to evaluating the potential of PmTKI in vivo and in vitro assays against insect serine protease were performed. The effect of PmTKI on the proteolytic activity of whole-gut extracts was measured by using BApNA (1.25 mM) as a substrate [34]. The assays were performed in 50 mM TriseHCl buffer, pH 7.5. PmTKI (0.5 mg mL1) was incubated with a 40 mL aliquot (20 mg of protein) of gut extracts at 37  C for 15 min before adding the substrate. Reactions were started with an addition of 500 mL of 1.25 mM BApNA solution, prepared in 1% DMSO (v/v) and 50 mM TriseHCl buffer, pH 7.5. After 30 min at 37  C, the reaction was stopped using an addition of 150 mL of 30% acetic acid solution. The color development was measured by absorbance at 405 nm. All assays were made in triplicate and the results of each series were expressed as the average value  SD. 3.5. Antichymotrypsin activity The inhibitory activity against bovine chymotrypsin was determined at standard concentration of 0.2 mg mL1. Enzyme was pre-incubated with 50
103 mol L1 TriseHCl buffer, pH 7.5 containing 2.0
102 mol L1 CaCl2 and PmTKI for 15 min at 37  C. After this time the reaction was initiated by addition of 200 mL of 1% azocasein (w/v). After 30 min, the reaction was stopped by adding 300 mL of 20% TCA (w/v). The reaction mixture was centrifuged at 12,000
g for 10 min and the supernatant was alkalinized with 2 mol L1 NaOH 1:1 (v:v), and absorbance was measured by spectrophotometer at 440 nm [33]. Assays were performed in triplicate and three independent experiments were conducted. 3.6. In vitro inhibitory activity of PmTKI forward elastase and papain assays In order to determine the in vitro porcine elastase activity, 30 mL of enzyme solution at a standard concentration of 100 mg mL1 dissolved in 50 mM TriseHCl buffer, pH 7.5 were pre-incubated with 350 mL of 50 mM TriseHCl buffer at pH 7.5 and 30 mL of PmTKI (at a standard concentration of 0.1 mg mL1) for 15 min at 37  C followed by the addition of 500 mL of 1% azocasein. The reaction was stopped after 30 min pre-incubation by adding 150 mL of 20% TCA solution (w:v). The reaction was centrifuged for 10 min at 12,000 g and aliquots of 500 mL from supernatants were added to 500 mL of 2 mol L1 NaOH 1:1 (v:v). Optical density was further measured at 440 nm [35]. In order to determine the inhibitory activity against papain, 10 mL of papain solution (100 mg mL1 dissolved in 25 mM sodium phosphate buffer, pH 6.0) was pre-incubated with 20 mL of activation solution (2 mM EDTA and 3 mM DTT in 25 mM sodium phosphate buffer, pH 6.0), 10 mL of PmTKI (0.1 mg mL1) and 250 mL of 25 mM sodium phosphate buffer, pH 6.0 for 10 min at 37  C

followed by the addition of 200 mL BANA 1 mM in 1% (v/v) DMSO and 25 mM sodium phosphate buffer, pH 6.0). After 20 min at 37  C, the reaction was stopped by adding 500 mL of 2% HCl dissolved in ethanol (v:v) and the colored product was developed by the addition of 500 mL of 0.06% p-dimethylaminocinnamaldehyde solution dissolved in ethanol. BANA hydrolysis was measured at 540 nm [36]. Assays were performed in triplicate and three independent experiments were conducted. 3.7. Polyacrylamide gel electrophoresis Denaturant sodium dodecyl sulfate polyacrylamide gel electrophoresis (12% SDS-PAGE) was performed as described by Laemmli [37]. Protein utilized as molecular weight markers were b-Galactosidase (116 kDa), BSA (66 kDa), ovalbumin (45 kDa), lactate dehydrogenase (35 kDa), restriction endonuclease Bsp981 (25 kDa), b-lactoalbumin (18.4 kDa), and lysozyme (14.4 kDa) purchased from Fermentas Inc. (Burlington, CA). 3.8. Mass spectrometry analyses PmTKI molecular mass was determined by using MALDI-ToF MS/MS analysis (UltraFlex III, Bruker Daltonics, Billerica, MA). Purified inhibitor was dissolved in a minimum volume of water that was mixed with an a-cyano-4-hydroxycinnamic acid saturated matrix solution (1:3, v:v), spotted onto a MALDI target plate and dried at room temperature for 5 min. The a-cyano-4-hydroxycinnamic acid matrix solution was prepared at 50 mM in H2O:ACN:TFA (50:50:0.3, v:v:v). Protein monoisotopic mass was obtained in the reflector mode with external calibration, using the Protein Calibration for mass spectrometry (up to 50 kDa mass range, Bruker Daltonics, Billerica, MA). 3.9. Determination of primary sequence N-terminal amino acid sequence analysis of PmTKI was determined at the Biochemistry and Molecular Biology of the Federal University of Ceará, Brazil using an automated protein sequencer from Shimadzu PPSQ-23 (Shimadzu Co., Kyoto, Japan) performing Edman degradation. A reversed-phase C18 column (4.6 mm
2.5 mm) under isocratic conditions was used for analysis of the PHT-amino acids retention times monitored at 269 nm. The sequence obtained was aligned with non-redundant protein database using the Blastp tool in order to determine similarity with others inhibitors. In addition, alignments among the sequences obtained were carried out by using ClustalW [38] in order to analyze the amino acids conserved. 3.10. Thermal, pH and reduction environment stabilities The thermal stability of PmTKI (1 mg mL1) was tested by incubation of the protein at different temperatures (37, 40, 60, 70, 90 and 100  C) for 30 min [6]. After heat shock at 4  C for 10 min the trypsin inhibitory was performed against trypsin. The stability in a broad range of pH was also checked where samples of PmTKI (1 mg mL1) were prepared in different buffers conditions including, 100 mM glycineeHCl (pH 2e3), 100 mM sodium phosphate (pH 6e 8) and 100 mM glycineeNaOH (pH 11e12) [39]. After incubation in each buffer, the samples were evaluated against trypsin using BApNA as a substrate. For activity evaluation of PmTKI in reduction environment, dithiothreitol (DTT) was utilized as a reducing agent in agreement with the methodology of Mello et al. with minor modifications [24]. PmTKI was pre-incubated in several final concentrations (1, 10 and 100 mM) and the reaction was stopped with an addition of iodoacetamide (1:2, v:v). The controls were

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performed in the absence of DTT. All assays were done in triplicate and the results of each series were expressed as the average value  SD.

[9]

3.11. Kinetic studies The inhibition constant was determined for bovine trypsin and chymotrypsin enzyme, pre-incubating the enzyme with crescent concentrations of PmTKI (3.75, 5.63, 7.50, 11.25 and 15.00 mM) in 50 mM TriseHCl buffer, pH 7.5 at 37  C. Two substrate concentrations for each enzyme were used. For trypsin, assays used BApNA (0.5 and 1.0 mM) and for chymotrypsin assays used BTpNA as a substrate (0.25 and 0.5 mM) [34]. The velocities of the reactions were expressed as 1/v (OD440/h/mL)1 and Ki value was determined by intersection of the two lines for each substrate concentration using a Dixon plot [40]. 3.12. Insect bioassay

[10]

[11]

[12]

[13] [14] [15]

The development of C. capitata in an artificial diet system was carried out in accordance with Macedo et al. [15]. Artificial diets (ca. 500 mg each) were prepared using 10.4% finely ground sugar cane fibers, 3% wheat germ, 6.5% wheat flour, 12% crystal sugar, 9.9% yeast, 0.3% sodium benzoate (VETEC), 0.9% HCl and 57% H2O. Concentrations of 0.05, 0.2, 0.5 and 1.0% (w/w) of PmTKI lyophilized were added to this diet. After preparation, the diets were presented to seven neonate larvae per diet in dark glass vial at a controlled temperature of 28  1  C and at 60e70% relative humidity in the growth chamber. After 4 days, the vials were opened and the mass and number of the larvae were measured. The experiments were carried out in six replicates and the average (SD) was calculated. Control artificial diets were made without PmTKI. Acknowledgments The authors are thankful for the grants and support from CAPES, CNPq, FAPDF, FAPERN and RENORBIO-FINEP. Our sincere acknowledgements to Mauricio Pereira de Sales, a great researcher and professor (in memoriam).

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