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Characterization of the AlTI13 protein from Indian siris (Albizia lebbeck)that inhibits the growth of cotton bollworm (Helicoverpa armigera)

Faiyaz K. Shaikh ⁎, Prafull P. Gadge, Ashok A. Shinde, Manohar V. Padul, Manvendra S. KacholeDepartment of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University Aurangabad, MS, India

a b s t r a c ta r t i c l e i n f o

Article history:Received 15 June 2013Revised 15 September 2013Accepted 8 February 2014Available online 19 February 2014

Keywords:Trypsin inhibitorsAlbizia lebbeckHelicoverpa armigeraGel X-ray film contact print techniqueInsect resistance

Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a simple and sen-sitive gel X-ray film contact print technique. About 17 AlTIs were detected in the seed extracts of A. lebbeck. Twogroups of AlTIs—1 major (10 AlTIs; slow migration on the gel) and 1 minor (7 AlTIs; fast migration on the gel)were identified. The former was specific only toward trypsin. However, the latter was specific toward both tryp-sin and Helicoverpa armigera gut proteinases (HaGPs). The most potent AlTI (AlTI13) was purified to assess itsin vivo bioefficacy toward HaGPs. Purification was achieved using (NH4)2SO4 fractionation, Sephadex G-100 col-umn chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependentbioefficacies of AlTIs in the (NH4)2SO4 F3 fractions (0.1%, 0.5%, and 1%) were approximately 79%, 83%, and 90%,respectively, resulting in reductions in the average larval weight of H. armigera. Artificial diet containing a singledose of AlTI13 (5 μg/g diet) reduced the larval weight by about 76%, with 60%mortality. The half-maximal inhib-itory concentrations (IC50) of AlTI13 for trypsin and HaGPs were 0.14 and 0.17 μmol/ml, respectively. The opti-mum conditions for AlTI13 were pH 8 and temperatures ranging from 35 to 40 °C. Reducing sodium dodecylsulfate-PAGE analysis indicated that ~28 kDa Kunitz-like trypsin inhibitor was present. Thus, we showed thatAlTIs, particularly, AlTI13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insectresistance in genetically engineered plants.© 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection

Society. Published by Elsevier B.V. All rights reserved.

Introduction

Proteinase inhibitors (PIs) are plant defensive proteins that havemolecular weights ranging from 4 to 85 KDa (Hung et al., 2003). In gen-eral, these inhibitors are grouped into families such as serine, cysteine,aspartic, and metallo PIs based on the target proteinase (Laskowskiand Kato, 1980). Among these, serine proteinase inhibitors are predom-inantly found in the plant kingdom, and have been categorized into 9subfamilies, namely, (1) Kunitz, (2) Bowman-Birk, (3) Cucurbitaceae,(4) potato I (PPI-1), (5) potato II (PPI-2), (6) superfamily of cereal inhib-itors, (7)mustard trypsin inhibitor, (8) serpin, and (9) amylase inhibitorof α-amylase (ragi) and trypsin of cereal (Shewry and Lucas, 1997;Christeller and Laing, 2005; Blanco and Casaretto, 2012). Kunitz-likePIs have been shown to have deleterious effects against pest insects,thus, representing promising candidates in the transgenic plant approach(Macedo et al., 2011). Structurally, Kunitz PIs have 1–2 polypeptidechains with low cysteine content and 4 cysteine residues organized into2 disulfide bridges. These PIs inhibit trypsin, chymotrypsin, subtilisin,and human plasma kallikrein.

Kunitz PIs, mainly trypsin inhibitors (TIs), inhibit proteinases in thegut of insects when consumed along with the diet or when expressedat certain levels in plants with which the insects are associated, thus,suppressing larval growth and development (Shewry and Lucas, 1997;Mosolov et al., 2001; Srinivasan et al., 2005; Délano-Frier et al., 2008).In addition, these inhibitors also play important roles on a broad rangeof biological activities, including the storage and regulation of endoge-nous proteinases in plants (Xavier-Filho, 1992; Shewry, 2003). These in-hibitors also form part of an effective component that may be used inthe treatment of blood clotting, hemorrhaging, inflammation, and can-cer (Chen and Shaw, 2003; Fook et al., 2005; Mello et al., 2006). Theseinhibitors may be synthesized constitutively during normal develop-ment or may be induced in response to insect and pathogen attacks(Ryan, 1990).

Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is an insectpests that devastate many important agricultural crop plants, includingcotton, chickpea, pigeonpea, tomato, sunflower, okra, and corn(Patankar et al., 2001). This insect pest is able to wipe out entire crops,invade more than 300 plant species, and is one of themost serious agri-cultural insect pest worldwide (Pawar, 1998; Rajapakse and Walter,2007). This pest has a complicated serine proteinase system, with pre-dominantly trypsin-like activity in the gut. H. armigera is able to adaptPIs expressed in host plants by producing inhibitor-insensitive,

Journal of Asia-Pacific Entomology 17 (2014) 319–325

⁎ Corresponding author at: Dept. of Biochemistry, Dr. Babasaheb AmbedkarMarathwada University Aurangabad 431004 Maharashtra, India. Tel.: +91 9422993637.

E-mail address: faizbiochem@gmail.com (F.K. Shaikh).

http://dx.doi.org/10.1016/j.aspen.2014.02.0021226-8615/© 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.

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inhibitor-resistant, and/or inhibitor degrading proteinases in the gut(Johnston et al., 1991; Broadway and Villani, 1995; Jongsma andBolter, 1997;Wu et al., 1997; Giri et al., 1998; Patankar et al., 2001). En-zymes that were both susceptible and resistant to inhibitors have beenfound in this insect pest (Bown et al., 1997; Dunse et al., 2010). This typeof adaptation by the proteinase system of H. armigera to host plant PIsstimulates researcher for the identification of eco-friendly alternativesto control this pest (Wu et al., 1997). Many PI isoforms are resistant tothe proteinases of H. armigera, including PIs extracted from non-hostplants, such as the winged bean (Psophocarpus tetragonolobus), thegroundnut (Arachis hypogaea), and the bitter gourd (Momordicacharantia). Moderate to high inhibition potential has been reported forthewinged bean against HaGPs, fromwhich the resulting PIswere char-acterized (Harsulkar et al., 1999; Giri et al., 2003). Bitter gourd protein-ase inhibitors (BGPIs) influence fertility, fecundity, and protein uptakewhen added to the diet of H. armigera and Spodoptera litura (Telanget al., 2003). The PIs in Acacia nilotica seeds were found to be effectiveat inhibiting H. armigera development and gut proteinases (Babu et al.,2012). Furthermore, the bioefficacy of the mustard trypsin inhibitorMTI-2 expressed at different levels in transgenic tobacco, arabidopsis,and oilseed rape lines has been well established for 3 different lepidop-teran species; namely, Plutella xylostella (L.), Mamestra brassicae (L.),and Spodoptera littoralis (De Leo and Gallerani, 2002).

Albizia lebbeck (L.) Benth. (Leguminosae, Mimosoideae), commonlyknown as Indian siris, is a perennial tree. The seed extracts of thisplant are known to contain multiple isoforms of PIs and amylase inhib-itors (Sharma et al., 2012; Hivrale et al., 2013; Shaikh et al., 2013). ThesePIs significantly influence the growth and survival of H. armigera larvae(Sharma et al., 2012; Hivrale et al., 2013). Therefore, the PIs of A. lebbeckmay be of importance in the transgenic plant approach, because theyexpress a wide range of host and non-host plant PIs, which, if purified,could be separately characterized and evaluated. Within this frame-work, here, we identify 17 AlTI isoforms from the seed extracts ofA. lebbeck by the gel X-ray film contact print technique (Pichare andKachole, 1994). Out of these 17 AlTIs, 7 inhibited HaGPs. We studiedthe effects of all AlTIs and the most potent AlTI (AlTI13) on H. armigeragrowth and development in vivo. Quantitative estimations wereperformed using enzyme assays, while qualitative analyses were con-ducted by electrophoretic separation followed by the gel X-rayfilm con-tact print technique.

Materials and methods

Procurement of A. lebbeck seeds and chemicals

Dry A. lebbeck seeds were collected from the campus of Dr. BabasahebAmbedkar Marathwada University, Aurangabad (MS), India. Second andfourth instar larvae of H. armigera were collected from a chickpea fieldin Maliwada, Aurangabad (MS), India. Trypsin (bovine pancreas, E.C.3.4.21.4), N-α-benzoyl-DL-arginine-p-nitroanilide (BApNA), ammoni-um sulfate, acrylamide, bisacrylamide, tetramethylethylenediamine(TEMED), ammonium persulfate, hexane, and polyvinylpyrrolidone(PVP) were obtained from Sisco Research Laboratories (SRL), Mumbai,India. Sephadex G-100 gel and gelatin were purchased from Sigma-Aldrich, St. Louis, MO, USA. Molecular weight markers were purchasedfrom Genei, Bangalore, India. X-ray films were obtained from Fuji film,USA. All other chemicals used in this studywere of the available highestpurity.

Extraction of AlTIs

The mature, dried seeds of A. lebbeckwere pulverized to a fine pow-der by a mixer grinder. The fat was removed from the powder by hex-ane and acetone washes. Defatted powder was suspended in Milli-Qwater (1:6 w/v) containing 1% PVP and incubated overnight at 15 °C.The suspension was then centrifuged at 12,000 ×g for 20 min at 4 °C,

and the supernatant was termed as crude AlTIs (Pichare and Kachole,1994; Padul et al., 2012). Crude AlTIs (about 40 ml) were saturated by(NH4)2SO4 in 3 fractions (i.e., 30%, 60%, and 90% (F1 [0–30%], F2[30–60%], and F3 [60–90%]) at 4 °C overnight. The AlTI precipitate thatwas obtained at each stage was dissolved in a minimum quantity ofbuffer (0.1 M Tris–HCl, pH 7.8) and centrifuged at 12,000 ×g and 4 °C.The supernatant was dialyzed extensively against the same buffer at4 °C overnight. The obtained AlTI fractions were stored in small vialsfor the subsequent analyses.

Extraction of H. armigera gut proteinases (HaGPs)

For HaGPs extraction, the midgut tissue of the fourth instar larvaeof H. armigera was dissected and stored at −20 °C. Fresh or thawedmidgut tissue was homogenized in 0.1 M glycine–NaOH buffer(1:3 w/v pH 9.6) for 15min at 10 °C. The suspension was centrifugedat 12,000 ×g for 20 min at 4 °C. The resulting supernatant was usedas a source of HaGPs (Padul et al., 2012).

Dot-blot/spot test

The dot-blot/spot test was carried out to determine the potency ofAlTIs against trypsin and HaGPs using X-ray film (Pichare and Kachole,1994; Padul et al., 2012). Three different concentrations of the enzymeand inhibitorwere prepared: 1(1:3), 2(1:1), and 3(3:1) v/v. The volumeof the reaction mixture was adjusted by 0.1 M Tris–HCl (pH 7.8) fortrypsin and 0.1 M glycine–NaOH buffer (1:3 w/v pH 9.6) for HaGPs.The total volume was made up to 20 μl by using respective buffer andloaded onto X-ray film. The film with spots was incubated for 20 minat 37 °C, then washed with tap water and dried in air. Different ratiosof enzyme and inhibitor produced different patterns of gelatin hydroly-sis on theX-rayfilmdepending on the efficiency of inhibitor, whichmaybe observed visually and scanned at 300 dpi using an HP digital scanner.

Visualization of AlTIs by gel X-ray film contact print technique

Crude AlTIs and fractions (F1, F2, and F3) of (NH4)2SO4were analyzedqualitatively on 10% Native-PAGE by employing a discontinuous buffersystem (Davis, 1964). Samples of about 50 μg were loaded into eachwell of the gel. A constant current (20 mA) was supplied to the geluntil the tracking dye bromophenol blue (BPB) reached the bottom ofthe gel. After electrophoresis, the gel was processed for AlTI activity bythe gel X-ray film contact print technique (Pichare and Kachole,1994). The gel was equilibrated in 0.1M Tris–HCl (pH 7.8), and incubat-ed with 0.1 mg/ml trypsin solution for 10 min. Excess trypsin was re-moved by the buffer. The gel was overlaid on X-ray film for 5 to10 min. The film was then washed with tap water and the AlTI bandswere visualized as unhydrolyzed gelatin. The X-ray film was scannedat 300 dpi using an HP digital scanner. The gel was then washed andstained by 0.1% Coomassie brilliant blue R-250 (CBB R-250). The sameprocedure was carried out for HaGPs using 0.1 M glycine–NaOH(pH 9.6) buffer containing 0.3 M CaCl2. The experiment was repeated3 times with 3 replicates each.

Purification of AlTI13

AlTI13was purified by gel filtration chromatography and preparativeelectrophoresis. The dialyzed fraction (F3) of the (NH4)2SO4 showingmaximum inhibitory activity was loaded on a Sephadex G-100 column(30 × 1.8 cm, flow rate 1 ml/3 min) that was equilibrated with 0.1 MTris–HCl, (pH 7.8). Fractions of 1.5ml volumewere collected. The quan-tity of proteins thatwere presentwasmonitored on a PC-based spectro-photometer (JASCO 500 series) at 280 nm. The active fractions weretested for inhibitory activity by the dot-blot assaymethod. The fractions(18–28) showing inhibitory activity were pooled together and concen-trated by lyophilization. The fractionwas further purified bypreparative

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native-PAGE. After electrophoresis, a vertical strip of gel was cut andprocessed to detect the AlTI13 activity band on X-ray film, as previouslydescribed. A horizontal strip of the remaining gel corresponding to theAlTI13 activity band of the X-ray film was excised and stored overnightat−20 °C, and then the sample was eluted. Preparative electrophoresiswas repeated several times to obtain sufficient amounts of AlTI13 for fur-ther characterization. The molecular weight of the partially purifiedAlTI13 was analyzed by 12% reducing SDS-PAGE (Laemmli, 1970).

Assay of trypsin inhibitory activity

Trypsin inhibitory activity was assayed according to the method ofKakade et al. (1969), with slight modifications using BApNA as the sub-strate and bovine trypsin as the standard enzyme. The reaction mixturecontained diluted inhibitor extract and trypsin (prepared in 0.01 NHCL). This mixture was incubated for 10 min in 0.1 M Tris–HCL(pH 7.8), which contained 0.3 M CaCl2. To the above mixture, 0.3 mlBApNA (prepared in appropriate volume of DMSO to make 1 mm con-centration) was added. The final volume of the reaction mixture was1.2 ml. After incubating the reaction mixture at 37 °C for 30 min, the re-actionwas terminated by adding 0.3ml of 30% glacial acetic acid (v/v). Ablank and a trypsin control were run simultaneously. The absorbancewas recorded at 410 nm against the blank. The inhibition of HaGPs wasassessed by incubating the inhibitor with HaGPs for 20 min in 0.1 Mglycine–NaOH (pH 9.6), which contained 0.3 M CaCl2. One trypsin ac-tivity unit is equal to the release of 1.0 μmol of p-Nitroaniline per minunder specific assay conditions, with 1 inhibitor unit being defined asthe inhibition of 1 enzyme unit. Inhibitory activity is expressed as thepercentage of inhibited enzyme activity out of the total enzyme activity.

Effect of pH and temperature on AlTI13

The effect of pH on AlTI13 (5 μg) was studied by performing theinhibitor assay with trypsin (20 μg) in different buffer systems: 0.1 Mglycine–HCL (pH 2), 0.1 M sodium-citrate (pH 4) and (pH 6), 0.1 MTris–HCL (pH 8), 0.1 M glycine–NaOH (pH 10), and 0.1 M glycine–NaOH (pH 12) at 37 °C, as described previously. The effect of tempera-ture on AlTI13 activity was studied by performing the reaction at differ-ent temperatures (20 °C to 80 °C at 5 °C intervals) in a dry bath. Thereaction was terminated by determining the residual AlTI13 activity im-mediately after placing the pre-incubated samples on ice.

Insect bioassays

The field collected H. armigera second instar larvae were reared onan artificial diet containing different doses of AlTIs at 28 ± 2 °C undera 16:8 light/dark (L:D, respectively) photoperiod. Three replicates ofall experiments were performed. Bioassays were performed using100 ml sterilized artificial diet (Giri and Kachole, 1998). The fraction ofammonium sulfate (F3) with the highest inhibitory activity was inte-grated into the artificial diet at 3 different doses (0.1%, 0.5%, and 1%,w/w) as previously suggested (Johnston et al., 1993; Ramesh Babuand Subrahmanyam, 2010). The diet with no added AlTIs was used asthe control diet. All diets were incubated overnight at 4 °C beforebeing offered to the larvae. Starved second instar larvae of averageweight were kept in separate vials and fed daily. The food was changeddaily, and was not allowed to dry or be completely eaten. Larval weightwas recorded at the same time every day. To test the effectiveness ofAlTI13, a single dose (5 μg/g diet)was incorporated into the artificial diet.

Protein determination

Protein content was estimated by following the method of Lowryet al. (1951) using bovine serum albumin (BSA) as the standard.

Statistical analysis

All experiments were conducted and analyzed in triplicate. Themeans and standard deviations were calculated and compared. Thestatistical significance of the difference was examined using one-wayanalysis of variance (ANOVA) on Minitab statistical software (Version15).

Results

Detection, visualization, and specificities

When the 3 protein fractions and crude AlTIs were analyzed for AlTIactivity by the gel X-ray film contact print technique on 10% native-PAGE, 17 distinct AlTIs were identified (Fig. 1). The greatest number ofAlTI bands were detected in the crude and F3 fraction. The F1 fractioncontained the least number of AlTI bands. Among the 17 AlTIs, 7 AlTIsinhibited HaGPs (Fig. 2). Based on the band specificities, the AlTIswere divided in 2 groups. The AlTIs (10) that migrated slowly on thegel and specifically inhibited trypsin belonged to the major group. Incomparison, the AlTIs (7) that migrated quickly on the gel and inhibitedboth trypsin and HaGPs belonged to the minor group. Among the fastmoving AlTI bands, AlTI13 exhibited the strongest inhibitory activitytoward both trypsin and HaGPs. Hence, the present studywas primarilyfocused on the characterization of this distinguished candidate.

Purification of AlTI13

The F3 protein showed strong inhibitory activity against trypsin andHaGPs, as shown by the gel X-ray film contact print technique. In com-parison, the other fractions exhibited low trypsin inhibitory activity. TheF3 fraction containing inhibitory protein was loaded on a Sephadex G-100 column equilibrated with 0.1 M Tris–HCl, (pH 7.8). The fractions(18–28) showing high inhibitory activity were pooled, lyophilized,and further purified by preparative native PAGE. A single band showingactivity toward both trypsin and HaGP was visualized on the gel and X-rayfilm (Figs. 1, 2). TheAlTI13wasmade upof a single polypeptide chainwith a molecular weight of ~28 KDa, analyzed on 12% reducing SDS-

Fig. 1.Detection of Albizia lebbeck trypsin inhibitors (AlTIs) from A. lebbeck by using the gelX-ray film contact print technique; 50 μg protein was electrophoresed on 10% native-polyacrylamide gel electrophoresis (PAGE), after which the AlTI activity bands werevisualized by incubating the gel in trypsin solution and then exposing it to an X-ray film.Lane 1: Crude seed extracts; Lane 2: F1 (0–30% (NH4)2SO4 precipitation [ppt]); Lane 3:F2 (30–60% (NH4)2SO4 ppt); Lane 4: F3 (60–90% (NH4)2SO4 ppt); and Lane 5: AlTI13.

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PAGE (Fig. 3). This finding indicates that the inhibitor might belong tothe Kunitz family. Fig. 4 presents a photograph of the spot test analysisshowing the potency of AlTI13 against trypsin and HaGPs. The spot testanalysis of AlTI13 activity, whichwas performed on X-ray film, indicatedthat the AlTI13 blocked gelatin hydrolysis by trypsin and HaGPs underdifferent concentrations.

Quantitative estimation of inhibitory activity

By using specific synthetic substrates (i.e., BApNA), the inhibitory ac-tivity of the AlTIs and AlTI13 toward trypsin and HaGPs was estimated(Fig. 5 and Table 1). Crude soluble protein extract and 3 protein fractions(F1 [0–30%], F2 [30–60%], and F3 [60–90%]) were tested. The F3 proteinfraction showed strong inhibitory activity against trypsin and HaGPs,whereas the other fractions (F1 and F2) exhibited low inhibitory activity(data not shown). The F3 fraction, which had the highest inhibitory

activity toward trypsin and HaGPs, was subjected to further analysis.Equal amount (about 10 μg) of 3 different doses of the F3 fraction(i.e., 0.1%, 0.5%, and 1%) and a single dose of AlTI13 were all foundto be effective against trypsin and HaGPs (Fig. 5). Dose dependent inhi-bition by protein from the F3 fraction increased the inhibition of trypsinfrom 70.39% ± 2.3 (0.1% AlTIs) to 98.05% ± 1.3 (1% AlTIs). In compari-son, 94.70% ± 2.1 inhibition of HaGPs occurred in the presence of 1%AlTIs. Electrophoretic purified AlTI13 inhibited trypsin by 76% andHaGPs by 67%. A concentration of about 5 μg of AlTI13 was found to besufficient to cause about 60% inhibition of trypsin and HaGPs comparedto the crude extracts, where only about 12% inhibition was achieved(Table I). The IC50 of AlTI13 for trypsin and HaGPs was 0.14 μmol/mland 0.17 μmol/ml, respectively.

Biochemical characterization with reference to pH and temperature

The data presented in Fig. 6 indicated that AlTI13 was active over awide pH scale (6–12), with optimum inhibitor activity being recordedat pH 8 (62.6% inhibition). The inhibitor lost its activity when the pHwashighly acidic (pH2, 16.0% inhibition). Considerable inhibitory activ-ity was retained, even in highly alkaline pH conditions (pH 12, 46.8% in-hibition). From the data presented in Fig. 7, it can be concluded thatAlTI13 was moderately heat stable, as activity declined at temperaturesabove 60 °C, with the optimum temperature falling in the range of30–40 °C (54–56% inhibition). Above 70 °C (30% inhibition), activity de-clined, indicating that the inhibitor was denatured.

Feeding bioassay

The efficiency of A. lebbeck AlTIs against larval digestive enzymeswas assessed by incorporating 3 different doses (i.e., 0.1%, 0.5% and1.0% w/w) of (NH4)2O4 from the F3 AlTIs fraction into the artificial

Fig. 2. Detection of Helicoverpa armigera gut proteinase (HaGP)-specific Albizia lebbecktrypsin inhibitors (AlTIs) by using the gel X-ray film contact print technique; 10%native-polyacrylamide gel electrophoresis (PAGE) of A. lebbeck seed samples was per-formed, after which the AlTI activity bands were visualized by incubating the gel inHaGP solution and then exposing it to an X-ray film. Lane 1: Crude seed extracts; Lane2: F3 (60–90% (NH4)2SO4 precipitation [ppt]); and Lane 3: AlTI13.

Fig. 3. Protein profile of Albizia lebbeck seed extracts by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE). Lane 1: Standardmolecularweightmark-er; Lane 2: Crude seed extracts; and Lane 3: Albizia lebbeck trypsin inhibitor (AlTI)13.

Fig. 4. Spot test assay of Helicoverpa armigera gut proteinase (HaGP) interactions withAlbizia lebbeck trypsin inhibitor (AlTI)13. Different HaGPs and AlTI13 ratios were incubatedand loaded on X-ray film.

Fig. 5. Percent inhibition of trypsin andHelicoverpa armigera gut proteinase (HaGP) activ-ity by 0.1%, 0.5%, and 1% Albizia lebbeck trypsin inhibitors (AlTIs) and AlTI13. Residual en-zyme activity was determined using N-α-benzoyl-DL-arginine-p-nitroanilide (BApNA) asthe substrate. The results are presented as the means ± standard deviation (SD), n = 3.

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diet. A single concentration (5 μg/g diet) of AlTI13was used for the feed-ing assays. Larvae that were fed the control diet weighed considerablymore compared to those fed AlTIs and AlTI13 incorporated diets(Figs. 8 and 9).

The bioassay results showed a vital reduction in growth with in-creased mortality. The average weight of larvae that were fed controldiet after the experimental period (10 days) was 650.5 ±45.57 mg,whereas the diets containing 0.1%, 0.5%, and 1% AlTIs produced approx-imately 79%, 83%, and 90% decreases inweight, respectively (Figs. 8A, 9).The insert in Fig. 8B shows the variation in the size of H. armigera larvaeand pupae fed AlTI13 and the control diet. AlTI13 caused larval weight tobe reduced by about 76% compared to the control, with individualsweighing only 151.2 ± 22.90 mg. These effects were significant at pb 0.001 by ANOVA. Eighty percent mortality was observed in the dietcontaining 1.0% AlTIs on the 10th day of the feeding assay (Fig. 9). Mor-tality on the artificial diet containing AlTI13was 10% on day 4 and 60%onday 10 of the feeding assay. Pupae were underweight in all of the testpopulations. Pupal weight was reduced in a dose-dependent manner,to about 80% at the highest dose of AlTIs (1%), compared with the con-trol. AlTI13 caused pupalweight to be reduced by 52.5% compared to thecontrol (Fig. 9).

Discussion

Many TIs have been identified and purified from the Leguminosaesubfamily Mimosoideae (Bhattacharyya et al., 2006; Macedo et al.,2007). The purification and biopotency of 1 TI with a low molecularweight from the crude seed extract of A. lebbeckwere previously report-ed by Sharmaet al. (2012). In addition, Hivrale et al. (2013) reported thepresence of 10 TIs in the seed extracts of A. lebbeck. By using 6% native-PAGE co-polymerized with 0.5% gelatin, these TIs were detected andfound to have insecticidal properties when added to the diet of H.armigera. As part of an ongoing effort by our research team to searchfor potent TIs against H. armigera, here we report the presence ofnewly identified AlTIs in the seed extracts of A. lebbeck by using 10%native-PAGE followed by the gel X-ray film contact print technique.This paper also describes the biochemical characterization and in vivobiopotency of 1 potent AlTI obtained from A. lebbeck seeds, namedAlTI13, which has high inhibitory activity against the gut proteinases ofH. armigera, considered insect pests of agronomic importance.

Over the last 2 decades, the gel X-ray film contact print techniquehas been effectively used to detect and characterize natural proteinasesand proteinase inhibitors (Pichare and Kachole, 1994; Giri et al., 2003;Damle et al., 2005; Parde et al., 2010; Padul et al., 2012). Gel X-rayfilm contact print technique greatly enhances the separation and iden-tification of TIs, which co-migrate with non-TI proteins. About 17 AlTIs(Fig. 1) were identified in this study using 10% native polyacrylamidegel electrophoresis followed by the gel X-ray film contact print tech-nique. The AlTIs were divided into 2 groups based on their specificity.The major group containing 10 AlTIs exhibited specificity to trypsin,while the minor group containing 7 AlTIs exhibited specificity to bothtrypsin and HaGP (Fig. 2). Similar types of PIs with various specificitieshave been previously reported (Harsulkar et al., 1999). Six trypsin spe-cific PIs and 3 HaGPs specific PIs were described from the winged bean.Furthermore, 4 PIs obtained from groundnut seed extracts, which ex-hibited activity toward both trypsin and HaGPs, were also explored(Harsulkar et al., 1999).

Mimosoideae usually contain Kunitz TI formed from 2 polypeptidechains linked by a single disulfide bridge; thereby, differing them fromthe other Kunitz-type single chain inhibitors within the Caesalpinioideaeand Papilionoideae subfamilies (Araujo et al., 2005). In the current study,electrophoretically purified potent AlTI13 revealed a single band(28 KDa) on reducing SDS-PAGE (Fig. 3), indicating the presence of just1 polypeptide chain. Hence, AlTI13 is similar to other TIs extracted fromMimosoideae that have just 1 polypeptide chain (Macedo et al., 2000;Mello et al., 2001; Lingaraju andGowda, 2008). Furthermore, thisfindingis similar to that of Giri et al. (2003), who reported the presence of highmolecular weight Kunitz PIs (WBTI 1–4, MW20–28 KDa) that had HaGPinhibition potential from the winged bean. Inhibitory activity was con-firmed by spot test analysis (Pichare and Kachole, 1994; Padul et al.,2012), inwhich the clearing zone formed by gelatin hydrolysis of trypsin

Table 1Inhibitory activity of Albizia lebbeck trypsin inhibitors (AlTIs) and AlTI13 on trypsin andHelicoverpa armigera gut proteinases (HaGPs).

Enzyme type Amount ofinhibitor (μg)

Inhibitor and % inhibitiona IC50c (μmol)

AlTIs/AlTI13 AlTI13

Trypsin 1 5.68 ± 0.28b/19.7 ± 1.57 0.142 6.92 ± 0.17/37.9 ± 2.483 9.10 ± 0.13/44.2 ± 0.924 11.64 ± 0.23/50.8 ± 1.525 12.78 ± 0.48/62.2 ± 1.63

HaGPs 1 5.08 ± 0.24b/17.6 ± 1.31 0.172 6.08 ± 0.07/36.2 ± 0.253 7.02 ± 0.23/40.6 ± 2.084 8.24 ± 0.12/44.7 ± 0.805 9.10 ± 0.12/58.5 ± 1.45

a 100% activity of enzyme and buffer alone reacting with the substrate. Values b100indicate the inhibition of enzyme activity.

b Values are mean ± standard deviation for at least 3 replicates (p ≤ 0.001).c IC50= concentration of inhibitor, which reduces the enzyme activity to 50% of the ac-

tivity in the absence of inhibitors. μmol = AlTI13 (μg) divided by the molecular weight ofAlTI13.

Fig. 6. Effect of pH on Albizia lebbeck trypsin inhibitor (AlTI)13. An inhibitor assay was per-formed using trypsin (20 μg) in different buffer systems at 37 °C, as described in theMaterials and methods. The results are presented as the mean ± standard deviation(SD), n = 3.

Fig. 7. Effect of temperature on Albizia lebbeck trypsin inhibitor (AlTI)13. The assay wasperformed at different temperatures for 10 min by using trypsin (20 μg). The results arepresented as the mean ± standard deviation (SD); n = 3.

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and HaGPs was compared against that formed by trypsin and HaGPs in-cubated with an inhibitor. AlTI13 was found to be potent against variousconcentrations of trypsin and HaGPs (Fig. 4). The purification stepsemployed in the present study increased the specific activity of AlTI13from the crude form (93.33 trypsin inhibitor units [TIUs]/mg protein)to purified form (266.66 TIUs/mg protein) (data not shown), alongwith the inhibition of HaGPs from the crude form (25.40%) to the puri-fied form (58.50%) (Table 1). The low levels of purification that were ob-tainedmay be attributed to the high concentration of AlTIs, as suggestedpreviously (Prabhu and Pattabiraman, 1980). The IC50 of AlTI13 for tryp-sin and HaGPs was 0.14 μmol/ml and 0.17 μmol/ml, respectively.

AlTI13was optimally active at a pH of 8, with fair activity in the alka-line region, whereas activity was reduced in an acidic environment(Fig. 6). This finding was similar to that obtained by Godbole et al.(1994), who found that the trypsin inhibitor in the pigeon pea was op-timally active between pH 7.0 and 10, with activity being lost at pH 3.The stability of AlTI13 in the alkaline region indicates that it may bepotentially used as a pesticide, because it is able towithstand the highlyalkaline conditions of the insect gut. AlTI13 was also optimally activeat temperatures ranging from 30 to 40 °C, with a decline in activity

above 70 °C (Fig. 7). Similar results were obtained by Kansal et al.(2008), who observed that TIs purified from the chickpea were stableuntil 80 °C, above which activity declined. This property of AlTIs is es-sential when raising transgenic plant. After transgenic expression, TIsshould be degraded by heating for use as human food. Therefore, wefound that AlTI13 might be inactivated by heating it above 70 °C, if itwere to be transgenically expressed in plants that host H. armigera.

It is important to study the in vitro and in vivo effects of AlTIsagainst particular insect pests to evaluate their bioinsecticidal activ-ity. Figs. 8 and 9 show how the AlTIs obtained from A. lebbeck influ-enceH. armigera larval weight at 3 different inhibitor concentrations(i.e., 0.1%, 0.5%, and 1.0% w/w) of ammonium sulfate when using theF3 fraction compared to a single concentration (5 μg/g diet) of AlTI13.Larvae that were fed artificial diet containing AlTIs and AlTI13 couldnot attain a comparable body weight to that of the control; instead, in-creased mortality rates were observed after 10 days of feeding (80%mortality when fed 1% AlTIs). This result might be due to the toxic ef-fects of the AlTIs. The larvae that were fed an artificial diet containingAlTI13 had a 10% mortality rate at day 4 and a 60% mortality rate atday 10 of the feeding assay. A similar study was undertaken by Gomeset al. (2005), who observed 45%mortality when 1.5% (w/w) of chickpeaTI was fed to Anthonomus grandis larvae. In addition, H. armigera larvaeexhibited about 33%mortality when fed a diet impregnated with mungbean TIs (Kansal et al., 2008).

An array of PIs is present in host plants; however, the protective roleof these PIs in natural plant defense mechanisms has yet to be deter-mined in many cases because of the induced complexity of insect gutproteinases and the weakened expression of PIs. Overexpressed heter-ologous serine PI genes are considered attractive tools for use in trans-genic crops, with plant engineering programs focusing on enhancingthe levels of insect resistant plants (Harsulkar et al., 1999; Telanget al., 2003). Therefore, the screening and characterization of such PIproteins from non-host plants are essential; for instance, A. lebbeckexhibits strong inhibitory activity against target insect pests.

In combination with this objective, our study indicates several newAlTIs. Electrophoretically purified AlTI13 strongly inhibits gut protein-ases and the growth of H. armigera. The potent inhibitory effects of theAlTI13 protein on the activity of proteinases and larval growth make ita promising candidate toward enhancing insect resistance in engineeredplants.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.aspen.2014.02.002.

Fig. 8. Effect of Albizia lebbeck trypsin inhibitor (AlTI)13 on the growth of Helicoverpa armigera larvae. H. armigera larvae were allowed feeding on an artificial diet containing AlTI13 andwithout AlTI13 (control). Larval weight was recorded every 24 h. A. Effect of dietary AlTI13 on the growth of H. armigera larvae. B. Effect of dietary AlTI13 on larva size and pupa formation.Each mean represents 3 replicates ± standard deviation (SD), and a difference of p ≤ 0.001 was considered significant.

Fig. 9. Effect of Albizia lebbeck trypsin inhibitors (AlTIs) and AlTI13 on the growth anddevelopment of Helicoverpa armigera. The weight is the mean weight of 20 larvae. Threereplicates were completed. The results were considered significant at p ≤ 0.001.

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Acknowledgments

We gratefully acknowledge Dr. A. N. Salve, Associate Professor,Department of Botany, Government Institute of Science, Aurangabad,India, for carefully reading the manuscript and providing assistancewith the language revision.We thank the University Grant Commission(UGC) and Department of Ministry of Minority Affairs, Government ofIndia, New Delhi for providing financial assistance in the form of theMaulana Azad National Fellowship (MANF) to Faiyaz K. Shaikh.

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