A novel 8.7 kDa protease inhibitor from chan seeds (Hyptis ... - UAM

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82 C. Aguirre et al. / Comparative Biochemistry and Physiology Part B 138 (2004) 81–89 proteolytic enzymes. Most of these inhibitors are small molecules with relative molecular masses ranging from 5 to 25 kDa, with compact structures and in many cases with a high content of disulfide bridges, characteristics that might contribute to their high thermal stability (Singh and Rao, 2002). Protease inhibitors participate in the defense against the attack of insects, documented in part by in vitro assays indicating that these proteins are responsible for the inhibition of a protease activity in the insect gut. Bioassays using purified proteins have also shown detrimental effects on insect growth and some of them also have insecticidal activities (Gatehouse and Gatehouse, 1998; Fabrick et al., 2002; Alfonso-Rubi et al., 2003). Different genes coding for protease inhibitors have been expressed in different plants, producing an increase in their resistance to the attack of some specific insects (Estruch et al., 1997; Altpeter et al., 1999; Fabrick et al., 2002). Mustard trypsin inhibitor 2 (MTI-2) expressed in transgenic tobacco did not affect the development of Spodoptera littoralis larvae but had a significant negative effect on their fertility (De Leo and Gallerani, 2002). The larger grain borer (Prostephanus truncatus) was accidentally introduced into Africa in the late 1970s from its area of origin in Central America, where it had long been recognized as an important pest of stored maize. The beetle appeared first in East and then West Africa (Dunstan and Magazini, 1981; Krall, 1984) and later spread to at least 15 countries (Bell et al., 1999; Farrell, 2000). It has recently been recognized as the most destructive pest of farm-stored maize and dry cassava in Africa (Boxall, 2002). Losses in maize due to P. truncatus were consistently higher than those produced by indigenous pest species, such as Sitophilus spp (5–10%, compared to 15–45% for P. truncatus) in Ghana. According to Boxall (2002), this situation threatens not only this important cash crop but also the food security of vulnerable rural products. The goal of the present work was to purify and characterize a novel protease inhibitor from chan seeds, with potential for the control of the insect P. truncatus. 2. Materials and methods Chan (H. suaveolens) seeds were provided by Dr Martha Vergara-Santana (Herbarium, University of Colima, Mexico). Insects (P. truncatus) were provided by the insectary at Cinvestav-Irapuato, Mexico. Bovine pancreas trypsin (type I; EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), papain (EC 3.4.22.2), elastase (EC 3.4.21.36)and N a- benzoyl-L-arginine ethyl ester (BAEE), were from Sigma-Aldrich (St. Louis, MO, USA). Sephadex G-75 and molecular mass markers were from Pharmacia (Uppsala, Sweden). Econo-Pac High Q cartridge was from Bio-Rad (Hercules, CA, USA). All chemicals were analytical grade. Deionized water was used throughout. 2.1. Inhibitor purification 2.1.1. Extraction Chan flour was defatted with a mixture of chloroform:methanol (2:1 vyv) in a 4:1 wyv ratio. Extraction of the inhibitor was carried out by stirring a suspension of Chan flour in water (1:5 wyv) for4hat48C. Insoluble materials were removed by centrifugation at 10 000=g for 60 min. The crude extract was precipitated by adding ammonium sulfate from 30 to 70% saturation. The precipitate was separated and dialyzed against water. The dialyzed solution was used for further purification. 2.1.2. Gel filtration A concentrated solution of H. suaveolens trypsin inhibitor (HSTI) after dialysis, was passed through a Sephadex G-75 gel filtration column (2.25=167 cm), equilibrated with 0.02 M ammonium bicarbonate, pH 7.8. It was eluted using a flow rate of 0.3 mlymin, collecting 4 ml fractions. Fractions with inhibitory activities (42–52, Fig. 2) were pooled and concentrated by lyophilization. 2.1.3. Ion-exchange chromatography Ion-exchange chromatography was carried out at RT (approx. 25 8C). A concentrated inhibitor sample from the pooled gel filtration samples (2 ml) was loaded into an Econo-Pac high Q cartridge column (1=5 cm) equilibrated with 0.01 M Tris–HCl pH 8.0. After washing the column with 30 ml of buffer solution, elution was carried out with a linear gradient of 0.0–0.4 M NaCl in 0.01 M Tris–HCl buffer (pH 8.0), with a flow rate of 1 mlymin, collecting 2 ml fractions. Fractions with trypsin inhibitory activities (67–81, Fig. 3) were pooled, dialyzed against deionized water and lyophilized.
C. Aguirre et al. / Comparative Biochemistry and Physiology Part B 138 (2004) 81–89 83 2.1.4. Reverse-phase HPLC A concentrated inhibitor sample from the pooled ion-exchange chromatography (100 ml) after dialysis, was separated by a reverse-phase Vydac C 18 HPLC column (22.5 mm ID=250 mm length and 10 mm particle size), with a gradient of 0–80% (by vol.) acetonitrile in water (Fig. 4). Peak fractions were frozen and lyophilized to remove the acetonitrile. Fractions were evaluated by electrophoresis (Fig. 5), and those containing a single band were used for protein characterization. 2.1.5. Protein determination Protein concentration was determined by the Bradford protein assay (Bradford, 1976). Bovine serum albumin (BSA) was used as standard. 2.2. Characterization 2.2.1. Molecular mass determination Molecular mass was determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) using a 13% separating gel, following procedure Schagger ¨ and von Jagow (1987). Globin (16 949 Da), Globin IqII (14 404 Da), Globin IqIII (10 700 Da), Globin I (8159 Da), Globin II (6214 Da) and Globin III (2512 Da), were used as molecular mass markers. 2.2.2. Isoelectric point Isoelectric focusing (IEF) was performed at RT in a Pharmacia LKB electrophoresis PhastSystem using a Phastgel 3-9. 2.2.3. Stability Stability of the inhibitor was evaluated by exposing the sample for 60 min at temperatures ranging from 4 to 94 8C, at five different pH values, from pH 3 to 10.7. The inhibitor (0.02 mg) was dissolved in 1 ml of the appropriate buffer (0.025 M) solution. Buffers included citrate solution pH 3 and pH 5, phosphate solution pH 7 and carbonate solutions pH 9.2 and pH 10.7. For residual trypsin inhibitory activity at the end of the thermal treatment, the solution was cooled in an ice bath and the activity determined at RT in 0.15 M Tris–HCl, CaCl 2, 0.05 M, pH 8.1 buffer solution, using bovine trypsin and BAEE as substrate to monitor the activity (Schwertz and Takenaka, 1955). 2.2.4. N-terminal sequence determination The N-terminal sequence of the purified protein was determined using repeated cycles of Edman degradation. Analysis was performed in an automatic sequencer (Beckman–Porton Protein sequencer, model LF 3000). 2.2.5. Circular dichroism spectroscopy analysis This analysis was performed using a JASCO J- 715 spectropolarimeter calibrated with (q)10- camphorsulfonic acid (Hennessey and Johnson, 1982). Samples were dialyzed overnight against water, and spectra were obtained in a quartz cell of 1 mm pathlength at a protein concentration of 0.1 mgyml. Circular dichroism spectra were deconvoluted in the DichroWeb server (Lobley et al., 2002) with four different methods: k2d, Selcon, Contin and CDSSTR (Andrade et al., 1993; Sreerama and Woody, 2000). The former is a neural network algorithm that interprets the region from 200 to 240 nm of CD spectra in terms of three different secondary structure contributors: a-helix, b-strand and others. The latter methods use a broader region of the spectra (185–240 nm) and estimates the a-helix, b-strand, turn and irregular fractions of secondary structure; also, they can use different sets of CD spectra as reference. In this work we used sets 3, 4, 6 and 7 from the DichroWeb server and results were averaged and are presented in Table 3. 2.3. Extraction of larval enzymes Proteolytic enzymes were extracted from third instar larvae for the insects: P. truncatus (Horn) (Coleoptera: Bostrichidae), Sitotroga cerealella (Olivier) (Lepidoptera: Gelechiidae), Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae), Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae) and Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae). For Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae), fifth instar larvae were used. The extraction was done according to the method described by Valdes-Rodrıguez ´ ´ et al. (1993), using crude extracts. 2.3.1. Zymograms For the assay of protease inhibitor activity in polyacrylamide gel electrophoresis, we followed the method described by Garcıa-Carreno ´ ˜ et al.
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A novel 8.7 kDa protease inhibitor from chan seeds (Hyptis ... - UAM

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