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

Comparative Biochemistry and Physiology Part B 138 (2004) 81–89 

A novel 8.7 kDa protease inhibitor from chan seeds (Hyptis 

suaveolens L.) inhibits proteases from the larger grain borer 

Prostephanus truncatus (Coleoptera: Bostrichidae) 

a a b 

Cesar Aguirre , Silvia Valdes-Rodrıguez ´ ´ , Guillermo Mendoza-Hernandez ´ , 

c a, 

Arturo Rojo-Domınguez ´ , Alejandro Blanco-Labra * 

a 

Departamento de Biotecnologıa ´ y Bioquımica, ´ 

Centro de Investigacion ´ y de Estudios Avanzados (Cinvestav) Unidad Irapuato. Km 9.6 Libramiento Norte Carretera Irapuato-Leon, ´ 

C.P. 36500 Irapuato Gto, Mexico 

b 

Departamento de Bioquımica, ´ Facultad de Medicina, Universidad Nacional Autonoma ´ de Mexico, ´ Apartado Postal 70-159, 

Mexico D.F. 04510, Mexico 

c 

Departamento de Quımica, ´ Universidad Autonoma ´ Metropolitana-Iztapalapa, San Rafael Atlixco 186, Col. Vicentina, 

Iztapalapa 09340, Mexico 

Received 28 October 2003; received in revised form 12 February 2004; accepted 28 February 2004 

Abstract 

A novel trypsin inhibitor purified from chan seeds (Hyptis suaveolens, Lamiaceae) was purified and characterized. Its 

apparent molecular mass was 8700 Da with an isoelectric point of 3.4. Its N-terminal sequence showed a high content 

of acidic amino acids (seven out of 18 residues). Its inhibitory activity was potent toward all trypsin-like proteases 

extracted from the gut of the insect Prostephanus truncatus (Coleoptera: Bostrichidae), a very important pest of maize. 

This activity was highly specific, because among proteases from seven different insects, only those from P. truncatus 

and Manduca sexta (Lepidoptera: Sphingidae) were inhibited. This inhibitor has potential to enhance the defense 

mechanism of maize against the attack of P. truncatus. 

2004 Elsevier Inc. All rights reserved. 

Keywords: Chan; Hyptis suaveolens; Larger grain borer; Maize; Manduca sexta; Plant defense mechanisms; Prostephanus truncatus; 

Proteases; Protease inhibitor 

1. Introduction 

A wild plant known as Chan or Colima’s Chia 

(Hyptis suaveolens L.) has played an important 

role as a source of food and medicinal plant for 

the pre-Hispanic tribes that inhabited the tropical 

and subtropical regions of the Central-West part of 

Mexico (Martınez, ´ 1959; Vergara-Santana and 

*Corresponding author. Tel.: q52-462-6239600; fax: q52- 

462-6245996. 

E-mail address: ablanco@ira.cinvestav.mx 

(A. Blanco-Labra). 

Bravo-Magana, ˜ 1992). From a reference to Chan 

in the Mendocino codex (Kingsborough, 1964), it 

was established that these seeds were cultivated 

and highly appreciated since the pre-Hispanic cultures, 

most probably due to their high resistance 

to insect and fungal attack, as well as for their 

high nutritional and medicinal characteristics (Hernandez, 

´ 1959; Kingsborough, 1964; Weber et al., 

1991; Singh and Upadhyay, 1993; Fatope et al., 

1995). 

Protease inhibitors, broadly distributed in nature, 

are proteins that form very stable complexes with 

1096-4959/04/$ - see front matter 2004 Elsevier Inc. All rights reserved. 

doi:10.1016/j.cbpc.2004.02.011

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.


Fig. 1. Zymogram of the crude extract in native PAGE. A: 

Crude extract of chan (H. suaveolens) seeds, B: Amaranth 

trypsin inhibitor used as positive control. 

(1993) using casein as substrate. For protease 

activity on X-ray film, we used the method 

described by Machhandra and Manavendra (1994). 

2.3.2. Protease inhibitor activity determination 

Inhibitor activity against trypsin and trypsin-like 

enzymes of P. truncatus was assayed by monitoring 

the initial rate of hydrolysis of BAEE at 253 

nm in 0.15 M Tris–HCl, CaCl2 

0.05 M, pH 8.1 

after 3 min of incubation (Schwertz and Takenaka, 

1955). Inhibitor activity against proteases of T. 

castaneum, C. maculatus, A. obtectus and S. zeamais 

was assayed by monitoring the hydrolysis of 

casein at 280 nm in 0.04 M succinic acid, 0.06 M 

NaCl, pH 6.5 after 3 min of incubation (Kakade 

et al., 1969). At pH 2.5, it was assayed by 

monitoring the hydrolysis of hemoglobin at 280 

nm in 0.2 M citric acid, 0.1 M NaCl, after 3 min 

of incubation (Lenney, 1975). One unit of proteolytic 

activity was defined as an increase in 0.01 

absorbance units under the assay conditions 

described. Inhibitor activity was defined as the 

difference between the proteolytic activity measured 

in the absence and in the presence of the 

inhibitor. Inhibition Units (IU) were calculated as 

follows: 

IUymls 

Enzyme ODyŽ EnzymeqInhibitor. 

OD 

0.01=Sample 

Ž ml. 

Samples of the inhibitor obtained from the 

purification procedure were screened for inhibitory 

Fig. 2. Gel filtration chromatography after (NH ) SO precip- 

4 2 4 

itation. Fractions were monitored for absorbance at 220 nm 

(line) and trypsin inhibitor activity (diamonds). 

activity against trypsin, chymotrypsin, papain and 

elastase. 

3. Results 

3.1. Purification 

When subjected to electrophoresis and stained 

for inhibitory activity, crude extract of Chan flour 

contained a single protein inhibitor (Fig. 1). Proteins 

were precipitated and separated by gel filtration 

(Fig. 2). The peak containing the inhibitory 

activity was collected and resolved by ion 

Fig. 3. Ion exchange chromatography of the fraction obtained 

after gel filtration. Fractions were monitored for absorbance at 

220 nm (line), trypsin inhibitor activity (diamonds) and NaCl 

gradient (broken line).


85 

Table 1 

H. suaveolens trypsin inhibitor (HSTI) purification 

Procedure Specific Yield Purification 

activity (%) fold 

(IUymg 

protein) 

Crude extract 138 100 1.00 

(NH 4) 2SO4 156 84 1.14 

precipitated 

fraction 

G-75 Filtration 441 50 3.21 

fraction 

Ion-exchange 3800 61 27.80 

fraction 

RP-HPLC 7900 10 57.40 

fraction 

Fig. 4. RP-HPLC chromatography of the fraction containing 

the ion-exchange chromatography fraction containing inhibitor. 

The asterisk indicates the peak corresponding to inhibitor 

activity. 

Fig. 6. HSTI isoelectric point determination was 3.4. 

Fig. 5. Purification steps of H. suaveolens trypsin inhibitor 

(HSTI) isolated from chan (H. suaveolens) seeds; M: Molecular 

mass markers, A: Crude extract, B: Precipitated fraction, 

C: G-75 chromatography fraction, D: ion-exchange chromatography 

fraction, E: RP-HPLC. 

exchange chromatography (Fig. 3). The inhibitory 

activity eluted as a single peak. However, the 

fraction containing the inhibitory activity after ion 

exchange chromatography was heterogeneous, 

containing minor non-inhibitory proteins, which 

were removed by a final step of reverse phasehigh 

performance liquid chromatography (Fig. 4). 

From the two protein peaks obtained, the major 

peak contained the inhibitory activity, which was 

homogeneous by SDS-PAGE (Fig. 5). The recovery 

and relative fold purification at different stages 

of purification are given in Table 1.


Fig. 7. HSTI stability curve. Samples of inhibitor were treated 

for 60 min at different pH and temperature conditions. After 

treatment, samples were assayed for trypsin inhibitory activity 

at room temperature at pH 8.1. 

3.2. Characterization 

The HSTI approximate molecular mass was 

8700 Da by SDS-PAGE (Fig. 5). Fig. 6 shows an 

acidic isoelectric point of 3.4. 

The heat stability of HSTI was highly dependent 

on the pH. The inhibition actually was stable at 

low and neutral pH (3, 4 and 7) over the temperature 

range 4–95 8C; however, as the pH increased, 

a substantial loss of activity was observed, beginning 

at 85 8C at pH 9.2 and 60 8C at pH 10.7 

(Fig. 7). 

3.2.1. N-terminal sequence 

The N-terminal primary sequence was: 

RGWGSDEKKDREEEXEQQ. This part of the 

Fig. 9. Zymogram for trypsin-like proteases from the whole 

intestinal extract of (A) the larger grain borer (P. truncatus) 

and (B) same extract after 15 min incubation with H. suaveolens 

trypsin inhibitor (HSTI). 

protein sequence contained a remarkably high 

content of acidic residues (7 out of 18), which is 

in accordance with the low isoelectric point (3.4) 

determined. On analyzing this partial sequence in 

the SwissProt data bank, no significant homology 

was found with any other protein. 

Fig. 8. Comparison of protease inhibitors extracted from different 

sources and measured against trypsin and trypsin-like 

a 

activity of P. truncatus. ( Valdes-Rodrıguez ´ ´ 

b 

et al., 1993; Men- 

´ 

dez-Moran, ´ 

c 

d 

1999; Campos et al., 1997; Blanco-Labra et al., 

1995). 

3.2.2. Circular dichroism spectroscopy analysis 

In the CD HSTI spectrum, the negative signal 

at 210 nm with a less intense peak approximately 

220 nm strongly suggested that this protein belongs 

to the aqb structural class (separate a-helix- and 

b-sheet-rich regions) (Fig. 10, Table 3). Also, the 

spectrum shows some similarities to that of ubi-


87 

Table 2 

H. suaveolens trypsin inhibitor (HSTI) activity measured 

against proteases from different insects 

Fig. 10. Far-UV circular dichroism spectrum of HSTI (line) 

corrected for water baseline and normalized to mean residue 

ellipticity. Spectra reconstructed with different methods are also 

shown: k2d (triangles), Selcon (circles), Contin (diamonds) 

and CDSSTR (squares). 

quitin, a protein of similar molecular mass (8.6 

kDa, and 76 amino acid residues) and known 

structure (PDB file 1UBI). Deconvolution of this 

spectra using four different methods, yielded similar 

helical and strand contents, approximately 30% 

and 18%, respectively (Table 3). In all four cases, 

a good reconstruction of spectra is achieved, 

although better with Contin and CDSSTR methods 

as judged from Fig. 10 and from the RMS values 

in Table 3. However, caution should be taken with 

the numerical results since protein concentration 

was estimated by a colorimetric method, and the 

ellipticity value may change once the extinction 

coefficient of HSTI is determined. 

3.2.3. Specificity 

The HSTI activity against insect proteases, was 

highly specific for trypsin-like serine proteases, it 

was not able to recognize chymotrypsin, papain or 

elastase (data not shown). Only two out of seven 

proteolytic activities from insects were recognized 

Protease source 

pH 8.1 

P. truncatus 30900 

M. sexta 7600 

S. cerealella N.D. 

pH 6.5 

T. castaneum N.D. 

C. maculatus N.D. 

A. obtectus N.D. 

S. zeamais N.D. 

pH 2.5 

T. castaneum N.D. 

C. maculatus N.D. 

A. obtectus N.D. 

S. zeamais N.D. 

IUymg protein 

N.D.: Not Detected. HSTI concentration was 37.5 mgyml. 

by this inhibitor, those from P. truncatus and M. 

sexta (Table 2). As shown in Fig. 9, all the trypsinlike 

protease activities from P. truncatus were 

highly susceptible to this inhibitor. HSTI had the 

largest inhibitory activity so far reported of any 

proteinaceous protease inhibitor against the 

enzymes from this important maize consumer 

(Table 2, Fig. 8). HSTI was at least twice as 

potent as the one reported from quinoa, and is 

much higher than those of amaranth, tepary beans 

and maize. All assays were measured under the 

same conditions. 

4. Discussion 

In the constant search for new sources of proteins 

with potential activities in the defense mechanism 

of plants, it has been important to investigate 

different plants, including those that grow in 

restricted areas. In this way, we have been able to 

identify several interesting protease and amylase 

Table 3 

Estimated secondary structure content of H. suaveolens trypsin inhibitor (HSTI) from deconvolution of its circular dichroism spectrum 

Helix Strand Other Sum RMS 

K2d 0.260 0.160 0.580 ("0.080) 1.000 600 

Helix Strand Turn Irregular 

Selcon 0.300 ("0.005) 0.182 ("0.003) 0.219 ("0.004) 0.287 ("0.007) 0.988 1450 

Contin 0.294 ("0.004) 0.193 ("0.021) 0.186 ("0.025) 0.331 ("0.045) 1.004 360 

CDSSTR 0.310 ("0.010) 0.175 ("0.015) 0.195 ("0.015) 0.315 ("0.025) 0.995 250 

RMS is a measure of the difference between experimental and reconstructed spectra. Uncertainity in k2d method is the sum of 

maximum expected errors in the estimated contents. Uncertainities in the other methods correspond to the intervals containing results 

from the four different reference sets of spectra used in the analysis.


inhibitors, such as, those present in tepary beans 

(Phaseolus acutifolius; Campos et al., 1997), amaranth 

(Amaranthus hypochondriacus; Valdes- ´ 

Rodrıguez ´ et al., 1993) and in quinoa (Chenopodium 

quinoa; Mendez-Moran, ´ ´ 1999). In the 

present report, a novel protease inhibitor was 

purified and characterized. The protein in this 

study was isolated from the seeds of chan, an 

ancient, not very widely used plant (Martınez, ´ 

1959) known to be highly resistant to insect and 

fungal attack (Singh and Upadhyay, 1993; Fatope 

et al., 1995). 

HSTI shows some similarities with the Bowman–Birk 

family of inhibitors, such as its apparent 

molecular mass of 8700 Da, and an isoelectric 

point of 3.4. The inhibitor also had a high thermal 

stability, particularly at acidic pH, which decreased 

at higher pH values. However, its N-terminal 

sequence (18 amino acids) was not similar to other 

protease inhibitor sequences previously reported. 

Similarities were found in the circular dichroism 

spectrum with that of ubiquitin, whose molecular 

weight is also very similar. However, there is not 

enough information on the primary sequence of 

HSTI to establish structural similarities. 

The main characteristic of this new inhibitor is 

its specific activity against particular trypsin-like 

enzymes from insects. It was shown to recognize 

only two out of the seven tested enzymatic extracts 

from insects. One of them was the trypsin-like 

protease activity obtained from the insect P. truncatus, 

a very important pest of maize in Latin- 

America. In the last 10 years it has been the main 

constraint for maize production in central Africa. 

The other insect proteases inhibited by HSTI, were 

those from M. sexta. 

Acknowledgments 

We would like to express our gratitude to 

Elizabeth Mendiola-Olaya and Yolanda Rodrıguez ´ 

for their technical assistance. We also thank CON- 

ACYT for a Ph.D. fellowship to C.A., and for 

grant 41348-F; SIGHO project N8 19990201013 

and Javier Luevano-Borroel ´ 

for his assistance in 

the insectary. 

References 

Alfonso-Rubi, J., Ortego, F., Castanera, ˜ P., Carbonero, P., Diaz, 

I., 2003. Transgenic expression of trypsin inhibitor CMe 

from barley in indica and japonica rice, confers resistance 

to the rice weevil Sitophilus oryzae. Transgenic Res. 12, 

23–31. 

Altpeter, F., Diaz, I., McAuslane, H., Gaddour, K., Carbonero, 

P., Vasil, I.K., 1999. Increased insect resistance in transgenic 

wheat stably expressing trypsin inhibitor CMe. Mol. Breeding 

5, 53–63. 

Andrade, M.A., Chacon, ´ P., Mertelo, J.J., Moran, ´ F., 1993. 

Evaluation of secondary structure of proteins from UV 

circular dichroism spectra using an unsupervised learning 

neural network. Protein Eng. 6, 383–390. 

Boxall, R.A., 2002. Damage and loss caused by the larger 

grain borer Prostephanus truncatus. Integ. Pest Manage. 

Rev. 7, 105–121. 

Bell, A., Muck, ¨ O., Mutlu, P., Schneider, H. 1999. Integrated 

post-harvest protection is worth its money. German Agency 

for Technical Co-operation. wDeutsche Gesellschaft fur ¨ 

Technische Zusammenarbeitx (GTZ), Eschborn, Germany 

(http:yywww.gtz.de). 

Blanco-Labra, A., Chagolla-Lopez, ´ A., Martınez-Gallardo, ´ 

N., 

Valdes-Rodrıguez, ´ ´ S., 1995. Further characterization of the 

12 kDa proteaseyalpha amylase inhibitor present in maize 

seeds. J. Food Biochem. 19, 27–41. 

Bradford, M.M., 1976. A rapid and sensitive method for the 

quantification of microgram quantities of protein utilizing 

the principle of protein-dye-binding. Anal. Biochem. 72, 

248–252. 

Campos, J.E., Martınez-Gallardo, ´ 

N., Mendiola-Olaya, E., 

Blanco-Labra, A., 1997. Purification and partial characterization 

of a proteinase inhibitor from tepary beans (Phaseolus 

acutifolius) seeds. J. Food Biochem. 21, 203–218. 

De Leo, F., Gallerani, R., 2002. The mustard trypsin inhibitor 

2 affects the fertility of Spodoptera littoralis larvae fed on 

transgenic plants. Insect Biochem. Molec. 32, 489–496. 

Dunstan, W.R., Magazini, I.A., 1981. Outbreaks and new 

records in Tanzania. The larger grain borer on stored 

products. FAO Plant Prot. Bull. 29, 80–81. 

Estruch, J.J., Carozzi, N.B., Desai, N., Duck, N.B., Warren, 

G.W., Koziel, M.G., 1997. Transgenic plants: an emerging 

approach to pest control. Nat. Biotechnol. 15, 137–141. 

Fabrick, J., Behnke, C., Czapla, T., Bala, K., Rao, A.G., 

Kramer, K.J., et al., 2002. Effects of a potato cysteine 

proteinase inhibitor on midgut proteolytic enzyme activity 

and growth of the southern corn rootworm, Diabrotica 

undecimpuctata howardi (Coleoptera: Chrysomelidae). 

Insect Biochem. Molec. 32, 405–415. 

Farrell, G., 2000. Dispersal, phenology and predicted abundance 

of the larger grain borer in different environments. 

African Crop Sci. J. 8, 337–343. 

Fatope, M.O., Nuhu, A.M., Mann, A., Takeda, Y., 1995. 

Cowpea weevil bioassay: a simple prescreen for plants with 

grain protectant effects. Int. J. Pest Manage. 41, 84–86. 

Garcıa-Carreno, ´ ˜ F.L., Dimes, L.E., Haard Norman, F., 1993. 

Substrate-gel electrophoresis for composition and molecular 

weight of proteinases or proteinaceous proteinase inhibitors. 

Anal. Biochem. 214, 65–69. 

Gatehouse, A.M.R., Gatehouse, J.A., 1998. Identifying proteins 

with insecticidal activity: use of encoding genes to produce 

insect-resistant transgenic crops. Pestic. Sci. 52, 165–175.


89 

Hennessey, J.P., Johnson Jr, W.C., 1982. Experimental errors 

and their effect on analyzing circular dichroism spectra of 

proteins. Anal. Biochem. 125, 177–188. 

Hernandez, ´ F., Historia natural de la Nueva Espana, ˜ 1959. II, 

III,, Universidad Nacional Autonoma ´ de Mexico, ´ Mexico. ´ 

Kakade, M.L., Simons, N., Liener, I.E., 1969. An evaluation 

of natural vs. synthetic substrates for measuring the antitryptic 

activity of soybean samples. Cereal Chem. 46, 

518–521. 

Kingsborough, E.K., 1964. Antiguedades ¨ de Mexico. ´ In: 

Corona Nunez, ˜ J. (Ed.), Codice ´ Mendocino, Vol. 1. Secre- 

taria de Hacienda y Credito ´ Publico, ´ Mexico, ´ pp. 1–49. 

Krall, S., 1984. A new threat to farm-level maize storage in 

West Africa: Prostephanus truncatus (Horn) (Coleoptera: 

Bostrichidae). Trop. Stored Prod. Inf. 50, 26–31. 

Lenney, J.F., 1975. Three yeast proteins that specifically inhibit 

yeast proteases A, B and C. J. Bacteriol. 112, 1265–1274. 

Lobley, A., Whitmore, L., Wallace, B.A., 2002. DICHROWEB: 

an interactive website for the analysis of protein secondary 

structure from circular dichroism spectra. Bioinformatics 18, 

211–212. 

Machhandra, M.P., Manavendra, S.K., 1994. Detection of 

electrophoretically separated protease inhibitors using X-ray 

film. J. Biochem. Bioph. Meth. 28, 215–224. 

Martınez, ´ M., 1959. Plantas utiles ´ de la flora mexicana. 

Edciones Botas, Mexico. ´ 

Mendez-Moran, ´ ´ L. 1999. Estudio de una proteına ´ purificada a 

partir de semillas de Chenopodium quinoa Willd. con 

actividad inhibitoria de proteasas tipo tripsina. M.Sc. Thesis. 

CINVESTAV-IPN Unidad de Biotecnologıa ´ e Ingenierıa ´ 

Genetica ´ de Plantas. Mexico. ´ 

Schagger, ¨ H., von Jagow, G., 1987. Tricine-sodium dodecyl 

sulfate-polyacrylamide gel electrophoresis for the separation 

of protein in the range from 1 to 100 kDa. Anal. Biochem. 

166, 368–379. 

Schwertz, G.W., Takenaka, Y., 1955. A spectrophotometric 

determination of trypsin and chymotrypsin activity. Biochim. 

Biophys. Acta 16, 571–575. 

Singh, G., Upadhyay, R.K., 1993. Essential oils: a potent 

source of natural pesticides. J. Sci. Ind. Res. India 52, 

676–683. 

Singh, R.R., Rao, A.G., 2002. Reductive unfolding and oxidative 

refolding of a Bowman-Birk inhibitor from horsegram 

seeds (Dolichos biflorus): evidence for ‘hyperreactive’ 

disulfide bonds and rate-limiting nature of disulfide isomerization 

in folding. Biochim. Biophys. Acta 1597, 280–291. 

Sreerama, N., Woody, R.W., 2000. Estimation of protein 

secondary structure from CD spectra: comparison of CON- 

TIN, SELCON and CDSSTR methods with an expanded 

reference set. Anal. Biochem. 287, 252–260. 

Valdes-Rodrıguez, ´ ´ S., Segura-Nieto, M., Chagolla-Lopez, ´ A., 

Vargas-Cortina, A., Martınez-Gallardo, ´ 

N., Blanco-Labra, 

A., 1993. Purification, characterization, and complete amino 

acid sequence of a trypsin inhibitor from amaranth (Amaranthus 

hypochondriacus) seeds. Plant Physiol. 103, 

1407–1412. 

Vergara-Santana, M.I., Bravo-Magana, ˜ F., 1992. Memorias. III 

Reunion ´ Nacional Selva baja Caducifolia. Universidad de 

Colima, Mexico. ´ 

Weber, C.W., Gentry, H.S., Kohlhepp, E.A., McCrohan, P.R., 

1991. The nutritional and chemical evaluation of chia seeds. 

Ecol. Food Nutr. 26, 119–125.

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

Create successful ePaper yourself

Delete template?

Save as template?