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

C. Aguirre et al. / Comparative Biochemistry and Physiology Part B 138 (2004) 81–89
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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.