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108

Protein & Peptide Letters, 2014, 21, 108-114

Purification and Physicochemical Characterization of a Trypsin Inhibitor

from Cassia absus Linn
Girijesh K. Patel1, Amit K. Gupta2, Akshita Guptaa, Manisha Mishra3, Pradhyumna K. Singh3,
Anil K. Saxena2*, Ashwani K. Sharma1,*
1

Department of Biotechnology, Indian Institute of Technology Roorkee-247667, India; 2Medicinal and Process Chemistry Division. CSIR-Central Drug Research Institute; Lucknow-226001, India; 3Plant Molecular Biology Division, CSIRNational Botanical Research Institute, Lucknow-226001, India; Present Address: Department of Biotechnology,
Graphic Era University Dehradun-248002, India
Abstract: A thermotolerant protein with trypsin inhibitory activity designated as CaTI was purified to homogeneity from
seeds of Cassia absus. Gel filtration chromatography and SDS-PAGE analysis showed the apparent molecular mass of
~20 kDa. Partial internal sequences indicate that CaTI belongs to Kunitz-inhibitor family. CaTI inhibits the bovine trypsin
in 1:1 molar ratio and exhibited a competitive-type inhibitory activity with Ki = 5.6  10-9 M. The inhibitory activity was
retained over a broad pH range (2-12). Thermal stability study showed that it is stable up to 80 C and inhibition activity
reduced at and above 90 C which might be due to the presence of predominantly -sheets revealed by the CD study. The
proteolysis studies of CaTI exhibited strong resistance to proteolysis by different proteases tested. The studies show that
CaTI can be used as potential candidates for the development of the transgenic plant against the microbes and insect pests.

Keywords: Cassia absus, Caesalpiniaceae, Kinetic study, Stability studies, Trypsin inhibitor.
INTRODUCTION
Plant Kunitz protease inhibitors (PIs) are widely distributed in plant and are involved in controlling endogenous
proteolysis. These PIs are small proteins and act as potent
natural defence agent against pathogens and the predators [1,
2]. Generally, they are present at higher concentration in the
storage tissues, but also induced in other plant tissues by
biotic stress like herbivory or wounding [3]. The inhibition
activity of the PIs is due to their capacity to form stable
complex with the target protease by blocking, altering or
preventing to access the enzyme active sites leading to unavailability of the amino acid necessary for the growth and
development of the insects and microbes [4,5]. The plant PIs
could also play important endogenous roles to regulate the
uncontrolled proteolysis during seed dormancy, reserve protein mobilization, and protection from foreign proteolytic
enzymes of pests and pathogens. Moreover, PIs may also act
as storage or reserve proteins [6]. Besides blocking the protease action, few PIs play a critical role in the modulation of
the programme cell death in soybean [7], growth factor activities, receptor clearance signalling [8]. It is reported that
consumption of proteins isolated from the plant source constituted with PI, reduces the incidence of breast, colon, prostate, oral and pharyngeal cancers [9].
*Address correspondence to these authors at the Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247 667, India; Tel:
+91-1332-285657; Fax: +91-1332-273560; E-mail: aksbsfbs@yahoo.co.in,
aksbsfbs@iitr.ernet.in
Medicinal and Process Chemistry Division, Central Drug Research Institute,
CSIR, Lucknow, 226001, India; Tel: +9152226124112/ex 4386; Fax:
+9152226123405; E-mail: anilsak@gmail.com

The authors have contributed equally.

-5/14 $58.00+.00

Based on amino acid sequence homology, binding sites

and inhibitory mechanisms, PIs are grouped into Kunitztrypsin inhibitors (TIs), Bowman-Birk TIs, potato I and II,
squash, barley, ragi 1 and 2, rapeseed trypsin inhibitor (RTI)/
mustard trypsin inhibitor-2, thaumatin families and others
depending on their molecular mass and cysteine content [1012]. The plant Kunitz-inhibitors possesses one or two polypeptide chain of approximately 20 kDa having single reactive site. Kunitz-type trypsin inhibitor reported from the subfamilies Caesalpinoideae and Papilionoideae, are monomeric in nature [13]. Most of the Kunitz-inhibitors have four
conserved cysteine residues resulting two disulphide bonds
in a single or double chain polypeptide but the literature survey reveals that plant Kunitz-inhibitors may not have any
cysteine residue or may have one, two and more than two.
This indicates the evolutionary diversion from Kunitz-type
ancestral protein precursor [14]. Due to the involvement of
proteases in several pathological events, characterizing the
structure and function of new protease inhibitors may discover useful information in knowing disease mechanism and
help in drug designing [15,16].
Cassia absus is a small, flowering, glandular viscid plant
belonging to the family Caesalpiniaceae and are used in the
verity of traditional medicines. Locally, it is known as Lal
Chakwar and Chaksu in traditional medicine. Its stem exhibits the anti-bacterial, anti-fungal, as well as anti-tumor activities. The seeds are used in the treatment of ophthalmia and
skin infections. Seed extract possesses attenuant, astringent,
hypertensive properties [17,18]. To date, no protein has been
reported from this plant and here, we for the first time describe the isolation and physicochemical characterization of

2014 Bentham Science Publishers

Purification and Physicochemical Characterization of a Trypsin

the Cassia absus trypsin inhibitor (CaTI) from the mature

seeds.
MATERIALS AND METHODS
Materials
Seeds of C. absus were collected from the Botanical garden, DDU Gorakhpur University, India. DEAE-sepharose,
trypsin, N-benzoyl-L-arginine ethyl ester (BAEE), Nbenzoyl-L-arginine p-nitroanilide (BAPNA), chymotrypsin,
N-benzoyl-L-tyrosine ethyl ester (BTEE), acrylamide, bisacrylamide, -mercaptoethanol, SDS, protein molecular
weight standards and BSA were purchased from SigmaAldrich (USA), Amicon Ultra from Millipore (Bedford, MA,
USA), dialysis membrane-70 (HiMedia, Pvt. Ltd, India). All
other chemicals were purchased from Merck and HiMedia
with analytical grade.
Extraction and Purification of CaTI
The mature seeds (10g) were crushed and soaked overnight at 4C in 50 mM potassium phosphate buffer pH 6.0
and filtered through muslin cloth. The filtrate was cleared by
centrifugation at 20,000  g for 30 min and supernatant was
loaded on DEAE column (1.5  20 cm Econo-column, BioRad) pre-equilibrated with 50 mM potassium phosphate
buffer, pH 6.0. The unbound proteins were washed and
bound proteins were eluted with step gradient of NaCl (0 to
0.5 M). Fractions having Trypsin inhibitory activity were
pooled, dialysed and concentrated on Amicon Ultra Cut-off
device (10,000 MWCO, Millipore) by spinning at 4500  g.
The concentrated protein was subjected to gel filtration on a
HiLoad 16/60 Superdex-75pg column (120 mL, GE
Healthcare) equilibrated with 50 mM potassium phosphate
buffer, pH 6.0. The protein was eluted with the same buffer
at ow rate of 0.5 mL/min and the fractions having inhibitory activity were concentrated and stored for further analysis.
Determination of Protein Concentration
The protein concentration in crude extract and purified
samples were estimated by dye binding method [19]. Bovine
serum albumin was used as the standard protein. The presence of protein during the different purification steps was
also monitored by measuring absorbance at 280nm.
SDS-PAGE Analysis and Molecular Mass Determination
Purified protein was analysed under reducing conditions
on a 12% SDS-PAGE gradient gel according to the method
of Laemmli [20]. The relative molecular mass of CaTI was
determined by performing SDS-PAGE with molecular
weight standards. The molecular weight standards used were
97.4 kDa, phosphorylase B; 66 kDa, bovine serum albumin;
43 kDa, ovalbumin; 29 kDa, carbonic anhydrase; 20.1 kDa,
soybean trypsin inhibitor and 14.3 kDa, lysozyme. The Protein bands were visualized by staining the gel with 0.15%
Coomassie brilliant blue R-250.

Protein & Peptide Letters, 2014, Vol. 21, No. 2

109

Identification of CATI by Mass Spectroscopy

The purified CaTI was resolved on 12% reducing SDSPAGE and stained with 0.15% CBB R-250. Gel piece containing CaTI band was excised, washed thoroughly with the
sterile water. Gel piece was destained with 45% methanol
containing 10% acetic acid. After destaining, gel piece was
dehydrated with acetonotrile (ACN) and 50 mM ammonium
bicarbonate (ABC) in ratio 2:1 with alternate washing with
25 mM ABC. The gel piece was reduced with 10 mM DTT
at 56C for 1 h. It was again dehydrated and rehydrated
again as described earlier and alkylated with the 50 mM iodoacetamide solution in dark at room temperature for 30
min. The protein was digested with trypsin (sequencing
grade, Porcine, Promega, USA) in 50 mM ammonium bicarbonate buffer pH 8.0 at 37C for 16 h in 1:20 enzyme, substrate ratio(w:w). The digested peptides were extracted by
sonication in extraction solution (60% ACN containing 1%
trifluoroacetic acid; TFA) for several times. The extracted
peptides were dried by centrifugal evaporation and suspended in 5
l 50% ACN containing 0.1% TFA). 0.5
l suspended peptides were spotted on MALDI plate followed by
0.5
l matrix (-cyano-4-hydroxycinnamic acid, CHCA),
concentration 10 mg/mL (prepared in 50% ACN containing
0.1% TFA). Spots were dried completely and subjected to
peptide mass fingerprinting (PMF) on MALDI-TOF-TOF
platform (model 4800, ABsciex, USA). The PMF data were
analyzed on the MASCOT search using MSDB, Swiss-Prot
and NCBI database with the following parameters: xed
precursor ion mass tolerance of 20 ppm, fragment ion mass
tolerance of 0.05 Da, calibration error of 0.005 Da, one
missed cleavage, carbamido methylation of cysteines and
possible oxidation of methionine. The spectra of common
contaminants were removed by searching against contaminant database and the remaining data were used for the identification of proteins.
Trypsin Inhibition Assay
The bovine pancreatic trypsin inhibition activity assay
was performed according to Shibata et al. [21] with slight
modification. The purified CaTI (10
g) was incubated for
10 min at 30C with 10
g trypsin solution in 50 mM TrsiHCl buffer, pH 8.0 containing 10 mM CaCl2. After 10 min
incubation 0.05 mL of 1 mM BAPNA was added and further
incubated at 37C for 10 min. The reaction was stopped by
adding 0.5 mL of 10% acetic acid and the residual trypsin
activity was measured by recording the absorbance at 410
nm against the reference solution containing same components except denatured enzyme using UV-Vis spectrophotometer (DU730, Backman).
To determine the molar inhibition ratio, different concentration of inhibitor was incubated with a fix concentration of
trypsin in the molar ratio of 0.129, 0.259, 0.388, 0.518, 648,
0.769, 0.972, 1.166, 1.31, 1.62 and 1.81 at 30 C for 15 min
and residual activity of trypsin was measured in 1 mL assay
volume in 50 mM Tris-HCl buffer, pH 8.0 containing 10
mM CaCl2. The amount of substrate (BAPNA) hydrolysed
by the enzyme was calculated using the molar extinction
coefficient of 8800 M-1 cm-1 at 410 nm. The trypsin inhibitory activity was also determined at different pH values (2.012.0).

110 Protein & Peptide Letters, 2014, Vol. 21, No. 2

Chymotrypsin Inhibition Assay

The chymotrypsin inhibitory assay was done according to
the Shee and Sharma [22]. Different concentrations of CaTI
was incubated with 10
g of chymotrypsin dissolved in 1.0
mM HCl containing 10 mM CaCl2 (nal concentration was
4.0  10-9 M in 1.0 mL assay volume) at 30 C for 15 min.
Enzymeprotein mixtures (100
l) were added to 900
l
buffered BTEE solution (1 mM BTEE in 50 mM Tris-HCl
buffer, pH 8.0 containing 10% methanol). Hydrolysis of the
substrate was monitored by measuring the absorbance at 256
nm for 10 min with spectrophotometer using molar extinction coefcient of 964 M-1cm-1.
pH and Thermal Stability Of CaTI
For the pH optima and stability studies, inhibitor was
assayed at pH range 2-12 with unit pH interval. CaTI was
incubated at 30 C for 30 min with different buffers glycineHCl (pH 2-3), sodium acetate (pH 4-5), potassium phosphate
(pH 6-7), Tris-HCl (pH 8-9) and Glycine-NaOH (pH 10, 11
& 12). After incubation, content was centrifuged and the pH
of the supernatant was brought to 8.0 and residual inhibition
activity was measured against the trypsin as described above.
At optimum pH, CaTI was incubated for 1, 2, 5, 10 and 20 h
and relative inhibition activity was determined.
To study the thermal stability, inhibitor solution was incubated at temperatures ranging from 20 to 100 C for 20
min. After incubation samples were kept on ice for 5 min
and centrifuged. The supernatant was used for residual inhibition activity measurement against trypsin using BAPNA.

Patel et al.

0.2 mg/mL purified protein in 20 mM potassium phosphate

buffer, pH 7.0 at 20 C. Far-UV CD spectra (180-260) were
recorded in a 1 mm quartz cell at bandwidth 1 nm and time
per point 0.5 second. Three consecutive scans were accumulated and average data was deduced. The average base line
spectrum of reagent blank was subtracted from the average
protein sample spectrum and resulted data was analysed
(190-240 nm) by online DichroWeb programme using
CDSSTR, K2D and CONTIN method with reference point 4
[24]. The results of the CD measurements were expressed as
mean residue ellipticity (MRE) in deg cm2 dmol-1.
RESULTS
Purification of CaTI
The protein with trypsin inhibition activity was purified
by the combination of anion exchanger DEAE-Sepharose
and Sephadex-75 gel filtration chromatography. The major
fraction of CaTI was eluted from DEAE column at 100 mM
NaCl with slight impurity and was further purified on gel
filtration column with the elution volume 68 mL which
showed a single peak indicating its purity. The molecular
weight of the protein was found to be ~21 kDa with the gel
filtration standards and 20 kDa with molecular weight standard on 12% reducing SDS-PAGE. The treatment of CaTI
with -mercaptoethanol did not alter the migration on SDSPAGE which revealed that CaTI is a monomeric protein with
apparent molecular mass 20 kDa (Fig. 1).
Identification of CaTI by Mass Spectroscopy

The inhibition equilibrium constant (Ki) of the inhibitor

was determined as described by Dixon [23]. The increasing
concentration of CaTI (12.5, 25, 37.5, 50, 75 and 100 nM)
was incubated with the fix concentration of the trypsin (final
assay concentration 1.07 nM) for 15 min at 37 C and the
residual inhibitory activity was monitored by using two different concentrations of BAPNA as a substrate (0.5 mM and
2 mM). After incubation at 37C for an additional 10 min the
reaction was stopped by addition of 200
l of 10% acetic
acid. The amount of product formed was calculated using a
molar extinction coefficient of 8800M-1 cm-1 at 410 nm.

The purified CaTI was subjected to the mass spectroscopic analysis in tandem mass spectrometer. The partial
internal sequencing revealed six different peptides with significant score. Out of these six, first two peptides were found
similar to the sequence present in other known Kunitz trypsin
inhibitors like Acacia confusa trypsin inhibitor [25] and Kunitz-like trypsin inhibitor-like 2 protein from Medicago truncatula (XP_003619553.1) shown in Figure 2. The other four
peptides (FSSXSR, TVVQPVI/LGDR, I/LI/LFI/LHETFR
and I/LVEEGEGTGFVGPFR) did not show sequence similarity to any known trypsin inhibitor. This indicated that the
protease inhibitor discovered in present study is a new entry
in Kunitz family.

Stability against Different Proteases

Inhibition Properties

The purified CaTI was used for proteolytic stability studies against different proteases. CaTI was incubated with different protease such as proteinase K, bovine trypsin and
chymotrypsin in the molar ratio 20:1(inhibitor: enzyme) in
50 mM Tris-HCl buffer, pH 8.0 while papain in 50 potassium phosphate buffer, pH 7.0 for different time interval (30,
60 and 90 min). After incubation, reducing sample buffer
was added and boiled for 5 min. The stability of CATI
against different protease was analyzed on 15 % reducing
SDS-PAGE gel using BSA as a control.

The CaTI possesses inhibition activity at wide pH range

(2-12). The maximum residual inhibition activity was found
at pH range (4-10). Thermal stability study showed that it is
stable up to 80 C and inhibition activity reduced at and
above 90 C when incubated for 20 min. The thermal stability of CaTI might be due to the presence of predominantly sheets revealed by the CD study. The purified protein inhibited trypsin completely at the molar ratio of 1:1 (Fig. 3).

Inhibition Kinetics of CaTI

Circular Dichroism (CD) Study of CaTI

CD study was performed on Chirascan CD spectrometer
(Applied Photophysics, UK). The CD study was done using

The Ki value of the CaTI was determined to be 5.6 10-9

for the BAPNA (Fig. 4). CaTI also inhibited the chymotrypsin activity slightly. A 20 kDa, glycosylated Bauhinia rufa
protease inhibitor from subfamily Caesalpiniaceae posses
highly specific elastase inhibition activity but did not possess
trypsin as well as chymotrypsin inhibition activity [26] indi-

Purification and Physicochemical Characterization of a Trypsin

Protein & Peptide Letters, 2014, Vol. 21, No. 2

111

a
b
Figure 1. a) Purity and molecular mass determination of CaTI protein under reducing condition on 12% SDS-PAGE gel. L1, molecular mass
standards; L2, crude extract; L3, 50 mM NaCl eluted fraction; L4, 100 mM NaCl fraction from DEAE; L6, 200 mM NaCl fraction; L5
Eluted fraction from HiLoad 16/60 Superdex-75 column (GE Healthcare).
b) Elution profile of CaTI protein from HiLoad 16/60 Superdex-75pg (120mL) column.
(a) Peptide 1

a
(b) Peptide 2

b
Figure 2. Amino acid sequence similarity of CaTI with other trypsin inhibitors.

cating CaTI exhibits different inhibitor specificity with trypsin and chymotrypsin inhibition activities. The stability study
of CaTI against the protease like trypsin, chymotrypsin and
proteinase K was also determined at different time (30, 60
and 90 min) with BSA as a control protein. The CaTI was
found completely stable against the trypsin and fairly stable
against chymotrypsin but sensitive against the proteinase K
and papain (Fig. 5). The resulted peptide after digestion with
proteinase K and papain show darker region in the gel which
are the less than 14 kDa. Such peptide bands are absent in
case of trypsin indicating stability of CaTI against trypsin
(Fig. 5). The stability of CaTI at pH 10.0 was determined
from 15 min to 20 h and was found stable until 20 h with 5%
loss in inhibition activity.

CD Study of CaTI
The CD spectrum of Far-UV region of a protein is the
most sensitive physical technique to determine and analyze
the secondary structure content of a protein. The result represents the negative maximum at 202 nm in CD spectrum of
CaTI, which is unusual for the most of the proteins but hallmarks for the Kunitz proteins (Fig. 6). The CD data recorded
at pH 7.0 in 20 mM potassium phosphate buffer was analysed by three different methods namely CONTIN, K2D and
CDSSTR. The absence of negative peak at 208 and 222 nm
indicate the lack of helical structure. CD spectrum analysis
of CaTI by CONTIN, K2D and CDSSTR methods showed
that CaTI contains 3.7%, 4% and 10% of
-helix respectively while -sheets were found to be 43% , 48% and 32%
by above methods respectively.

Residual inhibitory activity (% of Control)

112 Protein & Peptide Letters, 2014, Vol. 21, No. 2

Patel et al.

100

80

60

10

12

pH

a
100

Figure 5. Stability of CATI against the different protease at different time interval. L1, molecular mass standards; L2, BSA only; L3,
Incubation of BSA with Proteinase K for 90 min as control, L4-L6,
Incubation of CaTI with Proteinase K for 30, 60 and 90 min respectively; L7, Incubation of BSA with trypsin for 90 min as control;
L8-L10, Incubation of CaTI with Proteinase K for 30, 60 and 90
min respectively. L11, Incubation of BSA with chymotrypsin for 90
min as control; L12-L14, Incubation of CaTI with chymotrypsin for
30, 90 and 60 min respectively; L15, Incubation of BSA with papain for 90 min as control; L16-L19, Incubation of CaTI with papain for 30, 90 and 60 min respectively.

5000

60

MRE (deg cm2 dmol-1)

Residual trypsin activity (%)

80

40

20

-5000

-10000

-15000

0
0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Molar Ratio (Inhibitor : Enzyme)

-20000
190

b
Figure 3. a) Inhibition activity at different pH (Incubated at different pH for 30 min).
b) Molar Inhibition ratio of CaTI with trypsin.

200

210

220

230

240

Wavelength (nm)

Figure 6. Far-UV Circular dichroism study of CATI on Chirascan

CD spectrometer (Applied Photophysics, UK) in 20 mM potassium
phosphate buffer pH 7.0 at 20 C. The results of the CD measurements were expressed as mean residue ellipticity (MRE) in deg cm2
dmol-1.

DISCUSSION

Figure 4. Kinetics study of CaTI. Determination of the dissociation

constant (Ki) by Dixon method using BAPNA as a substrate at two
different concentrations, 0.5 mM (
) and 2 mM (). Velocity was
represented in OD/min.

Cassia absus is a well known plant and all parts possesses different medicinal properties [17]. The purification
employed simple two steps of ion exchange and gel filtration
chromatography. In the present study we have demonstrated
that CaTI is a single chain Kunitz inhibitor isolated from the
seed of Cassia absus. Reports revealed that single chain Kunitz-type inhibitors are characteristic feature of subfamily
Caesalpiniaceae [13, 27]. CaTI constitutes about 15-18% of
total mature seed proteins indicating to be a storage protein.
This is the main protein of the seed therefore; it may play a
pivotal role in germination, growth, development and defence. A numbers of the trypsin inhibitors have been characterised and possess storage as well as plant defence properties [27-29]. The apparent molecular weight of CaTI was
found to be 20 kDa by both, gel filtration chromatography
and SDS-PAGE analysis under reducing condition. These

Purification and Physicochemical Characterization of a Trypsin

results indicate the presence of the single polypeptide chain

like other trypsin inhibitor isolated from soybean [10], Inga
laurina [30], Pithecellobium dumosum seeds [31]. However,
some Kunitz inhibitors have two polypeptide chain linked by
disulfide bonds reported from seeds of Enterolobium contortisiliquum [32], Acacia confusa [25]. Kunitz-inhibitors may
not have any cysteine residue or may have one, two and
more than two which indicates the heterogeneous nature of
the subclass of Kunitz-type trypsin inhibitor [14]. The activities of the Kunitz inhibitors are highly variable some are the
highly potent inhibitor for trypsin and do not affect to chymotrypsin activity but some were found to be effective at
some level against the chymotrypsin [33]. CaTI completely
inhibited the bovine trypsin and moderately to chymotrypsin
but has very little inhibitory effect on proteinase K and papain. Some of the structural features are common in the Kunitz inhibitors like molecular mass, single binding site and
inhibition ratio. The molar inhibition ratio of CaTI (1:1) indicates its single active domain for the binding and inhibition
which is also supported by different reports [22,30].
MALDI/TOF/TOF study resulted six peptides sequence
among them only two have shown sequence similarity with
known plant inhibitors while other four did not show any
similarity with any known inhibitor which indicated that the
protease inhibitor discovered in present study is a new entry
in Kunitz family.
The inhibition activity of CaTI retains over broad pH
range (2-12) and up to 80C without any loss when incubated for 20 min. To the best of our knowledge Ki (5.6  10-9
M) value of CaTI towards the bovine trypsin is the smallest
among all known inhibitors from subfamily Caesalpiniaceae. The higher stability of CaTI in different physicochemical conditions and the lower Ki value can be utilize for
the development of the insect registrant transgenic plants
[34-36].
The CD spectrum of Far-UV region of a protein is the
most sensitive physical technique to determine and analyze
the secondary structure content of a protein. The shoulder in
215-225 nm regions of CaTI is a characteristic feature of the
Kunitz proteins which is due to the presence of the proline
residue towards the N-terminal regions [37,38]. The strong
positive spectrum at 195 nm is a feature of strong secondary
and tertiary structures. It is generally due to the presence of
the rigid conformation caused by benzene ring containing
amino acids in the hydrophobic core region of the Kunitz
domain [38]. CD spectra analysis revealed that CaTI is predominantly -sheets protein. Reports suggest that Kunitz
inhibitors possess predominantly twelve anti-parallel sheets and devoid of -helix (14, 37). Enterolobium contortisiliquum trypsin inhibitor (EcTI) possesses relatively high
-sheet content (59%) and 41% irregular structure [32].
CONCLUSION
The present communication describes the purification of
a new 20 kDa Kunitz inhibitor, from the seeds of Cassia
absus belonging to subfamily Caesalpiniaceae and its characterization. CaTI exhibits remarkable inhibition activity in a
broad pH and temperature range (2-12 and 0-80C, respectively). CaTI inhibits trypsin in a 1:1 molar ratio. CaTI is
found stable in different proteases like chymotrypsin and

Protein & Peptide Letters, 2014, Vol. 21, No. 2

113

bovine trypsin and less stable against proteinase K, and papain. CD analysis showed that CaTI primarily consists of sheets. To understand the high stability of CaTI, 3D structure
could be determined by crystallization. Plant Kunitz inhibitors are thought to be the important defence protein particularly against the insect pests as they inhibit the activity of the
digestive gut proteases leading to poor digestion of dietary
proteins and unavailability of important amino acids which
ultimately causes death. The protease inhibitors studied in
present study may be useful in the development of resistant
transgenic plants like the insect resistant cauliflower using
the trypsin inhibitor gene from the sweet potato.
ABBREVIATIONS
PIs

Protease inhibitors

CaTI

Cassia absus trypsin inhibitor

BTEE

N-benzoyl-L-tyrosine ethyl ester

BAPNA

N-benzoyl-L-arginine-p-nitroanilide

BSA

Bovine serum albumin

CONFLICT OF INTEREST
There are none.
ACKNOWLEDGEMENTS
CD studies were performed in NMR facility at Institute
Instrumentation Centre (IIC), IIT Roorkee and Mass Spec
analysis in Proteomics Facility at CSIR-National Botanical
Research Institute, Lucknow. We gratefully acknowledge Dr.
D.C. Saini, Scientist E, Birbal Sahni Institute of Palaeobotany, Lucknow, India for the identification of the
plant. G.K. Patel acknowledges MHRD, A.K. Gupta to
ICMR and M. Mishra to CSIR, Government of India for fellowships. The CDRI communication number allotted to this
manuscript is 8472.
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Received: April 26, 2013

Patel et al.

Revised: May 28, 2013

Accepted: May 31, 2013

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