A stable serine protease, wrightin, from the latex of the plant Wrightia tinctoria (Roxb.) R. Br.: Purification and biochemical properties

Article abstract of DOI:10.1021/jf0726536

Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the

Full text of DOI:10.1021/jf0726536

J. Agric. Food Chem. 2008, 56, 1479–1487 1479
A Stable Serine Protease, Wrightin, from the Latex of
the Plant Wrightia tinctoria (Roxb.) R. Br.:
Purification and Biochemical Properties
RITU TOMAR, REETESH KUMAR, AND M. V. JAGANNADHAM*
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India
Today proteases have become an integral part of the food and feed industry, and plant latex could
be a potential source of novel proteases with unique substrate specificities and biochemical properties.
A new protease named “wrightin” is purified from the latex of the plant Wrightia tinctoria (Family
Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular
mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (ꢀ1%280) of
36.4. Optimum activity is achieved at a pH of 7.5–10 and a temperature of 70 °C. Wrightin hydrolyzes
denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity;
for example, the Km value is 50 µM for casein as substrate. Wrightin showed weak amidolytic activity
toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-R-benzoyl-DL-arginine-p-nitroanilide
(BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by
serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine
protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably
thermostable, retaining complete activity at 70 °C after 60 min of incubation and 74% of activity after
30 min of incubation at 80°. Besides, the enzyme is very stable over a broad range of pH from 5.0
to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents,
and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and
biotechnological industries, especially in operations requiring high temperatures.
KEYWORDS: Plant protease; serine protease; wrightin; glycosylated; Wrightia tinctoria
INTRODUCTION
In addition, conducting thermal processes at higher temperatures
minimizes microbial contamination (2).
Proteases are one of the industrially most important enzymes
and account for nearly 60% of total worldwide enzyme market.
Proteases are extensively employed in various industries such
as food, detergent, pharmaceutical, and leather. In the food
industry the hydrolytic property of proteases is exploited for
degradation of the turbidity complex resulting from protein in
fruit juices and alcoholic liquors, the improvement of quality
of protein-rich foods, soy protein hydrolysis, gelatin hydrolysis,
casein and whey protein hydrolysis, meat protein recovery, and
meat tenderization (1). There are many operations in the food
industry that require the performance of proteolytic enzymes
at high temperatures (65–85 °C) such as hydrolysis of proteins
at high temperature, enzymatic production of aspartame and
other peptides, baking, and brewing. Thermostability is desirable
because the efficiency of enzymes is markedly improved at
higher temperatures. Also, holding time for specific processes
can be shortened, thereby minimizing undesirable chemical
reactions. This is particularly valuable in food processing, where
nutrients may be lost during processing at high temperatures.
Proteolytic enzymes or proteases are a class of proteins
ubiquitously found in all organisms from microbes to higher
organisms; they act as catalysts and perform diverse vital
functions. In plants, proteases are involved in all aspects of the
life cycle ranging from the mobilization of storage proteins
during seed germination to the initiation of cell death and
senescence programs (3). Different parts of plants are valuable
reservoirs of biomolecules, and proteases from plant latex
represent an important segment of them. Members of several
plant families such as Euphorbiaceae, Apocynaceae, Moraceae,
and Caricaceae exude latex on wounding, which constitutes
organic and inorganic compounds, waxy materials, and enzymes
mainly proteases. These extracellular proteases play a defensive
role for plants against herbivores, insects, and pests (4, 5).
Besides, plant latex could be a potential source of proteases
due to easy purification methods, low levels of interfering
substances during purification, and good yield of proteases.
Plant-derived proteases find promising use in food and
biotechnological industries due to broad substrate specificity,
high stability in extreme conditions, good solubility, and activity
over a wide range of pH and temperature. Thiol proteases from
plant latex such as papain, bromelain, ficin, and calotropins are
* Corresponding author (e-mail jvm@bhu.ac.in or jaganmv@
satyam.net.in; telephone 91-542-2367936; fax 91-542-2367568).
10.1021/jf0726536 CCC: $40.75
2008 American Chemical Society
Published on Web 01/26/2008

1480 J. Agric. Food Chem., Vol. 56, No. 4, 2008
Tomar et al.
flow rate of 5 mL/min, and fractions of 4 mL were collected. The
absorbances at 280 nm as well as caseinolytic activity of the proteins
in all the fractions were determined. Bound proteins were eluted in a
single peak with caseinolytic activity through out the peak, and all of
the fractions were heterogeneous on SDS-PAGE. Therefore, all of the
fractions in the peak were pooled, and such a pool was used for further
purification. In subsequent purifications, crude latex was subjected to
a SP-Sepharose column and bound proteins were eluted directly with
0.8 M NaCl and pooled.
Step 3. Cation-Exchange Chromatography on CM-Sepharose. The
pool of fractions after SP-Sepharose were dialyzed extensively against
the 0.01 M acetate buffer, pH 4.9, and subjected to cation-exchange
chromatography on a CM-Sepharose fast flow column, which was pre-
equilibrated with 10 mM acetate buffer, pH 4.9. The column was
washed with the same buffer until no protein was detected in eluant,
and the bound proteins were eluted with a linear salt gradient of 0–0.5
M NaCl at a flow rate of 3 mL/min. Fractions of 3 mL were collected,
and each fraction was assayed for protein content as well as protease
activity.
utilized widely in several processes in the food and dairy
industries (6, 7). However, a limitation in the use of thiol
proteases is that their proteolytic activity is readily suppressed
by air oxidation and metal ions. Therefore, these proteases
require mild reducing and chelating agents for activation and
proper activity and, thus, are not so economical and handy. In
contrast, serine proteases from plant sources are both stable and
active under harsh conditions (at temperatures of 70–80 °C and
high pH), in the presence of surfactants or oxidizing agents;
thus, they are more useful and economical for industrial
applications. Therefore, the search for new potential plant serine
proteases continues.
Although there is high diversity of serine proteases in the
plant kingdom, as revealed by cDNA cloning experiments
performed on L. esculantum (tomato), L. longiflorum (lily), and
A. thaliana (8), to date most of the data relating to substrate
and cleavage specificities, kinetic parameters, and other enzy-
matic properties are limited to cucumisin (EC 3.4.21.25), the
first plant subtilase from the fruit of C. melo, and cucumisin-
like proteases isolated from fruits of other cucurbits (9–11).
Therefore, the present study aimed to purify a novel thermo-
stable serine protease from the latex of the plant Wrightia
tinctoria (Roxb.) R. Br. and investigate its physicochemical and
kinetic properties. W. tinctoria belongs to the Apocynaceae
family and is a small deciduous tree distributed in all parts of
India. W. tinctoria is a medicinally important plant, and its
extract shows antibacterial, antidandruff, and antipsoriasis
activities (12–14). It exudes copious amounts of latex upon
wounding of the leaf or stem throughout the year, and there is
no seasonal change in the protein levels or composition of the
latex. Thus, latex can be harvested at any time of the year for
protease purification. The purified enzyme was named wrightin
according to the nomenclature of proteases.
Protein Concentration. Protein concentration at different stages of
purification was determined by absorbance at 280 nm as well as
according to the method of Bradford (15) using BSA as a standard.
Protease Assay. The hydrolyzing activity of the protease was
determined using denatured natural substrates such as casein, hemo-
globin, and azoalbumin (chromogenic substrate) (16). Enzyme at a
concentration of 15 µg in total volume of 0.5 mL of 0.05 M Tris-HCl
buffer, pH 7.5, was incubated at 37 °C for 10 min. An equal volume
of 1% casein solution (w/v) prepared at the same pH was added to the
enzyme solution, making the final volume 1 mL, and the reaction
mixture was incubated further for 30 min at 37 °C. The reaction was
stopped by an addition of 0.5 mL of 10% TCA and allowed to stand
for 10 min at room temperature. Soluble peptides were separated by
centrifugation for 10 min using a tabletop centrifuge. The absorbance
of the TCA-soluble peptides in the supernatant was measured at 280
nm. In the case of azoalbumin (0.6%, w/v) as substrate, 0.5 mL of
supernatant after TCA precipitation was mixed with an equal volume
of 0.5 M NaOH and incubated for 15 min. The development of color
was measured spectrophotometrically by absorbance at 440 nm. With
hemoglobin (1% w/v) as a substrate, activity is taken in the same
manner as of casein. A control assay, without the enzyme, was done
and used as blank in all spectrophotometric measurements. One unit
of enzyme activity was defined as the amount of enzyme, under given
assay conditions, that gives rise to an increase of 0.01 unit of absorbance
at 280 at 440 nm per minute of digestion. The number of units of
activity per milligram of protein was taken as the specific activity of
the enzyme.
Assay for Amidolytic Activity toward Synthetic Substrates. The
enzymatic hydrolysis of different synthetic substrates peptidyl-pNA
(peptidyl p-nitroanilide) by the purified protease wrightin was studied
by a spectrophotometric method (16). Substrates used were N-R-
benzoyl-DL-arginine-p-nitroanilide (BAPNA), L-Ala-Ala-p-nitroanilide,
N-succinyl-Phe-p-nitroanilide, L-Glu-p-nitroanilide, L-Ala-p-nitroanilide,
and L-Leu-p-nitroanilde. In every case, a stock of 1–12 mM solution
of synthetic substrate was prepared by dissolving the required amount
of substrate in a minimum volume of DMSO and made up to the final
volume with 0.05 M Tris-HCl buffer, pH 7.5. The reaction mixture
contained approximately 15 µg of enzyme in 0.5 mL of Tris-HCl buffer,
pH 7.5, and 0.5 mL of peptidyl p-NA. After 60 min of incubation at
37 °C, the reaction was terminated by the addition of 0.2 mL of 30%
acetic acid and the liberated p-nitroanilide was determined by absor-
bance measurements at 410 nm using the extinction coefficient of 8800
M-1 cm-1 for p-nitroanilide as a measure of hydrolysis (20).
Electrophoresis and Gelatin Zymography. Homogeneity and
intactness of the enzyme during all stages of purification were assessed
by SDS-PAGE (17). For molecular mass determination, purified enzyme
is subjected to electrophoresis on 12.5% SDS-PAGE under both
reducing and nonreducing conditions. The protein bands on the gel
were visualized by staining with 0.2% Coomassie brilliant blue R-250.
Molecular mass markers used were phosphorylase b (97.40 kDa), BSA
(66.0 kDa), ovalbumin (43.0 kDa), carbonic anhydrase (29.0 kDa),
soybean trypsin inhibitor (20.1 kDa), and chicken egg white lysozyme
MATERIALS AND METHODS
Bovine serum albumin (BSA), hen egg white lysozyme, azoalbumin,
hemoglobin, DTNB (5,5′-dithiobis[2-nitrobenzoic acid]), DTT (dithio-
threitol), DFP (diisopropyl fluorophosphate), GuHCl (guanidine hy-
drochloride), urea, o-phenanthroline, EDTA (ethylenediaminetetraacetic
acid), EGTA (ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-
tetraacetic acid), SBTI (soyabean trypsin inhibitor), HgCl2 (mercuric
chloride), PCMB (p-chloromercuric benzoate), ꢀ-ME (ꢀ-mercaptoet-
hanol), PMSF (phenylmethanesulfonylfluoride), acrylamide, N,N-me-
thylene bisacrylamide, E-64 {L-trans-epoxysuccinylleucylamide(4-
guanidino)butane-N-[N-(L-3-transcarboxyirane-2-carbonyl)-L-
leucyl]agimatine}, Coomassie brilliant blue G-250, all synthetic
substrates, triethanolamine, agarose, o-phenylenediamine, and other
standard proteins were obtained from Sigma Chemical Co. (St. Louis,
MO). Coomassie brilliant blue G 250 was from Eastman Kodak.
Ampholine carrier ampholytes were from LKB. Acetonitrile, methanol,
ethanol, butanol and dioxan were of high-performance liquid chroma-
tography grade. All chemicals were of highest purity.
Purification of Protease Wrightin. All of the purification steps were
carried out at 4 °C unless stated otherwise.
Step 1. RemoVal of Gum. Latex was collected by making a superficial
incision on young stems of W. tinctoria into falcon tubes containing
chilled 0.01 M acetate buffer, pH 4.5, and subsequently stored at -20
°C for 24 h. Frozen latex was thawed to room temperature and
centrifuged at 17000g for 20 min to remove any insoluble debris. The
resulting clear supernatant called crude latex was used in the next step.
Step 2. Cation-Exchange Chromatography on SP-Sepharose. Chro-
matography was performed at room temperature. Supernatant obtained
in the above step was subjected to cation-exchange chromatography
on a SP-Sepharose fast flow column pre-equilibrated with 0.01 M
acetate buffer, pH 4.5. The column was washed with the same buffer
until no protein was detected in the eluant. The bound proteins were
eluted with a linear gradient of 0–0.8 M NaCl in the same buffer at a

Stable Serine Protease, Wrightin, from W. tinctoria
J. Agric. Food Chem., Vol. 56, No. 4, 2008 1481
(14.3 kDa). The molecular weight of the enzyme was extrapolated from
the plot of log molecular weight versus electrophoretic mobility of
molecular weight markers.
pH and Temperature Optima. The proteolytic activity of wrightin
was examined in the pH range of 1.0–11.5 to determine the optimum
pH. The buffers used were 0.05 M KCl-HCl (pH 1.0–1.5), 0.05 M
glycine-HCl (pH 2.0–3.5), 0.05 M sodium acetate (pH 4.0–5.5), 0.05
M sodium phosphate (pH 6.0–7.5), 0.05 M Tris-HCl (pH 8.0–10.0),
and 0.05 M sodium carbonate (pH 10.5–11.5). Because of the
insolubility of azoalbumin below pH 4.0, enzyme assays at lower pH
were carried out using hemoglobin as substrate. Substrate solution of
azoalbumin (0.6% w/v) or hemoglobin (1% w/v) was prepared in the
respective pH buffers, and activity was taken at the same pH as per
the method described earlier at 37 °C. For temperature optimum activity
of wrightin was assayed in the temperature range of 10–95 °C. Prior
to assay both protease and substrate (azoalbumin) solution were
incubated at the desired temperature for 15 min, and activity measure-
ments were done at the same temperature.
Stability. Stability of an enzyme dictates its utility and commercial
applicability. The stability of wrightin was assessed over a wide range
of pH and temperature and in the presence of chemical denaturants.
Wrightin was incubated for 24 h in given buffers in the pH range of
0.5–12.0 and assayed for residual activity. The different buffers used
were already mentioned in an earlier section. Similarly, enzyme was
incubated at various concentrations of the denaturants urea and GuHCl
for 24 h and assayed for residual activity, whereas enzyme was
incubated at different temperatures in the range of 10–95 °C for various
periods of time and an aliquot was utilized to determine the residual
activity. Although the enzyme was incubated over a range of temper-
atures, the activity measurements were carried out at pH 7.5 and 37
°C. In all of the above cases the residual activities of pretreated samples
were measured by the standard protease assay described earlier.
Proteases that degrade gelatin (denatured collagen) can be detected
directly in gels. Gelatin is incorporated prior to polymerization and
remains immobile during electrophoresis. Protease activity is indi-
cated by unstained (i.e., proteolytically degraded) regions against a
stained background, in contrast to conventional gels. To confirm the
proteolytic activity of purified protease in gel, gelatin zymography was
performed. Gelatin solution (1.5% w/v) was copolymerized with a
12.5% resolving gel. Protein sample was prepared in nonreducing
sample buffer and heated to 37 °C before loading on the gel. After
electrophoresis, the gel was washed for about 1 h with two changes of
2.5% Triton X-100 to remove SDS and placed in zymogram develop-
ment solution (50 mM Tris buffer, pH 8.0) for 15 h at 37 °C. Gel was
stained with 0.2% Coomassie brilliant blue R-250 and destained until
a
clear region against
a dark background appeared (negative
staining).
Mass Determination by Matrix-Assisted Laser Desorption/
Ionization Time-of-Flight (MALDI-MS). The purity and molecular
weight of the purified protein were also checked by mass spectrometry.
Enzyme at a concentration of 7 mM was mixed with saturated sinapinic
acid solution in 0.1% trifluoroacetic acid in water/acetonitrile (2:1)
mixture. One microliter was spotted onto the sample plate and allowed
to dry. Data were collected on a Shimadzu Axima CFR-plus MALDI-
TOF mass spectrometry in linear mode. MALDI-TOF standard BSA
was used for calibration of the instrument prior to data collection.
Isoelectric Focusing. The isoelectric point of the purified enzyme
was determined by isoelectric focusing in tube gels as described for
ervatamin B (18) with some modifications. Electrophoretic runs were
carried out with ampholine, in the pH range of 5.0–8.0. A 5%
polyacrylamide gel containing 2% desired ampholine was cast in tube
gels. Anodic and cathodic chamber buffers were 0.1 M phosphoric acid
and 0.1 M sodium hydroxide, respectively. The gels were subjected to
a prerun at a constant current of 1 mA per rod for 2 h to develop the
pH gradient. Protein sample (100 µg) containing 10% (v/v) ampholine
and 25% glycerol was loaded on each gel and electrophoresed at a
constant current of 2 mA per rod for 4 h. After the run, tube gels were
fixed in fixing solution (30 mL methanol, 70 mL of distilled water,
3.45 g of sulfosalicylic acid, and 11.5 g of trichloroacetic acid in 100
mL of distilled water) for 1 h. The gels were stained with 0.1% (w/v)
Coomassie brilliant blue R-250 dissolved in 100 mL of staining solution
(25% ethanol, 8% acetic acid in distilled water). Gels were destained
with repeated changes of destaining solution (25% ethanol, 8% acetic
acid in distilled water) until a clear background was obtained.
Carbohydrate Content. Carbohydrate moieties in glycoprotein have
been known to impart protein stabilization, protection from degradation,
control of protein solubility, and transport inside the cells. In view of
the occurrence of a few plant serine proteases as glycoproteins and
also the fact that wrightin is a secreted protein, determination of the
carbohydrate content of wrightin became essential. The carbohydrate
content, if any, of the protease was determined using the phenol-sulfuric
acid method (19). A solution of enzyme in water in the concentration
range of 1–10 µg per 10 µL was taken in a microtiter plate. To each
well 25 µL of 4% aqueous phenol was added. After 5 min 200 µL of
H2SO4 was added, and the increase in absorbance was measured at
492 nm. A standard plot was generated with known concentrations of
galactose (absorbance at 492 nm vs concentration), and the unknown
values of the sample were extrapolated.
The stability of enzyme activity was also analyzed in the presence
of organic solvents, SDS, oxidizing agent (H2O2), bleaching agent
(sodium perchlorate), and metal ions. In these studies, enzyme was
incubated in 0.5 mL of 0.05 M phosphate buffer, pH 7.5, containing
different concentrations of a given additive. Such a pretreated enzyme
sample is added to 0.5 mL of substrate (casein) solution prepared in
the same buffer, and the residual activity was determined. Enzyme
assays were done as described earlier. Effective concentration of the
organic solvent in the solvent composition did not alter the solubility
of casein, which is confirmed by light scattering measurements in
control experiments.
Effect of Substrate Concentration on Enzyme Kinetics. The effect
of increasing substrate concentration on the velocity of the enzyme-
catalyzed reaction was studied using casein as substrate at pH 7.5 at
37 °C. In each case, 15 µg of wrightin was used, and the concentration
of casein was in the range of 1–150 µM. A Lineweaver–Burk plot was
plotted, and the value of the Michaelis–Menten constant (Km) was
calculated. Proteolytic assays were performed as described
previously.
Autodigestion. Proteases, in general, are prone to autodigestion, and
the extent of autolysis depends on enzyme concentration, pH, time of
incubation, temperature, and type of activator, if any. In the present
study different concentrations of wrightin in the range of 0.05–0.45
mg/mL were incubated with 0.05 M Tris-HCl, pH 7.5, at room
temperature for 48 h. An aliquot of enzyme was used to determine the
protease activity with casein as substrate. The activity of the enzyme
after the first 10 min was taken as 100% for the calculation of the
residual activities.
Extinction Coefficient. The extinction coefficient of wrightin was
determined using dry weight as well as spectrophotometric methods
as described for procerain (21). The average value of the extinction
coefficients obtained from the two methods was used for all other
practical purposes.
Effect of Various Inhibitors on Activity of Wrightin. The effect
of increasing concentration of various protease inhibitors on the
proteolytic activity of the purified protease was examined to identify
the mechanistic class of enzyme. The inhibitors used in the present
study were specific to cysteine proteases (IAA, E-64, mercuric chloride
NEM, PCMB), serine proteases (PMSF, DIFP, SBTI), and metallo-
proteases (EDTA, EGTA and O-phenanthroline). A control assay of
the enzyme activity was done without inhibitors, and the resulting
activity was taken as 100%. The enzyme was incubated with an
inhibitor, at a given concentration, for 30 min at room temperature,
and an aliquot was used for the activity measurement. The activity
assay was done using azoalbumin as a substrate as described earlier.
Tyrosine and Tryptophan Contents. Total numbers of tryptophan
and tyrosine residues in purified protein were determined according to
the method of Goodwin and Morton (22), where absorbance spectra of
the purified enzyme in 0.1 M NaOH were recorded between 300 and
220 nm using a Beckman DU 640B spectrophotometer. Under similar
conditions, tyrosine and tryptophan contents of BSA and lysozyme were
also determined to validate the current measurements.
Free and Total Sulfhydryl Content. Estimation of free and total
cysteine residues of wrightin were carried out using Ellman’s method

1482 J. Agric. Food Chem., Vol. 56, No. 4, 2008
Tomar et al.
(23). For the free sulfhydryl group measurement, the purified enzyme
was activated with 0.01 M ꢀ-mercaptoethanol in 0.05 M Tris-HCl, pH
8.0, for 15 min and then dialyzed against 0.1 M acetic acid. Similarly,
for the measurement of total sulfhydryl content, the enzyme was reduced
in the presence of 6 M GuHCl (24). The free cysteine and total
sulfhydryl contents were measured by the DTNB reaction (25), where
the liberated TNB anion after reaction of sulfhydryl group with DTNB
was monitored spectrophotometrically at 412 nm. The molar extinction
coefficient of TNB anion at 412 nm is 14150 M-1 cm-1. Under similar
conditions, free and total sulfhydryl contents of BSA and lysozyme
were carried out to validate the current measurements.
Polyclonal Antibodies and Immunoasays. Antibodies to the
purified enzyme were raised in a male albino rabbit (weight about 1.0
kg). The enzyme (200 µg) was emulsified with Freund’s complete
adjuvant in 0.01 M sodium acetate buffer, pH 5.5, and the emulsion
was injected subcutaneously at multiple sites. After 1 week, a booster
dose of 350 µg of protein was given as emulsion with Freund’s
incomplete adjuvant. Two similar doses were administered at intervals
of 7 and 15 days as emulsions with Freund’s incomplete adjuvant. At
7 days after the last booster dose, the rabbit was bled through its
marginal ear vein. Blood was allowed to clot initially for 1 h at room
temperature and later for 12 h at 4 °C. Supernatant was collected as
antisera, by centrifuging at 5000 rpm, and stored at -20 °C until further
use. Preimmune serum was collected prior to the immunization of the
rabbit and used as a control for immunoassays. The presence of
antibodies was confirmed by Ouchterlony’s double-immunodiffusion
assay (26). One percent agarose in phosphate buffer saline containing
0.02% sodium azide was solidified in a Petri dish, and appropriate holes
were punched into it. Wrightin, trypsin, and protienase K (each 40 µg)
were loaded into the peripheral wells, and 100 µL of the antiserum
was added in the central well. The plate was left at room temperature
for 24 h in a moist environment. A control assay was carried out with
preimmune serum.
Figure 1. Elution of wrightin on cation-exchange chromatography on CM-
Sepharose. Protein pool from SP-Sepharose after dialysis was applied to
a CM-Sepharose column pre-equilibrated with 0.01 M acetate buffer, pH
4.9. The bound proteins were eluted with a linear gradient of 0–0.5 M
NaCl. The fractions of 3 mL volume were collected at a flow rate of 3
mL/min and assayed for protein content (b) and activity (O) using casein
as a substrate. The fractions from 147 to 168 were pooled and are
indicated by a horizontal line.
Table 1. Purification of Wrightin from Latex of W. tinctoria
total protein total activity specific activity
(units)a
step
(mg)
(units/mg)
% recovery
1. crude extract
2. SP-Sepharose
3. CM-Sepharose
341.5
276.4
24
6660
5640
753
19.5
20.4
31.3
100
84.6
11.3
a Definition of 1 unit: the amount of enzyme under the assay conditions described
giving rise to an increase of 0.01 unit absorbance at 280 nm per minute of digestion.
Casein was used as substrate.
RESULTS AND DISCUSSION
Purification of Wrightin. A novel serine protease from the
latex of W. tinctoria (family Apocynaceae) was purified to
homogeneity by a simple procedure involving cation-exchange
chromatography. Crude latex (devoid of gum and any insoluble
matter) was applied to a SP-Sepharose Fast Flow column. In
initial experiments the bound proteins were eluted with a linear
gradient of 0–0.8 M NaCl, which resulted as a single peak
having caseinolytic activity throughout the peak (data not
shown), but the fractions were heterogeneous when submitted
to SDS-PAGE; therefore, all of the active fractions were pooled
and dialyzed extensively with several changes of 0.01 M acetate
buffer, pH 4.9. In all subsequent preparations of the protein,
the SP-Sepharose column was eluted with 0.8 M NaCl directly.
The protein pool from SP-Sepharose was rechromatographed
on a CM-Sepharose Fast Flow column, and the bound proteins
were eluted using a linear gradient of 0–0.5 M NaCl. The
resulting elution profile showed two resolved peaks, A and B
(Figure 1). Most of the fractions of the two peaks exhibited
some caseinolytic activity, whereas most of the activity loaded
to the column appeared in peak B. Proteolytic activity observed
in the separated peaks suggests the existence of multiple
proteases in the latex of W. tinctoria. Such multiple proteases
in latex are not an uncommon feature as evidenced by the
presence of multiple proteases in the latex of Euphorbia tirucalli,
Euphorbia lacteal (27) EraVatamia caronaria (18), and others.
All of the fractions of the two peaks were assessed for
homogeneity by SDS-PAGE. Fractions 147-168 of the ascend-
ing limb of peak B are homogeneous with higher magnitude of
the activity (Figure 1). The active fractions from CM-Sepharose
other than the homogeneous ones (147–168) when subjected
to SDS-PAGE result in multiple bands, which are distinctly
different in molecular size from the fractions, which are
homogeneous between 147 and 168. Preliminary experiments
such as pH and temperature optima and kinetics showed some
differences between the pools, confirming that the crude latex
contains multiple proteases with distinct properties. Therefore,
only the pure fractions from CM-Sepharose were chosen for
further studies. The pool of fractions was concentrated by an
Amicon membrane concentrator using a membrane with a
molecular weight cutoff of 10 kDa followed by dialysis against
0.05 M phosphate buffer, pH 7.5, and stored at 4 °C for further
use. Typically, the yield of purified protein is 11 ( 1% and
specific activity is 31 ( 0.5 units/mg. The low yield of protease
activity might be due to the presence of multiple proteases in
the latex. The purification results of a typical batch are
summarized in Table 1. The purification has been carried out
many times. The purification protocol is highly reproducible,
and the yields as well as specific activity of the enzyme
preparation were consistent. It is worth mentioning that ap-
plication of crude latex after removal of gum (step 1), directly
to CM-Sepharose at pH 4.9 or 4.5 bypassing chromatography
on SP-Sepharose (step 2), resulted in a single peak, without
any resolution, where all of the fractions are active but
heterogeneous. However, no loss of activity relative to the total
activity loaded to the column is observed. Therefore, step 2 is
necessary. It appears that some unknown constituents present
in the latex either might have been removed in the unbound
while loading on SP-Sepharose, leaving only proteins bound to
the column, or remained tightly bound to the column even in
the presence of 0.8 M NaCl. To the best of our knowledge, this
is the first report on the identification and purification of a serine

Stable Serine Protease, Wrightin, from W. tinctoria
J. Agric. Food Chem., Vol. 56, No. 4, 2008 1483
Table 2. Specific Activity of Wrightin with Natural and Synthetic Substrates
substrate
specific activitya
casein
31.3 ( 0.5
20.8 ( 0.5
22.4 ( 0.5
2.1 ( 0.5
0.8 ( 0.5
no activity
no activity
no activity
azoalbumin
hemoglobin
L-Ala-p-nitroanilide
LAla-Ala-p-nitroanilide
LLeu-p-nitroanilide
N-R-benzoyl-DL-arginine-p-nitroanilide (BAPNA)
N-succinyl-Phe-p-nitroanilide, L-Glu-p-nitroanilide
a Specific activity is defined as the number of enzyme units per milligram of
protein with natural substrates. Specific activity is defined as the number of moles
of p-nitroaniline liberated per minute of synthetic substrate digestion per mole of
enzyme.
serine proteases. The proteolytic nature of wrightin was also
confirmed by gelatin zymography, where a digested gelatin
appeared as a white band corresponding to the position of the
protein in the gel (Figure 2). A comparison of physicochemical
properties of wrightin with other plant serine proteases is
summarized in Table 3.
Carbohydrate Content. Wrightin is a glycoprotein having
a detectable amount of carbohydrate in its molecular structure.
The amount of carbohydrate content was about 8% as estimated
by the phenol-sulfuric acid method. Furthermore, on staining
with Schiff’s reagent after SDS-PAGE, the wrightin band
appeared with a magenta color (data not shown), which confirms
the glycosylation of wrightin. Glycosylation to varying degrees
was seen in serine proteases purified from the Euphorbiaceae
family (27) as well as in cucumisin (32) and taraxalisin (33).
However, the biological role of such glycans has not been well
documented yet. In general, glycan chains played a role in
protecting the proteins from degradation and were as well
responsible for the thermal stability and solubility of the
glycoproteins. Glycans influence the conformation, stability, and
biological activity of the glycoprotein as well.
Figure 2. Electrophoretic analysis of purified protease: (A) SDS-PAGE
(lane 1, protein markers; lanes 2 and 3, wrightin under reduced and
nonreduced conditions, respectively; lane 4, crude latex after removal of
gum); (B) zymogram (in gel activity) of serine protease wrightin (clear
region shows the hydrolysis of gelatin by enzyme); (C) isoelectric focusing
(electrophoresis was performed using 5% polyacrylamide disk gels with
ampholine carrier ampholytes, pH 5–8; 100 µg of samples was loaded
and electrophoresed for 3 h at a constant voltage of 300 V).
Effect of Inhibitors on the Activity of Wrightin. Inhibition
of a protease by inhibitors specific for different class of proteases
is a useful tool to establish the mechanistic class of purified
unknown protease (28). The effect of various inhibitors on the
activity of wrightin is summarized in Table 4. The activity of
the enzyme was effectively blocked, by almost >93%, by serine
protease inhibitors such as DFP and PMSF. At the same time,
inhibitors of metalloprotease (EDTA and o-phenanthroline) and
cysteine protease (iodoacetic acid, E-64, PCMB) failed to inhibit
the proteolytic activity of wrightin. However, HgCl2 inhibited
the enzyme activity to some extent (about 20%). Such inhibition
was reported for cucumisin (32). The observed inhibition profile
places wrightin in the class of serine proteases. The absence of
inhibition by soybean trypsin inhibitor (SBTI), which is
abundantly present in many protein-rich foods, rules out the
possibility that the protease reported here was a trypsin-like
protease. Thus, wrightin can be a potential enzyme in the food
industry, where it may be effectively used in the processing of
protein-rich food.
pH and Temperature Optima. The hydrolyzing activity of
wrightin as a function of pH is shown in Figure 4A. Wrightin
exhibited full activity over a broad range of pH from neutral to
alkaline, pH 7.5-10.0, with a slow decrease afterward. The
protein retained 75% of the activity even at pH 11.5 and 73%
of activity at pH 5.0 as well. The protein was inactive below
pH 2.0, and such loss of activity of the protein at lower pH
could be due to incorrect protonation state of amino acid side
chain in the active site of enzyme or altering protonation state
of substrate, hence resulting in improper binding of the substrate
Figure 3. Mass spectrometry of wrightin. MALDI-TOF standard BSA was
used for calibration of the instrument, and data were collected in linear
mode.
protease from the genus Wrightin of the Apocynaceae family.
Hence, it is important to study the biochemical properties of
wrightin and compare them with those of other plant serine
proteases.
Homogeneity and Physical Properties of Purified Protease.
Wrightin appeared as a single band on SDS-PAGE under
reducing and nonreducing conditions with an estimated molec-
ular mass of 57 kDa (Figure 2). Mass spectrometric analysis
of the protein confirms the homogeneity as well as monomeric
nature of the enzyme with a single peak of molecular mass of
57.9 kDa (Figure 3). The average value of extinction coefficient
of the enzyme was 36.4 by dry weight and spectroscopic
methods. Similarly, purified protein appeared as a single band
on isoelectric focusing with an approximate isoelectric point
of pH 6.0 (Figure 2). Molecular masses of known plant serine
proteases are in the range of 19-110 kDa, the majority of
proteases falling in the range of 60-80 kDa (28) with some
exceptions such as MRP (proteinase I) and RSIP from Zea mays
L. (29, 30), which were reported to have molecular masses of
59 and 54 kDa, respectively. Most of the serine proteases derived
from plant sources have isoelectric points in the range of 4.0–7.0
and rarely have highly basic pI values, as reported for endopro-
tease from melon fruit (31). The molecular mass and isoelectric
point of wrightin were similar to those of other known plant

1484 J. Agric. Food Chem., Vol. 56, No. 4, 2008
Tomar et al.
Table 3. Comparison of Physicochemical Properties of Wrightin with Other Plant Serine Proteases
optima
stability
plant source
mol mass (kDa)
pH
temp (°C)
pH
temp (°C)
glycoprotein
pI
W. tinctoria (wrightin)
Z. maysa (RSIP)
T. kirrilowib
57.9
59
7.5–10
6.5–7.0
10.0
8.0
70
nd
70
40
70
58
65
60
60
30
5.5–11.5
5–9
75–80
nd
yes
nd
6.0
4.5
nd
4.5
nd
nd
nd
7.2
nd
nd
50
67
54
4–12.5
6–9
50
40
50
yes
yes
yes
yes
nd
yes
nd
nd
T. officinalec
C. melod (cucumisin)
7.1
4–11
7.0–9.0
4–10
5–12
7–11
nd
M. pomiferae
65
67
51.4
67
8.5
11
8
nd
65
65
C. trigonusf
E. millig (Milin)
B. hispidah
10.0
6.5–10.5
60
nd
P. hindsiii
82.0
i
a Reference 30. nd, not determined. b Reference 37. c Reference 33. d Reference 32. e Reference 38. f Reference 9. g Reference 39. h Reference 11. Reference 40.
Table 4. Effect of Inhibitors on the Activity of Wrightin
inhibitor type
inhibitor (I)
PMSF
DFP
[I]a
% residual activity
serine protease
100 µM
20 µM
1 mM
5.0
6.7
99
SBTI
cysteine protease
E-64
100 µM
100 µM
100 µM
100 µM
100 µM
96
98
80
100
100
iodoacetic acid
HgCl2
PCMB
NEM
metalloprotease
EDTA
o-phenanthroline
5 mM
5 mM
100
99
a Minimum amount of inhibitor required for maximum inhibition.
to enzyme. The wide range of optimum pH of wrightin is similar
to other serine proteases that have, in general, optimum pH
values of 7.0–11.0 with the exceptions of hordolisin and SEP-1
from barley (optimum pH of 6.0 and 6.5, respectively), as well
as Ara12 from A. thaliana and protease C1 from soybean (which
acts best in the pH range 3.5–6.5) (28). However, the pH
optimum of wrightin is different from that of proteases of the
Euphorbiaceae family, which is pH 6.0–8.0 (27).
The protease activity of wrightin was also monitored at
different temperatures in the range of 10–95 °C and is shown
in Figure 4B. The enzyme activity increased linearly from 20
to 70 °C with a rapid decrease of activity afterward and was
negligible by 90 °C. Therefore, the optimum temperature for
the activity of wrightin is around 70 °C. Such a high temperature
optimum is not common as most of the plant serine proteases
are reported to act best in the range of 30–60 °C (Table 3).
pH and Temperature Stability. The remarkable feature of
wrightin is its stability with respect to pH as well as high
temperature. Wrightin is stable with full activity in a broad range
of pH from 5.5 to 11.5 (Figure 4A) when exposed to a given
pH for 24 h. It also showed a considerable amount of activity
even at low pH of 3.0. The thermal stability of wrightin was
examined by measuring the residual activity after the enzyme
had been subjected to different temperatures for various times.
The protease exhibited residual activities of 95% and 74% at
80 °C when the time of exposure of the enzyme was 15 and 30
min, respectively (data not shown). The protease retained full
activity up to a temperature of 70 °C followed by a decrease of
activity to 50% at 80 °C and complete loss by 90 °C when the
time of exposure to a given temperature was increased to 60
min (Figure 4B). These results show that wrightin is a
thermostable protease, and its heat stability is higher than that
of other known plant serine proteases when compared as in
Figure 4. (A) Effects of pH on activity (O) and stability (b) of wrightin.
Fifteen micrograms of enzyme was used for all activity measurements.
For pH optima enzyme activity was determined at various pH values in
the range of 0.5–12.0. Enzyme was equilibrated at given pH, and activity
was taken at the same pH using substrate prepared in the corresponding
buffer. For pH stability enzyme was incubated for 24 h at various pH
values and activity was taken at pH 7.5 with azoalbumin as substrate. In
both cases the specific activity of 20.8 ( 0.5 units/mg determined at pH
7.5 and 37 °C is taken as 100%. (B) Effect of temperature on activity
(O) and stability (b) of wrightin. Temperature optimum was determined
by incubating the enzyme at different temperatures from 10 to 95 °C for
15 min and taking activity at the same temperature. The specific activity
of 68.33 ( 0.5 units/mg determined at pH 7.5 and 70 °C is taken as
100%. For temperature stability measurements, enzyme was incubated
for 60 min at different temperatures and activity was measured at 37 °C
at pH 7.5. The specific activity of 20.8 ( 0.5 units/mg determined at pH
7.5 and 37 °C is taken as 100%.

Stable Serine Protease, Wrightin, from W. tinctoria
Table 5. Stability of Wrightin under Various Conditions
J. Agric. Food Chem., Vol. 56, No. 4, 2008 1485
condition
stability
% residual activity
pH
temperature
urea
methanol
isopropanol
acetonotrile
dioxan
5.5–11.5
75–80 °C
8 M
50%
50%
100
100
100
100
86
50%
50%
64
57
ethanol
butanol
SDS
50%
50%
0.1%
5%
4%
10 mM
1.5
53
20
86.7
100
100
100
100
H2O2
sodium perchlorate
metal ions (Ca2+, Mg2+ and K+)
GuHCl
Table 3. Besides, wrightin was also stable with 100% activity
in 8 M urea and 1.5 M GuHCl when incubated for 24 h (Table
5). Thus, wrightin may be a potential enzyme in processes of
the food industry, which require elevated pH or tem-
peratures.
Effect of Organic Solvents, Metal Ions, SDS, and Oxidiz-
ing and Bleaching Agents. The stability of wrightin was
examined as a function of increasing percentage of different
organic solvents (Figure 5). At 50% concentration of organic
solvents wrightin retained full activity in methanol and isopro-
panol and half of the activity in ethanol, dioxan, and acetonitrile,
whereas the activity was reduced to one-third in butanol. Hence,
the protein is most stable in methanol and least in butanol.
Besides, the enzyme retained full activity in the presence of
0.1% SDS (detergent), 5% H2O2 (oxidizing agent), and 4%
sodium perchlorate (bleaching agent). Metal ions such as K+,
Ca2+, and Mg2+ did not affect the activity of wrightin up to 10
mM concentration. Thus, wrightin could be advantageous to
microbial proteases, which require metal ions, mainly Ca2+, as
activator for proteolytic activity (34, 35). The stability of
wrightin under different conditions is presented in Table 5. The
retention of considerable activity by wrightin under adverse
conditions as described above may be useful to explore the
possibilities of utilization in biotechnology, the food industry,
and others.
Figure 6. Effect of substrate concentration on reaction velocity of wrightin.
(A) Effect of substrate concentration on the activity of wrightin. At the
higher substrate concentrations the activity of wrightin attains saturation
in accordance with the Michaelis–Menten equation. Different concentrations
of substrate in the range from 0.0 to 50 µM and 15 µg of enzyme were
used in the measurements. Casein was used as a substrate. (B)
Lineweaver–Burk plot. Km was calculated according to the Michaelis–
Menten equation. The Km value of wrightin was found to be 50 ( 0.5
µM.
velocity of the enzyme obeyed the Michaelis–Menten kinetics
when denatured casein was used as substrate as shown Figure
6A. At higher concentration of the substrate, enzyme activity
was found to reach saturation, resulting in a typical hyperbolic
curve. The value of Km as estimated from the Lineweaver–Burk
plot (Figure 6B) is 50 ( 0.5 µM.
Effect of Substrate Concentration on the Reaction Veloc-
ity. The effect of increasing substrate concentration on reaction
Substrate Specificity. The activity of wrightin was examined
against denatured natural substrates as well as chromogenic
synthetic substrates. The specific activity of wrightin against natural
and synthetic substrates is summarized in Table 2. Wrightin
hydrolyzes denatured natural substrates such as casein, hemoglobin,
and azoalbumin with significant activity. Wrightin showed weak
amidolytic activity toward synthetic substrates such as L-Ala-p-
nitroanilide and L-Ala-Ala-p-nitroanilide. The chain length of the
substrate as well as amino acids at positions P1 and P2 may have
strong influence in the catalytic action of the enzyme. The lower
activity of wrightin toward L-Ala-Ala-pNA compared to L-Ala-
pNA might be due to a preference for alanine at the P1 position by
the enzyme, not at the P2 position. Wrightin failed to hydrolyze
other synthetic substrates such as L-Leu-p-nitroanilide, N-R-
benzoyl-DL-arginine-p-nitroanilide (BAPNA), N-succinyl-Phe-p-
nitroanilide, and L-Glu-p-nitroanilide used in this study. Besides,
lack of hydrolysis of BAPNA, a synthetic substrate for trypsin, by
wrightin makes it distinct from trypsin-like enzymes (36).
Figure 5. Effect of organic solvents on the activity of wrightin. The
enzyme was incubated at given percentage of each organic solvent
for 30 min at room temperature, and the residual activity was measured
with casein as substrate. The specific activity of 31.3 ( 0.5 units/mg
determined at pH 7.5 and 37 °C in the absence of organic solvent
was taken as 100%.

1486 J. Agric. Food Chem., Vol. 56, No. 4, 2008
Tomar et al.
Figure 7. Autodigestion of wrightin as a function of protein concentration.
Wrightin at different concentrations in the range of 0.05-0.5 mg/mL was
incubated at room temperature for 48 h. An aliquot was used to determine
residual activity (b). The activity of enzyme (O) after 10 min of incubation
of the protein at every concentration was taken as 100%.
Figure 8. Ouchterlony’s double immunodiffusion. Assay was carried out
in (1%) agarose in phosphate-buffered saline containing 0.02% sodium
azide. Anti-wrightin serum (100 µL) was added in the central well, and
40 µg of wrightin was added in the lower two peripheral wells. Forty
micrograms of proteinase K and 40 µg of trypsin were added in the
remaining upper wells. One merging precipitin band between antisera
and wrightin appeared in 24 h.
Autodigestion. Generally protease undergoes autolysis, which
is dependent on protein concentration, temperature, time, and
activators, if any. Therefore, it is important to check the
conditions for the storage of the enzyme without any loss of
proteolytic activity. Autocatalyis, if any, of wrightin was
monitored as a function of increasing protein concentration in
the range of 0.05–0.45 mg/mL at room temperature under neutral
conditions (Figure 7). The magnitude of loss of activity
decreases with increase in enzyme concentration from 0.05 to
0.45 mg/mL, and no loss of activity was seen at higher
concentrations. The enzyme retains >70% of activity even at a
very low protein concentration of 0.05 mg/mL. Most proteases
undergo autodigestion at low protein concentrations, for ex-
ample, cysteine proteases such as procerain (21). Wrightin is
less susceptible to autodigestion, which in turn indicates its high
stability. In our experience, the enzyme is stable for 6 months
at 4 °C without any loss in activity.
Specific Amino Acid Residues. The total sulfhydryl content
of the wrightin was found to be 9 (measured value ) 9.15 (
0.01) with one free (measured value ) 1.09 ( 0.01) cysteine
and the other eight forming four disulfide bonds. The tryptophan
and tyrosine contents of the protein were 20 (mesured value )
20.16 ( 0.1) and 75 (measured value ) 75.2 ( 0.5),
respectively. The total cysteine content of wrightin is comparable
to that of serine proteases such as Euphorbains y3 (total cysteine
) 10) and Euphorbains y1 (total cysteine ) 9) (27).
peratures in the food industry. Wrightin could also be success-
fully employed in protein-rich foods containing soybean trypsin
inhibitors. Besides, other properties of the enzyme such as its
low susceptibility to autodigestion and its ability to withstand
the harsh conditions of organic solvents and various chemical
denaturants may allow various other applications to be explored.
Moreover, wrightin can also be used as a model system to
understand thermostability or thermoactivity, which would be
useful for protein engineering.
ABBREVIATIONS USED
SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel
electrophoresis; TEMED, N,N,N,N-tetramethylethylenediamine;
UV, ultraviolet; HEPES, N-2-hydroxyethylpiperazine-N-2-
ethanesulfonic acid; PCMB, p-chloromercuribenzoate; NEM,
N-ethylmaleimide; GuHCl, guanidine hydrochloride; DTNB,
5,5µ-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; EDTA,
ethylenediaminetetraacetic acid; EGTA, ethylene glycol bis(R-
aminoethyl ether)tetraacetic acid; SBTI, soybean trypsin inhibi-
tor; PMSF, phenylmethanesulfonyl fluoride; TFA, trifluoroacetic
acid; TCA, trichloroacetic acid; BAPA, NR-benzoylarginine
p-nitroanilide.
Antigenic Properties. Polyclonal antibodies against wrightin
were raised in the male albino rabbit. The presence of antibodies
in the anti-rabbit serum was checked by Ouchterlony’s double-
immunodiffusion method. The precipitin line became distinctly
visible by approximately 24 h. One merging precipitin band
devoid of spur was observed (Figure 8) between antiwrightin
serum and wrightin. This band not only indicates the purity of
wrightin but also that the antibodies are specific to the enzyme.
At the same time other serine proteases such as trypsin and
proteinase K did not cross-react with antiwrightin serum,
revealing the antigenic distinction of wrightin from the latter
two serine proteases. Control experiments with preimmune
serum did not show any cross-reactivity (data not shown).
Easy availability of the latex and the simple purification
procedure reported in this paper make large-scale production
of wrightin possible and economical. The stability of the enzyme
at higher temperatures and over a broad range of pH also make
it a potential enzyme for industrial applications at high tem-
SAFETY
Acrylamide is a potent neurotoxin and carcinogen, and it was
handled with safety gloves. The handling of phenol and TCA
was done carefully because of their highly corrosive nature to
skin. All other experiments were carried out with the utmost
precaution and care.
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Received for review September 7, 2007. Revised manuscript received
November 26, 2007. Accepted December 4, 2007. Financial assistance
in the form of a research fellowship to R.T. from the Indian Council
of Medical Research, Government of India, is highly appreciated.
Infrastructure facilities are funded by the University Grants Commis-
sion and the Department of Biotechnology, Government of India.
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