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Research article

Purication and physicochemical characterization of a serine protease with

brinolytic activity from latex of a medicinal herb Euphorbia hirta

Girijesh Kumar Patel, Ashish Ashok Kawale, Ashwani Kumar Sharma*

Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667, India

a r t i c l e i n f o

Article history:

Received 9 November 2011

Accepted 7 December 2011

Available online 13 December 2011

Keywords:

CD studies

Euphorbia hirta

Euphorbiaceae

Fibrinogenolytic activity

Fibrinolytic activity

Kinetic studies

a b s t r a c t

A 34 kDa serine protease, designated as hirtin, with brinolytic activity was puried to homogeneity

from the latex of Euphorbia hirta by the combination of ion exchange and gel ltration chromatography.

The N-terminal sequence of hirtin was found to be YAVYIGLILETAA/NNE. Hirtin exhibited esterase and

amidase activities along with azocaseinolytic, gelatinolytic, brinogenolytic and brinolytic activities. It

preferentially hydrolyzed Aa and a-chains, followed by Bb and b, and g and geg chains of brinogen and

brin clot respectively. The optimum pH and temperature for enzyme activity was found to be pH 7.2 and

50 C respectively. Enzymatic activity of hirtin was signicantly inhibited by PMSF and AEBSF. It showed

higher specicity for synthetic substrate p-tos-GPRNA for thrombin. The CD spectra of hirtin showed

a high content of b-sheets as compared to a-helix. The results indicate that hirtin is a thrombin-like

serine protease and may have potential industrial and therapeutic applications.

2011 Elsevier Masson SAS. All rights reserved.

1. Introduction

Blood coagulation and brinolysis, involving a series of serine

proteases, are two important processes associated with haemo-

stasis and wound healing [1]. Fibrinogen is a heterotrimer protein

having three subunits namely Aa, Bb and g. The Aa and Bb subunits

are specically hydrolysed by the thrombin resulting the insoluble

brin clot formation (a, b and geg) at the nal step of blood

coagulation in response to an injury [2]. Fibrinolysis dissolves the

brin clot through the action of plasmin resulting in wound heal-

ing. Fibrin clot formation and brinolysis are tightly regulated

processes. However, thrombotic disorders may occur in a condition

when brin hydrolysis does not take place. Intravascular throm-

bosis resulting from brin aggregation in arteries is the main cause

of cardiovascular disorders including myocardial infarction [3]. The

brinolytic agents currently available for treatment of thrombosis

are plasminogen activators such as tissue-type plasminogen acti-

vators and urokinase-type plasminogen activators and all these

agents exhibit undesirable side effects. Therefore, the search for

other brinolytic enzymes as therapeutic agents for treatment of

thrombotic disorders from diverse sources is needed. Previously,

many proteases affecting coagulation and brinolysis have been

studied from various sources including plants [4e6], annelids [7,8],

insects [9], snake venom [10,11], bacteria [12] and recently from

Korean mushroom [13].

Plant latex is rich source of multiple proteases belonging mainly

to cysteine or serine super family [5,14]. They have been reported

from different plant families including Asclepiadaceae, Apocynaceae,

Moraceae, and Euphorbiaceae [15]. Earlier, many proteases from the

latex of different species of Euphorbia genus have been reported

[16]. In recent years, proteases affecting coagulation and brino-

lysis have been isolated and characterized from plant latex partic-ularly from Euphorbiaceae family [4,14]. Traditionally, the lattices

from different plants have been used to stop bleeding which has

been attributed to presence of proteases having ability to affect

blood coagulation.

Euphorbia hirta, belonging to Euphorbiaceae family, is a small,

herbaceous wild plant with great medicinal value. Traditionally, the

plant has been used for the treatment of respiratory ailments and

gastrointestinal disorders [17]. Many organic compounds have

been isolated and characterized from this plant such as avonoids,

polyphenols, tannins, triterpenes, phytosterols [18]. It is reported

that the plant extract have anti-allergic, anti-inammatory [19],

Abbreviations: BSA, bovine serum albumin; BAEE, N-benzoyl- L -arginine ethyl

ester; BTEE, N-benzoyl-L -tyrosine ethyl ester; BAPNA, N-benzoyl-L -arginine- p-

nitroanilide; BTNA, N-benzoyl-L -tyrosine p-nitroanilide; p-tos-GPRNA, N- p-tosyl-L -

Gly-L -Pro-L -Arg p-nitroanilide; AAPF, N-succinyl-L -Ala-L -Ala-L -Pro-L -Phe- p-nitro-

anilide; AAPL, N-succinyl-L -Ala-L -Ala-L -Pro-L -Leu- p-nitroanilide; AAA, N-succinyl-L -

Ala-L -Ala-L -Ala- p-nitroanilide; AAV, N-succinyl-L -Ala-L -Ala-L -Val- p-nitroanilide;

PMSF, phenylmethylsulfonyluoride; AEBSF, Aminoethyl-benzene sulfonyl uoride

hydrochloride; EDTA, Ethylenediaminetetraacetic acid; IAA, Iodoacetic acid; E64, 1-

trans-epoxysuccinylleucylamide(4-guanidino)butane-N-[N-( L -3-trans-carboxyir-

ane-2-carbonyl)- L -leucyl] agimatine; SDS, Sodium dodecyl sulphate; CBB R-250,

Coomassie brilliant blue R-250.

* Corresponding author. Tel.: þ91 1332 285657; fax: þ91 1332 273560.

E-mail addresses: [email protected], [email protected] (A.K. Sharma).

Contents lists available at SciVerse ScienceDirect

Plant Physiology and Biochemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m/ l o c a t e / p l a p h y

0981-9428/$ e see front matter 2011 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.plaphy.2011.12.004

Plant Physiology and Biochemistry 52 (2012) 104e111

mailto:[email protected]:[email protected]://www.sciencedirect.com/science/journal/09819428http://www.elsevier.com/locate/plaphyhttp://dx.doi.org/10.1016/j.plaphy.2011.12.004http://dx.doi.org/10.1016/j.plaphy.2011.12.004http://dx.doi.org/10.1016/j.plaphy.2011.12.004http://dx.doi.org/10.1016/j.plaphy.2011.12.004http://dx.doi.org/10.1016/j.plaphy.2011.12.004http://dx.doi.org/10.1016/j.plaphy.2011.12.004http://www.elsevier.com/locate/plaphyhttp://www.sciencedirect.com/science/journal/09819428mailto:[email protected]:[email protected]


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antimicrobial [20], diuretic and antidiabetic activity [21]. Till date,

no protein has been characterized and this is the rst protein re-

ported from this plant. The present work describes the purication

and characterization of a novel thrombin-like serine protease,

designated as hirtin, with brinolytic and brinogenoltic activities

from the latex of E. hirta.

2. Result and discussion

2.1. Puri cation and SDS-PAGE analysis of hirtin

A new brinolytic serine protease named as hirtin was puried

from the latex of E. hirta by the combination of Q sepharose anion

exchange and Superdex 200 gel ltration chromatography. The

crude milky latex, full of the gum and waxy contents, was frozen

at 20 C for 20 h and thawed on ice and centrifuged. The cleared

supernatant of latex, after centrifugation and ltration with

0.45 mm syringe lter, was used as crude enzyme solution and

loaded on to a pre-equilibrated Q sepharose fast ow column.

Bound proteins were eluted with a linear salt gradient of 0e0.5 M

NaCl. The fractions having proteolytic activity were eluted at

150 mM NaCl in 50 mM potassium phosphate buffer pH 7.2 withslight impurity. For further purication and molecular weight

determination, dialysed and concentrated protein sample was

loaded onto a pre-equilibrated HiLoad 16/60 Superdex 200 column

(16 600 mm, GE Healthcare). The protein was eluted with the

same buffer at a ow rate of 0.5 mL min1 with the retention

volume of 75.25 mL. The protein was concentrated and stored for

further use. The purication results of hirtin are summarized

(Table 1). The activity yield of the puried enzymewas calculated to

be 38.4% with the specic activity of 8.24 activity units mg1 min1

under the optimal assay conditions using BAEE as a substrate. The

fold purication of enzyme is less (3.39) than the expected value

which may be due to presence of the other proteases in the crude

extract of latex and also due to the loss of the activity during

purication, dialysis and concentration of hirtin. The fold puri-cation and yield of hirtin are comparable to other serine proteases

like Dubiumin, Milin and Cryptolepain [22e24]. The relative

molecular mass and purity of hirtin was analysed on 12% SDS-PAGE

under reducing condition. The apparent molecular mass was found

to be 34 kDa on SDS-PAGE (Fig. 1a) while on gel ltration column,

the molecular weight was determined to be 37.4 kDa by comparing

with gel ltration molecular weight standards. SDS-PAGE and gel

ltration chromatography results indicate that hirtin is a mono-

meric protein (Fig. 1b) as it exhibit single band indicating high

purity. Literature survey revealed that nearly all plant serine

proteases are glycoprotein. The difference in molecular weight as

determined by reducing PAGE and gel ltration chromatography

may be either due to the destruction of glycosylated moiety during

boiling before loading protein sample onto SDS-PAGE or thedestruction of weak interactions which facilitate the increased

mobility under reducing condition on SDS-PAGE. So, the apparent

molecular mass of hirtin, used in different biochemical calculations

was assumed to be 34 kDa. The molecular mass of the known serine

proteases from plant origin vary from 19 to 110 kDa and majority of

them are found to be in between 60 and 80 kDa. The molecular

mass of most serine proteases reported from the Euphorbiaceae

family has been found in the range 43e74 kDa [25]. Some of the

brinolytic proteases isolated from different sources represent the

molecular mass range 23e43 kDa and composed of single mono-

meric polypeptide like hirtin [4,6,8,13].

2.2. N-Terminal and partial internal sequencing

The puried hirtin was electrophoresed and electroblotted onto

a PVDF membrane for the N-terminal sequence analysis by Edman

degradation method. The N-terminal sequence of 15 amino acids of

hirtin was found to be Y-A-V-Y-I-G-L-I-L-E-T-A-A/N-N-E. The NCBI

blast did not show any match with known serine protease amino

acid sequences in database. However, the literature survey revealed

that N-terminal amino acids sequence of hirtin showed consider-

able identity only to that of reported N-terminal sequence of Milin

[25] isolated from the latex of Euphorbia milli (Table 2) but not to

other plants proteases reported from Euphorbiaceae family as Miliin

Table 1

Summary of purication of hirtin from Euphorbia hirta latex.

Purication

steps

Total

protein

(mg)

Total

activity

Unitsa

Specic

activity

(Units/mg/min)

Fold

purity

Activity

yield (%)

Crude latex 12.0 29.17 2.43 1.00 100

Q Sepharose 3.3 17.6 5.33 2.19 60.3

Superdex 200 1.36 11.2 8.24 3.39 38.4

a One unit of enzyme activity was dened as the amount of enzyme which gives

rise to an increase in unit absorbance at 253 nm/min of BAEE digestion in described

assay conditions.

Fig.1. a) Purity and molecular mass determination of hirtin under reducing condition

on 12% SDS-PAGE: Lane 1, molecular mass standards; Lane 2, crude latex; Lane 3,

partially puried hirtin after Q sepharose chromatography. b) Elution prole of hirtin

on gel ltration chromatography (HiLoad 16/60 Superdex 200 gel ltration column) on

AKTA-Prime, Insert: SDS-PAGE of puried hirtin after gel ltration chromatography: L1,

puried hirtin, L2, molecular mass standards.

G.K. Patel et al. / Plant Physiology and Biochemistry 52 (2012) 104e111 105


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[26] and Eumilin [14] and other plant families [22,24]. It is to be

noted that unlike hirtin which is a 34 kDa protein possessing

brinolytic activity, Milin is a 51 kDa protein and does not possess

brinolytic activity. Furthermore, the N-terminal amino acid

sequence of the hirtin is completely different from reported bri-

nolytic enzymes isolated from different sources indicating that it is

a unique protease [4,5,13,27]. In partial internal sequencing, one

peptide (R.F-I-W-L-L-T-V-P-R.A) was obtained with higher MASCOT

score (52) than the signicant (48) but it did not showanysequence

similarity with known plant proteases. The N-terminal and partial

internal sequencing results revealed that hirtin is a unique

protease.

2.3. Proteolytic assay and inhibition study of hirtin

The hydrolytic activity of hirtin towards the azocasein, gelatine,

human brinogen, brin clot and other synthetic substrates was

determined. The enzyme ef ciently hydrolysed the azocasein as

measured spectrophotometrically. In the gelatinolytic assay, the

incorporated gelatine in the PAGE gel showed a clear zone in a blue

background of gelatine stained with CBB R-250 indicating the

proteolytic activity of hirtin. The gelatinolytic activity was per-

formed by incubating the semi-native gel of hirtin after removal of

Table 2

Comparison of N-terminal amino acids of hirtin with other plant serine protease.

Protease/plant sources N-terminal sequences Reference

Hirtin (Euphorbia hirta) YAVYIGLILETAA/NNE Present study

Milin (Euphorbia milii) DVSYVGLILETD Subhash et al. (2006)

Eumiliin (Euphorbia milii) AFLLQIIVTPPN Fonseca et al. (2010)

Proteases B (E.supina) TTRTPNFLGLVD Arima et al. (2000)

Fig. 2. a) In-gel gelatinolytic (zymography) assay of hirtin at pH 5.0, 7.0 and 8.0. b) Fibrinogenolytic activity of hirtin analysed on 12% SDS-PAGE under reducing conditions: Lane 1,

molecular mass standards; Lane 2, pure hirtin; Lane 3 and Lane 11, pure brinogen incubated for 120 min without hirtin, Lane 4e10, incubation of brinogen (150 mg) with hirtin

(2.0 mg) at 37 C for 5, 10, 20, 30, 60, 90 and 120; Lane 12e14, incubation of brinogen (150 mg) with hirtin (5 mg) at 37 C for 60, 90 and 120 min in 50 mM HEPES buffer pH 7.2. c)

Fibirinolytic activity of hirtin analysed on 12% SDS-PAGE under reducing conditions: Lane 1, brin clot incubated for 120 min without hirtin; Lane 2e6 brin clot incubated with

hirtin (2.0 mg) at 37

C for 10, 20, 30, 45 and 60 min in 50 mM HEPES buffer pH 7.2.

G.K. Patel et al. / Plant Physiology and Biochemistry 52 (2012) 104e111106


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SDS at different pH (5, 7 and 8). The in-gel gelatinolytic activity

(zymography) was found to be highest at neutral pH 7 (Fig. 2a). The

activity gels (zymography) of hirtin at different pH clearly

demonstrate its activity at a wide pH range.

The time dependent incubation of hirtin (2 mg) with human

brinogen showed that the enzyme hydrolysed Aa chain very

ef ciently within 5 min of incubation while the hydrolysis of Bb and

g chain is slower. The hydrolysis of Aa chain of hirtin is similar to

the Synadenium grantii latex protein [4] and Eumilin [14] which

hydrolyse the Aa chain within 5 min of incubation and more active

as compared to the other brinolytic enzyme like ‘Pergularain e I

which could not hydrolyse Aa chain completely even after 30 min

of incubation [6] and Korean Cordyceps militaris brinolytic enzyme

which takes 60 min for hydrolysing Aa chain [14]. The hydrolysis of

Bb chain of brinogen by hirtin (2 mg) starts at 30 min of incubation

while g chain was not completely hydrolysed even after 120 min

incubation and seems to be resistant for hydrolysis. When 5 mg

hirtin was incubated with brinogen, the Bb chains almost hydro-

lysed in 120 min at 37 C and g showed partial hydrolysis (Fig. 2b).

In case of Eumilin and ‘Pergularain e I’, hydrolytic activity was not

observed towards the Bb and g chains even at 120 min of incuba-

tion. These results clearly demonstrate that hirtin is more effective

than Eumilin and Pergularain e I. The hydrolytic pattern of humanbrinogen by hirtin is similar to that of human thrombin which is

a specic brinogenolytic enzyme and hydrolyses Aa and Bb

subunits to release the brinopeptide A and B to form brin clot

during blood coagulation [28]. The ef ciency of hirtin is also

comparable to other brinogenolytic and brinoltic enzymes iso-

lated from different organisms like metalloprotease from the Rattle

snake [10]. In contrast, the snake venom protease isolated from

Taiwan habu hydrolysed the Bb chain more ef ciently than Aa

chain while the g remains intact [29] (Hung et al., 2001).

To evaluate the brinolytic activity, partially cross-linked brin

clot was formed using human thrombin which was further incu-

bated with the hirtin (2 mg) for different time duration and the

hydrolytic pattern were visualised on 12% SDS-PAGE under

reducing condition (Fig. 2c). The result showed that hydrolysis of a chain starts at 10 min of incubation and is completely hydrolysed

in time dependent manner while b and g chains are partially

hydrolysed at 60 min incubation at 37 C.

Tomonitor the thrombin-like activity, the puried protein (5 mg)

protein was mixed with 100 ml of citrated human plasma solution

(50 mg mL 1) along with a positive control containing pure human

thrombin (1.5 U) in 100 mL of citrated human plasma solution and

negative control only contains buffer with citrated human plasma.

The positive control readily form the brin clot after addition of

thrombin while the hirtin takes more time (10 min) to form

a visible clot.

The evaluation of cleavage specicity of the hirtin towards the

various synthetic amide and ester substrates were also investigated

(Table 3). The amidolytic activity of the hirtin was investigated byusing various chromogenic substrates. The enzyme showed the

remarkable hydrolytic activity towards amide substrate p-tos-

GPRNA, specic for the thrombin protease but did not show

signicant activity towards other amide substrates as AAPF and

BAPNA. For the esterase activity, BAEE and BTEE were used. It was

found that hirtin ef ciently hydrolysed the only BAEE. These results

demonstrated that hirtin possess both esterase and amidase

activities. The enzyme exhibited extremely low or no hydrolytic

activity towards the AAV, AAPL and AAA when incubated for the

30 min at 37 C.

In order to explore the class of hirtin protease, the inhibition

study was carried out using different class of inhibitor such as

PMSF, chymostatin, leupeptin, IAA, E64, HgCl2, EDTA, and pestatin

A. The enzyme activity was signi

cantly inhibited by only PMSF

(73%) and AEBSF (85%) and not any other inhibitor (Table 4). The

leupeptin did not show inhibition of hirtin activity which indicates

that it is not a plasmin-like protease. These results demonstrated

that hirtin is a thrombin-like serine protease. It can have applica-

tions in food industry and can be developed as potential thera-

peutic agent for treating thrombotic disorders.

2.4. Kinetic study

The kinetic parameters were determined by using the BAEE and

p-tos-GRPNA as substrate. The apparent MichaeliseMenten

constant (K m) and maximum reaction velocity (V max) for the BAEE

at 37 C was calculated from the LineweavereBurk plot and found

to be 0.575 mM and 1.87 107 M s1 respectively. The turnover

number (K cat) and catalytic ef ciency (K cat/K m) were founded to be

5.3 s1 and 9.2 103 M1 s1 respectively for BAEE. The apparent

K m and V max for the p-tos-GRPNA were found to be 0.431 and

3.8 108 M s1 respectively and K cat and K cat/K m for the p-tos-

GRPNA were calculated to be 0.646 s1 and 1.5 103 M1 s1.

2.5. Effect of pH and temperature on enzyme activity

The effect of pH on the enzyme activity was monitored from pH

3e11. Hirtin was found to be active at broad pH range from 3 to 9

with the pH optima 7.2 (Fig. 3a). The relative activity at pH 5 and 8

were 51% and 80% respectively and reduced drastically above pH 9.

The pH optima and activity in broad pH range of hirtin is similar to

other brinolytic enzymes of latex glycoprotein [4] Korean

C. militaris [13], Neanthes japonica brinase [8], cucumicin-like

protease [30] whereas some of the brinolytic enzyme show

their optimum activity either at acidic [31] or basic pH [32]. The

report showed that the pH optima of euphorbain proteases are inpH range of 6e8 [16].

Table 3

Activity of hirtin towards different substrates.

Substrates Characteristic Hirtin

activity

N- p-tosyl-Gly-Pro-Arg p-nitroanilide Thrombin Yes

N-benzoyl-L -arginine- p-nitroanilide Cathepsin H/trypsin Very less

N-suc-Ala-Ala-Pro-Phe- p-nitroanilide Cathepsin

G/chymotrypsin

No

N-suc-Ala-Ala-Pro-Leu- p-nitroanilide Elastase NoN-suc-Ala-Ala-Ala- p-nitroanilide Elastase No

N-suc-Ala-Ala-Val- p-nitroanilide Elastase No

N-benzoyl-L -tyrosine p-nitroanilide Chymotrypsin No

N-benzoyl-L -tyrosine ethyl ester Chymotrypsin Yes

N-benzoyl-L -arginine ethyl ester Trypsin Yes

Gelatin General Yes

Human brinogen Thrombin Yes

Azocasein General Yes

Table 4

Effect of Inhibitors on hirtin activity.

Inhibitor type Inhibitor name Inhibitor conc mM Residual activity %

Control No inhibitor e 100

Serine protease PMSF 1 27

AEBSF 1 15

Serine/cysteine Leupeptin 1 98

Cysteine protease IAA 1 98.0

E64 1 97.0

HgCl2 1 58.0

Metalloprotease EDTA 1 68.5

Aspartic protease Pepstatin A 1 98.0



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For the pH stability study, the residual activity of the pre-

incubated enzyme at different pH (3e11) was measured using p-

tos-GPRNA as a substrate at 37 C in 50 mM HEPES buffer pH 7.2. It

was found that enzyme is completely stable between pH 4e8. The

activity at broad pH range and its pH stability conrms its potential

use in biotechnological applications. The puried hirtin was active

from temperature ranging from 30 to 70 C. The temperature

optima of hirtin was found to be 50 C (Fig. 3b) and stable for

40 min without any loss in enzyme activity while 35% activity

remains when incubated for 60 min at 50

C.

2.6. Effect of metal ions, reducing agents, organic solvent,detergents on enzyme activity

The effect of metals ions on hirtin activity was measured in

presence of various metal salts (Table 5). The metal ions such as

Ca2þ and Mg2þ were found to increase the enzyme activity. The

CaCl2 at 2 mM concentration increased the hirtin activity by 2 fold

and at 5, 10 and 20 mM it remains up to 1 fold. The results indicate

that hirtin activity is not absolutely dependent on metal ions and

could be advantageous to microbial proteases which are dependent

on Ca2þ for their activity. Moreover, some metal ions Co2þ, Hg2þ

and Ba2þ have negative effect on the enzyme activity. Similar

observation was also reported from Dubiumin [24]. The enhanced

activity of hirtin in presence of Ca2þ is similar to the most studied

proteases which interfere in blood coagulation are either serine or

metalloprotease and requires Ca2þ for the activity [11].

It was found that the organic solvents as methanol, ethanol, and

DMF at 5 and 10% concentration did not affect the enzyme activity.

The DMSO did not affect enzyme activity at 5% but reduce 50%

activity if present at 10%. The detergents as SDS, Triton X-100, and

Tween-80 slightly increase the enzyme activity when present at 1%

but at 5% concentration the activity reduce by 10e15%. The SDS

increases the activity even at 5% concentration by 50%. The DTT, b-

mecaptoethanol, Urea did not affect the enzyme activity at 1 and 5%

concentration incubated for the 20 min. The 1% HCl solution has no

effect on activity but reduced 50% when present at 5% which might

be due to alteration of the pH of the reaction mixture.

2.7. Circular dichroism study

Far-UV CD spectra (190e240 nm) were analysed by Dichroweb

programme using CDSSTR method which showed less content of

the a-helix (13%) and high (34%) content of b-sheets at pH 7.0

(Fig. 4) which revealed that hirtin is a, b protein. The results are

similar to other reported brinogenolytic proteases which showed

less content of a-helix (7%) and high (48%) of b-sheets [4].

3. Conclusion

In conclusions, a novel thrombin-like serine protease (hirtin)

having brinolytic activity was puried and characterized from the

latex of E. hirta. The enzyme ef ciently hydrolyzed brinogen and

brin clot. Inhibition studies and high speci

city to chromogenic

2 4 6 8 10 120

20

40

60

80

100

R e l a t i v e a c y i v i t y ( % )

pH

20 30 40 50 60 70 80 900

20

40

60

80

100

R e l a t i v e a c t i v i t y ( % )

Temperature °C

a

b

Fig. 3. a) pH optima of hirtin. b) Temperature optima of hirtin.

Table 5

Effect of metal ions on hirtin activity.

Metal ions Concentration (mM) Relative activity (%)

Control H2O 100

NaCl 1 102

CaCl2 1 150

2 200

MgCl2 1 105

MnCl2 1 95

CuSO4 1 87.4

BaCl2 1 86

NiSO4 1 91

HgCl2 1 52.5

CoCl2 1 83

190 200 210 220 230 240-6

-4

-2

0

2

4

6

8

M R E × 1

0 3 ( d e g

c m 2

d m o l -

1 )

Wavelength (nm)

Fig. 4. CD study of hirtin at pH 7.2 at 25 C.



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substrate for thrombin clearly indicate that hirtin is a thrombin-like

serine protease. The enzyme exhibited stability over a wide range of

pH and temperature. The results suggest that hirtin can be devel-

oped as potential therapeutic agent for thrombotic disorders and in

other biotechnological applications. The actual biological role of the

hirtin, a major protein in the E. hirta latex needed to be further

investigated.

4. Experimental

4.1. Materials

The latex of E. hirta was collected locally. Q sepharose, casein,

human brinogen fraction I: type I, citrated human plasma,

thrombin, BSA, azocasein, gelatin, haemoglobin, skimmed milk, 2-

[4-(2hydroxyethyl)1-piperazinyl)] ethane sulphonic acid (HEPES),

3-cyclohexylamino-1propan-sulphonic acid (CAPS), synthetic

substrates like N-benzoyl-L -arginine ethyl ester (BAEE), N-benzoyl-

L -tyrosine ethyl ester (BTEE), N-benzoyl-L -arginine- p-nitroanilide

(BAPNA), N-benzoyl-L -tyrosine p-nitroanilide (BTNA), N- p-tosyl-

Gly-Pro-Arg p-nitroanilide ( p-tos-GPRNA), N-succinyl-Ala-Ala-Pro-

Phe- p-nitroanilide (AAPF), N-succinyl-Ala-Ala-Pro-Leu- p-nitro-

anilide (AAPL), N-succinyl-Ala-Ala-Ala- p-nitroanilide (AAA), N-succinyl-Ala-Ala-Val- p-nitroanilide (AAV), Inhibitors like Phenyl-

methylsulfonyluoride (PMSF), 4-(2-Aminoethyl)benzenesulfonyl

uoride hydrochloride (AEBSF), leupeptin, EDTA, HgCl2, 1-trans-

epoxysuccinylleucylamide(4-guanidino)butane-N-[N-(L -3-trans-

carboxyirane-2-carbonyl)-L -leucyl] agimatine (E64), gel regents as

SDS, acrylamide, N,N-methylene bisacrylamide, b-mercaptoetha-

nol, coomassie brilliant blue R-250 (CBB R-250) and blotting

chemicals were purchased from Sigma Aldrich (St Louis, MO,USA).

Protein molecular weight standards and gel ltration standards

were purchased from Bio-Rad, USA. All other chemicals and

solvents trichloroacetic acid (TCA), dimethylsulfoxide (DMSO),

dimethylformamide (DMF), methanol and ethanol used were of

analytical grade.

4.2. Collection of latex and puri cation of hirtin

The latex of E. hirta is mainly present in the apical part of plant.

Latex was collected in 50 mM phosphate buffer pH 7.2 in fractions

of 1 mL on ice by breaking the apical tender part of plant. The

collected samples were stored at 20 C for overnight. The frozen

latex was thawed and centrifuged (Allegra X-22R, Bakeman) at

20000 g for 30 min at 4 C. The cleared supernatant was collected

and ltered through 0.45 mm syringe lter to remove all trace of

precipitated gum and theltrate was used as crude latex extract for

protein purication. All the purication steps were performed at

4 C in a cooling cabinet. The crude latex extract from above step

was loaded on Q sepharose fast ow column (1.5 20 cm Econo-

column, Bio-Rad), pre-equilibrated with 50 mM phosphate bufferpH 7.2. Column was washed extensively to remove the unbounded

proteins and other impurities. The bounded proteins were eluted at

a ow rate of 1 mL min1 with a linear gradient of NaCl (0e0.5 M)

in 50 mM phosphate buffer pH 7.2. Fractions having proteolytic

activity were pooled, dialysed against the 50 mM phosphate buffer

pH 7.2, concentrated and loaded onto a pre-equilibrated HiLoad 16/

60 Superdex 200 gel ltration column (16 600 mm, GE Health-

care). The protein was eluted with the same buffer at a ow rate of

0.5 mL min1 and the fraction having protease activity was pooled

for further analysis. The initial calibration of column to demon-

strates the good resolution in the separation of standard proteins

and relative molecular weight of hirtin was determined by using

the gel ltration standards (Bio-Rad) as 670 kDa, thyroglobulin;

158 kDa,g-globulin; 44 kDa, ovalbumin; 17 kDa, myoglobin; and

1.35 kDa, vitamin B12. The concentration of puried protein was

estimated spectrophotometrically by Coomassie blue dye binding

method [33] using BSA as the standard protein. The presence of

protein during the purication steps were also monitored by taking

absorbance at 280 nm using UV eVis spectrophotometer (DU730,

Bakcman).

4.3. SDS-PAGE analysis

The crude extract of latex, Q sepharose eluted fractions and

puried hirtin were electrophoresed on 12% polyacrylamide gel

containing 0.1% SDS under reducing conditions. The protein bands

were visualized by 0.15% staining with CBB R-250. The relative

molecular mass of hirtin was also determined by using a low range

SDS-PAGE molecular weight standards (Bio-Rad) as97.4 kDa,

phosphorylase b; 66.2 kDa, bovine serum albumin; 45 kDa, oval-

bumin; 31 kDa, carbonic anhydrase; 21.5 kDa, trypsin inhibitor and

14.4 kDa, lysozyme.

4.4. N-Terminal and partial internal sequencing

The puried hirtin was electrophoresed on 12% SDS-PAGE andelectroblotted onto a PVDF membrane (Immobilon-P, Millipore) at

350 mA using 100 mM CAPS buffer, pH 11.0 in mini trans-blot unit

(Bio-Rad) according to Matsudaria [34]. After electroblotting, the

membrane was rinsed 3e4 times with deionized water and stained

with 0.1% CBB R-250 in 1% acetic acid 50% methanol for a few

seconds. Membrane was destained with 50% methanol. Protein

spots were excised and rinsed for 10 min in deionized water and

air-dried. The N-terminal amino acid sequencing was performed by

Edman degradation method on an automated sequencer (Procise

491cLC; Applied Bio-systems) at the protein sequencing facility of

Institute of Microbial Technology (IMTECH), Chandigarh, India. A

database search for comparable peptides sequences was performed

by NCBI blastp program [35].

The partial internal sequencing of hirtin was done at proteomicsfacility of The Centre for Genomic Application (TCGA), New Delhi,

India. The puried protein was electrophoresed on a 12% SDS-PAGE

and protein bands were excised and partially digested with trypsin.

The resulted peptides were subjected to two dimensional liquid

chromatography ESI-MS (Agilent 1100 series 2DnanoLC-MS) fol-

lowed by reverse phase separation. The peptides get ionized in the

liquid phase in the Electrospray ionizer (Bruker Daltonics Ultraex

TOF/TOF) and enter the ion trap, get fragmented (MS/MS) and

detected. The data were analysed using MASCOT search engine.

4.5. Proteolytic assay and kinetic study

The hydrolytic activity of the hirtin towards different substrates

were monitored at 37

C using azocasein, gelatin, brinogen,synthetic peptides as p-tos-GPRNA, BAPNA, BTNA, AAPF, AAPL,

AAA, AAV, esters as BAEE and BTEE as substrate.

4.5.1. Azocaseinolytic assay

The azocaseinolytic activity of puried hirtin was assayed using

1% azocasein as substrate in 50 mM potassium phosphate buffer pH

7.2. The reaction was performed in 500 ml assay volume containing

200 ml of 1% azocasein (w/v) and HEPES buffer pH 7.2 using 5 mg of

enzyme and the mixture was incubated for 20 min at 37 C. The

reaction was terminated by adding one volume of 20% TCA and

placed on ice for 10 min and centrifuged at 15,000 g for 10 min.

The supernatant was mixed with the equal volume of 1 M NaOH

solution and absorbance was measured at 440 nm with the reagent

blank.



7/8

4.5.2. In-gel protease assay (Zymography)

In-gel protease assay was performed according to the method

described by Choi et al. [36] with slight modications using 0.1%

gelatin co-polymerised with the resolving gel. The puried enzyme

was mixed with non-reducing sample buffer and was run on 12%

SDS-PAGE without boiling. After electrophoresis, gel was washed

with 20% TritonX-100 for 1 h at4 C and rinsed with distilled water

for three times at the interval of 30 min to remove all trace of SDS.

Now gels were immersed in developing buffers at pH 5.0, 7.0 and

8.0 supplemented with 2 mM CaCl2, 100 mM NaCl and incubated

for 12 h at 37 C for the protease activity. The reaction was termi-

nated by adding ice cold 20% TCA for 20 min at room temperature.

Gel was stained with 0.15% CBB R-250 in water:methanol:acetic

acid (50:40:10) and destained with same solution without dye to

visualize the clear hydrolytic zone.

4.5.3. Fibrinogenolytic and brinolytic activity

The Fibrinogenolytic activity assay was performed as

described by Rajesh et al. [4] with slight modication. The reac-

tion mixture contained 150 mg of brinogen, 2 mM CaCl2 in

50 mM HEPES buffer pH 7.2, was incubated with 2.0 mg of enzyme

at 37 C for 5, 10, 20, 30, 45, 60 and 120 min and also by using

5.0 mg of enzyme for 30 60 and 90 min in different tubes. Acontrol reaction mix was also incubated for 120 min at 37 C in

absence of hirtin. The reaction was terminated by boiling after

addition of 4X reducing sample buffer (0.25 M TriseHCl pH 6.8,

5% b-mercaptoethanol, 8% SDS, 40% glycerol and 0.2% bromo-

phenol blue). The hydrolyzed products were analysed on a 12%

SDS-PAGE and protein pattern was visualized by staining with

0.15% CBB R-250.

For the brin clot degradation activity, partially cross-linked

brin clot was formed using 150 mg of brinogen in 50 mM

HEPES buffer pH 7.2 supplemented with 2 mM CaCl 2 in presence of

1.5 U of human thrombin after incubation for 20 min at 37 C.

Partially cross-linked clot was washed with HEPES buffer pH 7.0

and incubated with 2 mg hirtin in 50 mM HEPES buffer pH 7.0

containing 2 mM CaCl2 at 37 C for 10, 20, 30, 45 and 60 min indifferent tubes. The reaction was terminated and analysed on 12%

reducing SDS-PAGE as described above.

4.5.4. Thrombin-like activity

The lyophilised citrated human plasma (sigma) was dissolved in

sterile, nuclease and protease free distilled water. The 100 ml of

human plasma (50 mg mL 1) was incubated in different tubes with

different amount of hirtin (2 and 5 mg) in 50 mM HEPES buffer pH

7.2 having 2 mM CaCl2. The time for visible clot formation was

recorded.

4.5.5. Activity towards synthetic substrates

The enzymatic hydrolysis of different synthetic substrates

peptidyl- pNA (peptidyl p-nitroanilide) by the puried hirtin wasstudied by spectrophotometric method. The synthetic substrates

used were BAPNA, BTNA, p-tos-GPRNA, AAPF, AAPL, AAA, and AAV.

The 500 ml reaction mixture containing 1 mM substrates, 2 mM

CaCl2 in 50 mM HEPES buffer pH 7.2 was initially equilibrated at

37 C for 5 min and further incubated for 10 min after addition of

5 mg of enzyme. The reaction was terminated by addition of 500 ml

of 20% acetic acid. The rate of hydrolytic activity was determined by

measuring the released p-nitroaniline at 410 nm using the molar

extinction coef cient ε410¼ 8480 M1 cm1.

The hydrolytic activity towards the ester substrates, N-benzoyl-

L -arginine ethyl ester (BAEE) and N-benzoyl-L -tyrosine ethyl ester

(BTEE) were examined spectrophotometrically at 253 nm

(ε253 ¼ 1150 M1 cm1) and 259 nm (ε259 ¼ 964 M

1 cm1)

respectively.

4.5.6. Kinetic study

The kinetic parameters were calculated from the initial rate of

enzymatic hydrolysis of puried hirtin using BAEE and p-tos-

GPRNA as substrate. The enzymatic activity towards BAEE and p-

tos-GPRNA were assayed using 2.0 mg hirtin protease by continuous

and discontinuous method respectively. To determine the apparent

MichaeliseMenten constant (K m), the increasing BAEE concentra-

tion (0.05e1.5 mM) was incubated with the 2.0 mg of enzyme in

1 mL reaction volume and the initial rate of hydrolysis was deter-

mined by absorbance change per min at 253 nm by continuous

method. For p-tos-GPRNA, the 500 ml reaction mixture containing

a substrate range (0.01e2.0 mM) were incubated with enzyme at

37 C for different times. After incubation, the reactions were

terminated by addition of 20% acetic acid andabsorbance was taken

at 410 nm. Each assay was carried out in triplicate. The kinetic

parameters were calculated from LineweavereBurk plot.

4.6. Effect of pH and temperature on enzyme activity

To determine the optimum pH of hirtin, enzyme activity was

assayed at different pH (3e11), using p-tos-GPRNA as a substrate in

different buffers as citrate phosphate (3e8), TriseHCl (8e9) and

glycine-NaOH (10e

12). For the pH stability study puried hirtin(5 mg) was incubated for 1 h at 37 C in different buffers of pH range

(2e11) at the interval of 1 pH unit and the residual activity was

determined by further incubation for 10 min at 37 C at pH 7.2 as

described earlier.

To determine the optimum temperature, 5 mg of puried hirtin

enzyme was used in a 500 ml of reaction mixture containing 1 mM

p-tos-GPRNA in 50 mM HEPES buffer pH 7.2 with 2 mM CaCl2. The

reaction mixture was incubated for 10 min at temperature ranges

from 25 to 90 C at 5 C increment and the hydrolytic activity was

determined as describedabove. The stability of enzyme at optimum

temperature was determined by pre-incubating the enzyme at

temperature optima (50 C) for 0e90 min with 10 min increments

and the residual activity was determined using p-tos-GPRNA as

a substrate at 37 C. The non heated enzyme was considered aspositive control and assumed to have 100% activity.

4.7. Protease inhibition assay

The protease inhibition assay was done using the PMSF, AEBSF,

leupeptin, E64, HgCl2, EDTA, pestatin A to determine the class of

protease. The 1 mM inhibitor solution was incubated with 5 mg of

the puried hirtin in 50 mM phosphate buffer pH 7.2 and the

residual activity was determined using BAEE as substrate as

described above. The control assay was done in absence of the

inhibitor and the activity was taken as 100%.

4.8. Effect of metal ions on enzyme activity

The effect of metal ions on the enzyme activity was determined

using different salts in 50 mM HEPES buffer pH 7.2. The salt of Naþ,

Ca2þ, Mg2þ, Mn2þ, Hg2þ, Ba2þ and Co2þ were added as chlorides,

Cu2þ and Ni2þ were added as sulphates. The entire assay was done

in triplicate using p-tos-GPRNA as a substrate and average was

taken as data point.

4.9. Effect of denaturants, organic solvents and detergents on

enzyme activity

The effect of different surfactants like SDS, Tween-20, Tween-80,

Triton X-100, Urea (1 and 5%), organic solvents methanol, ethanol,

DMSO, DMF (1 and 10%) and reducing agents DTT, b-mercaptoe-

thanol (1and 5%) on enzyme activity was studied by pre-incubating



8/8

the puried hirtin at 37 C for 30 min with these effectors. The

relative activity was determined in 50 mM HEPES buffer pH 7.2

supplemented with 2 mM CaCl2 and 1 mM p-tos-GPRNA as

a substrate. The enzyme activity was assumed as 100% in absence of

any additive.

4.10. Circular dichroism study

Circular Dichroism study was performed on Chirascan CD

spectrometer (Applied Photophysics, UK). Far-UV CD spectra

(180e260) were recorded in a 1 mm quartz cell at bandwidth 1 nm

and time per point 0.5 s with three repeats. The CD study was done

using 0.2 mg mL 1 puried protein in 20 mM Citrate phosphate

buffer pH 7.0 at 25 C. Three consecutive scans were accumulated.

The average base line spectrum of buffer blank wassubtracted from

the average protein sample spectrum and analysed (190e240 nm)

by online DichroWeb programme using CDSSTR method [37]. The

results of the CD measurements were expressed as mean residue

ellipticity (MRE) in deg cm2 dmol1.

Acknowledgements

CD studies were performed at NMR facility at Institute Instru-

mentation Centre (IIC), IIT Roorkee. G. K. Patel and A. A. Kawale

gratefully acknowledge the nancial support from Ministry of

Human Resource Development (MHRD) and Department of

Biotechnology (DBT), Government of India, respectively.

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