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Purification and characterization of fibrinolytic enzyme from Kiwifruit

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International Journal of Biochemistry. Photon 108 (2013) 195-201
https://sites.google.com/site/photonfoundationorganization/home/international-journal-of-biochemistry
Original Research Article. ISJN: 4438-5728
International Journal of Biochemistry
Purification and characterization of fibrinolytic enzyme from Kiwifruit
Salem A. Habib, Entsar A. Saad*
Chemistry Department, Faculty of Science (New-Damietta), Damietta University, Damietta, Egypt
Article history:
Received: 14 May, 2013
Accepted: 26 May, 2013
Available online: 28 July, 2013
Keywords:
Fibrinase, Actinidia deliciosa, Kiwifruit, Fibrinolytic
enzyme, Thrombolytic agent, Fibrin
Corresponding Author:
Saad E.A.*
Lecturer of Biochemistry
Email: entsarsaad@gmail.com
Phone: +2057403866
Fax No: +2057 403868
Habib S.A.
Professor of Biochemistry
Email: salem_habib@yahoo.com
Abstract
When a blood vessel is injured, the body forms the
blood clot (thrombus). When a thrombus causes
more than 90% obstruction this can result in anoxia
and infarction. Therefore, searching for effective,
inexpensive and safe thrombolytic agents from
various natural sources is of great interest. Hence,
1. Introduction
Thrombosis is the formation of a blood clot
(thrombus) inside a blood vessel, obstructing
the flow of blood through the circulatory
system. When a blood vessel is injured, the
body uses platelets and fibrin to form a blood
clot. When a thrombus occupies more than
75% of surface area of the lumen of an artery,
blood flow to the tissue supplied is reduced
enough to cause symptoms because of
decreased oxygen (hypoxia) and accumulation
of metabolic products like lactic acid. More
than 90% obstruction can result in anoxia, the
complete deprivation of oxygen, and infarction,
a mode of cell death. Therefore, thrombi are
major causes of morbidity and mortality
(Corrado et al., 1994). Several lines of
research toward the improvement of
thrombolytic agents are being explored
including their production (Abdel-Fattah et al.,
1983; Abdel-Fattah et al., 1993). There has
been a great deal of interest in the search for
new thrombolytic agents from various sources
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fibrinolytic enzyme production from kiwifruit for
blood clot lysis that can be employed as a good
therapeutic agent is of importance. The present
investigation deals with purification, characterization
and therapeutic application of kiwifruit fibrinase.
Fibrinase was purified from concentrated
homogenat with specific activity of 160 U mg-1
protein. The optimal pH was 6.5. The enzyme pK1
was 5.2 and pK2 was 7.3. The km value for fibrin
was 1.5 g/l. The optimal temperature was 30° C.
The activation energy was 10.4 KJ/mol. Fibrinase
was inactivated at 60, 70 and 80° C with t1/2 values
of 34, 21 and 16 min at 60, 70 and 80° C,
respectively. Many compounds and metal ions like
2+
iodoacetamide, EDTA and Cu inhibited the
2+
fibrinase while others like G-6-P, AMP and Ca
showed enzyme activation. Fibrinase displayed
great affinity towards human blood clot and seems
to be a promising thrombolytic agent for clinical use.
Citation:
Saad E.A., Habib S.A., 2013. Purification and
characterization of fibrinolytic enzyme from Kiwifruit.
International Journal of Biochemistry. Photon 108, 195-
201.
with particular interest in microbial and higher
plant sources.
1.1 Fibrinolytic enzymes
Fibrin is the end product of blood clotting.
Biosynthesis of fibrinolytic enzymes was
studied in bacteria isolated from sea water and
from different aquatic habitats. Egorov et al.
(1986) found that strains with the highest
fibrinolytic activity belonged to Bacillus genus
and were isolated from mineral detritus and
ruff intestines in the Black Sea. The fibrinolytic
enzyme was purified from supernatant of
Bacillus sp. strain CK 11-14 culture broth and
showed thermostable, hydrophilic, and strong
fibrinolytic activity (Kim et al., 1996). A Bacillus
sp. producing a new fibrinolytic enzyme was
screened from a fermented fish known as
Jeat-Gal in Korea (Kim et al., 1997). The
fibrinolytic enzyme separated by Imshenetski
et al. (1991) from Pseudomonas genus
195
completely lysed, in vitro, human blood
thrombi within 50 min.
1.2 Kiwifruit
Actinidia deliciosa (Actinidiacene), the well-
known as just "kiwi" is a plant native to the
eastern Asia. Commercial planting began in
New Zealand in the late 1930 s, and for the
past three decades kiwifruit has been
increasingly available worldwide (Lucas et al.,
2003).
Kiwifruit contain many medicinally useful
compounds such as vitamin C, carotenoids,
flavonoids, minerals and others (Wills et al.,
1986). Kiwifruit contains a large amount of a
protease enzyme (actinidin) that was first
discovered by Arcus (Arcus, 1959).
Considerable portion of structural and
functional characteristics of actinidin has been
revealed by researchers (Brocklehurst et al.,
1984; Lewis and Luh, 1988). Kiwifruit has a
preventive effect against cardiovascular
diseases (Hertog et al., 1993). It has been
used for the treatment of many cancers
especially cancers of the digestive system,
lung, and liver (Block et al., 1992; Miller and
Rice-Evans, 1997; Motohashi et al., 2002).
Kiwifruit is also an effective and accessible
agent in debridement of dead tissue in burn
wounds (Hafezi et al., 2010; Kooshiar et al.,
2010). In addition, kiwifruit have anti-obesity
effects (Sung et al., 2013) and regulates
adipocyte differentiation and function (Abe et
al., 2010). Moreover, kiwifruit fibrinolytic
activity was observed by Jung et al. (2005).
According to the best of our knowledge
fibrinase production from kiwifruit for blood clot
lysis have not been reported.
2. Objective of Research
In view of the important role of fibrinase in the
therapeutic application, the present study
deals with purification and characterization of
fibrinase separated from kiwifruit. It can be
employed as a good and safe therapeutic
thrombolytic agent and hence help to reduce
the morbidity and mortality among patients
suffering from thrombi.
3. Materials and Methods
3.1 Preparation and purification of fibrinase
Kiwifruit homogenate was filtrated and the
clear supernatant which represents the crude
enzyme was collected. The crude enzyme was
precipitated from the supernatant by 60 %
ammonium sulfate. After centrifugation at 3500
rpm, the supernatant was discarded and the
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pellet, which contains the enzyme, was mixed
with calcium phosphate gel and centrifuged for
20 min at 2500 rpm. Most of the enzyme
activity appeared in the supernatant. The
supernatant was applied onto Sephadex G
200 column (1.5 x 50 cm). Elution was carried
out at a flow rate of 15 ml/h with a linear
gradient of 0.2 - 1.6 % NaCl in 0.1 M
phosphate buffer, pH 8.0. The most active
fractions of which were pooled dialyzed with
cold distilled water and concentrated with
polyethylene glycol. Fibrinase assay was
tested with 25.5 µg of the enzyme protein.
During the entire procedure the protein was
kept at 4° C or on ice.
3.2 Assay of fibrinase
The assay of fibrinase activity was carried out
using bovine fibrin as a substrate suspended
in 0.1 M phosphate buffer pH 8.0 (Basha and
Beevers, 1975). The peptides released were
measured using the method of Lowry et al.
(1951). One unit of fibrinase was defined as
the amount of the enzyme that produces, in 1
hr, 1 mg of soluble peptides as a product of
fibrin hydrolysis.
3.3 Effect of fibrinase on human blood clot
A clot of 1 ml plasma after washing with
phosphate buffer was incubated with the
purified fibrinase from kiwifruit at various time
intervals. After incubation, the produced free
proteins and polypeptides from the human
blood clot were followed by polyacrylamide gel
electrophoresis (PAGE).
3.4 Protein determination
The protein content of the supernatant was
measured using the method of Lowry et al.
(1951).
3.5 Polyacrylamide gel electrophoresis
(PAGE)
Electrophoresis was carried out as described
by Davis (1964). The gels were stained by
Coomassie blue and destained with a mixture
of methanol and galacial acetic acid.
4. Results
4.1 Fibrinase purification and its specific
activity determination
The purification of fibrinase from kiwifruit was
performed. The most effective purification step
was the elution from Sephadex G200. The
specific activity for the enzyme was 160 Umg-1
protein. Fibrinase activity was measured at
different periods of incubation ranging from 10
to 70 min. and the results showed the period
196
of 60 min seems to be the optimum time for Figure 2: Effect of temperature on fibrinase activity
maximal activity of the enzyme (Fig. 1).

Figure 1: Effect of incubation time on fibrinase

activity

Figure 3: Effect of pH on fibrinase activity.

4.2 Effects of temperature, pH and substrate

concentration on fibrinase activity & related
fibrinase constants determination
The effects of temperature and pH on the
enzyme activity were studied and the optimum
temperature was 30° C (Fig. 2) and the
optimum pH value was 6.5 (Fig. 3). The pK
values for the fibrinase were determined from
the relation between log V and pH (Fig. 4).
The pK1 was 5.2 and the pK2 was 7.3. On Figure 4: Determination of pK values of the
fibrinase enzyme
plotting the enzyme activity against fibrinase
concentration a linear relationship was
obtained (Fig. 5). The relation between
substrate concentration and enzyme activity
was shown in Fig. 6. Km value was 1.5 g/l and
Vmax was 120. Plotting log Vo/Vmax-Vo against
log S resulted in a straight line (Fig. 7). The
slope of the plot represents Hill coefficient
value, which is less than unity. The line
weaver-Burk plot was constructed from the
relation between log 1/S against log 1/V (Fig.
8). The Arrhenius plot for the fibrinase was
constructed as a straight line in the relation
between log V and the reciprocal of the
absolute temperature (Fig. 9), and the Figure 5: Effect of enzyme concentration on
calculated value of the fibrinase activation fibrinase activity
energy was 10.4 KJ mol-1.

The rate of heat inactivation of fibrinase was

studied at 60, 70 and 80° C (Fig 10). Most of
the enzyme activity was lost at 80° C. The
calculated t1/2 values are 34, 21 and 16 min for
the incubation of the enzyme at 60, 70 and 80°
C, respectively. The decay constant (Kd)
values (Fig. 11) were higher at 80° C than at
60° C or 70° C.

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Figure 6: Effect of substrate concentration on Figure 10: Heat inactivation of fibrinase at 60, 70
fibrinase activity and 80° C

Figure 7: Hill coefficient for fibrinase Figure 11: Relation between the time of fibrinase
inactivation and decay constant (Kd) at different
temperatures.

Figure 8: Line weaver-Burk plot for fibrinase

4.3 Activation and inhibition of fibrinase
The effect of-SH reagents and chelating
agents (Table 1) on fibrinase activity was
investigated. The results showed that
iodoacetamide, iodoacetic acid, sodium azide
and HgCl2 are completely inhibited fibrinase
activity after being included in the reaction
mixture at 2 mM. Some metabolites such as
G-6-P and AMP showed activation for the
enzyme and others such as glucose, fructose
2+
and NADP showed inhibition (Table 2). Co ,
2+ 2+ 2+ 2+ 3+
Cu , Ni , Zn , Mg , and Fe metal ions
2+
inhibited the enzyme while Ca ion activated
the enzyme (Table 3).
Figure 9: Arrhenius plot for fibrinase.
Figure 12 A: Effect of fibrinase on blood clot.

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Table 1: The effect of some-SH reagents and chelating agents on fibrinase activity
Inhibitor Iodoacetamide Iodoacetic acid Sodium azide HgCl2 EDTA Urea
Percent of inhibition (%) 100 100 100 100 80 72

Table 2: The effect of some metabolites on fibrinase activity

Metabolite Glucose Fructose G-6-P 10 G-6-P 40 AMP 10 NADP AMP 40 Control
Enzyme activity (%)
18 30 53 58 80 108 38 50

Table 3: Effect of some metal ions on enzyme activity

Metal salt CuCl2 CoCl2 CaCl2 ZnCl2 NiCl2 FeCl3 MgCl2 Control
Enzyme activity (%) 0.0 0.0 85 0.0 6.0 28 39 50

4.4 Therapeutic application of fibrinase as fibrinase from Codium intricqtuem (Cornish-Bowden

thrombolytic agent
To investigate the therapeutic application of
fibrinase as thrombolytic agent, the lysis of
human blood clot was studied (Fig. 12). The
purified enzyme displayed a great affinity
towards human blood clot.
Figure 12 B: Lysis of human blood clot by fibrinase
and Wharton, 1988).
5.3 Fibrinase constants
In the present work, linear relationship between
fibrinase activity and fibrinase concentration was
obtained. The enzyme pK1 was 5.2 and the pK2 was
7.3, these values represent the presence of
imidazolium group of histidine and ammonium
group of cystine. In the present investigation, Km
value was 1.5 g/l and Vmax was 120. Plotting log
Vo/Vmax-Vo against log S resulted in straight line.
The slope of the plot represents Hill coefficient
value, which is less than unity. This suggested that
the binding of the substrate is negatively
cooperative and thus confirms the similar results of
El-Naggar et al. (1997) for fibrinase from Penicillium
decumbans.

5.4 Fibrinase activation and inhibition

Bands 1 and 2 represent the polypeptides produced
as a result of the action of the enzyme on blood
clot, Bands 3 and 4 represent the enzyme and its
isozyme.
Lane 1 represents the enzyme and the blood clot
at zero time, Lanes 2, 3, 4, 5, 6, 7, 8, 9 represent
the enzyme and blood clot at 1/2, 1, 11/2, 2, 2 1/2,
3, 3 1/2, 4 hours of incubation respectively.
5. Discussion
The present results showed that iodoacetamide,
iodoacetate and HgCl2 completely inhibited
fibrinase activity. This inhibition indicates the
necessity of -SH group for enzyme catalysis. In
support, Ahmed et al. (1990) reported that fibrinase
disulfide bonds are important for its activity. Sodium
azide completely inhibited fibrinase activity. This
inhibition is similar to that obtained by El-Naggar et
al. (1997) for fibrinase from Penicillium decumbans.
Chelating agent EDTA and urea inhibited fibrinase
revealing that the fibrinase from kiwi is a
metalloenzyme. Description of fibrinase as a
metalloenzyme is in consistent with the results for
the enzyme from bacilli (Kim et al., 1997) and
Penicillium (El-Nagar et al., 1997).

5.1 Fibrinase specific activity
The present study showed that the specific activity
-1
for the enzyme was 160 U mg protein. Higher
-1
specific activity of 600 U mg protein was reported
for fibrinase from Codium latum (Matsubara et al.,
1999). The optimal time for maximal activity of
fibrinase enzyme was 60 min.
5.2 Fibrinase optimum temperature and
optimum pH
The maximum activity of the enzyme was reported
at pH 6.5 and at temperature 30° C. This coincided
with the optimum pH recorded for fibrinase from
Oidiodendron flavum (Ali et al., 1990). Higher
optimal temperature of 70° C was reported for
In the present work, some metabolites such as G-6-
P and AMP showed activation for the enzyme and
others such as glucose, fructose and NADP showed
inhibition. Habib et al. (2010) reported that, all
sugars decreased fibrinase productivity except
lactose and starch increased enzyme productivity
from S. violaceoruber and S. spiroverticillatus,
respectively. Previously, in this concept, galactose
increased asparaginase and protease productivity
from A. niger, while asparaginase productivity from
A. terreus was stimulated by starch, fructose,
glucose, galactose, cellulose and sucrose (El-
Waseef et al., 1993).

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 2+ 2+
The present study also showed that Co , Cu ,
2+ 2+ 2+ 3+
Ni , Zn , Mg , and Fe metal ions inhibited the
2+
enzyme while Ca ion activated the enzyme. Foda
et al. (1980) reported that CaCl2 stimulated the
production of asparaginase from Wp. Polymorpha,
while ZnSO4, FeSO4, CuSO4 and MgSO4 caused
slight decrease in the production, also Allison and
2+ 2+ 2+
Macfariane (1992) found that Ca , Mn and Co
stimulated the activity of protease from Clostridium
sporogenes. Also El-Waseef et al. (1993) reported
2+ 2+ 2+ 2+
that Co , Ni , Zn and Cu increased
asparaginase productivity from A. terreus.
5.5 Therapeutic application of fibrinase as
thrombolytic agent
Thrombolytic agents from various sources have
been extensively investigated. Enzymes, such as
urokinase, streptokinase and tissues plasminogen
activators have been widely used in the treatment of
thrombosis. However, these enzymes are often
expensive, thermolabile and can produce
undesirable side effects (Chitte and Dey, 2000). In
the present study, the fibrinase from kiwifruit
showed a great affinity towards human blood clot.
This result is in accordance with the findings of El-
Shora et al. (2002), Abdel-fattah et al. (1993), El-
Nagar et al. (1997) and El-Waseef et al. (1993).
This indicates that fibrinase, extracted from kiwifruit,
is able to lyze human blood clot and would have
future therapeutic applications.
Conclusion
The purified fibrinase enzyme from kiwi has a
specific activity of 160 U mg-1 protein. The
maximum activity of the enzyme was reported at pH
6.5 and at temperature 30° C. The enzyme pK1
was 5.2 and the pK2 was 7.3. Its Km value was 1.5
g/l and Vmax was 120. Iodoacetamide, iodoacetate,
HgCl2, sodium azide, EDTA and urea inhibited the
fibrinase activity. Some metabolites such as G-6-P
and AMP activated the enzyme and others such as
glucose, fructose and NADP showed inhibition. In
2+ 2+ 2+ 2+ 2+ 3+
addition, Co , Cu , Ni , Zn , Mg , and Fe
2+
metal ions inhibited the enzyme while Ca ion
activated the enzyme. The fibrinase from kiwifruit
showed a great affinity towards human blood clot
lysis and could have future therapeutic applications.
Acknowledgements
The authors would like to thank Rasha Badawy for
her help.
Conflict of Interest statement
The authors declare that there are no conflicts of
interest.
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