Advances in Bioscience and Biotechnology, 2011, 2, 20-26 ABB
doi:10.4236/abb.2011.21004 Published Online February 2011 (http://www.SciRP.org/journal/abb/).
Published Online February 2011 in SciRes. http://www.scirp.org/journal/ABB
Enzyme electrophoresis method in analysis of active
components of haemostasis system
Ludmila Ostapchenko, Oleksiy Savchuk, Nataliia Burlova-Vasilieva
Educational and Scientific Centre “Institute of Biology” of National Taras Shevchenko University of Kyiv, Kyiv, Ukraine.
Received 9 December 2010; revised 16 January 2011; accepted 20 January 2011
ABSTRACT
The novel modifications of substrate-containing so-
dium dodecyl sulfate-polyacrylamide gel electropho-
resis that can be used for the detection of proteases
and its activators are reported. The protease/acti-
vator samples were separated on a protein substrate-
SDS-polyacrylamide gel. To detect plasminogen acti-
vators fibrinogen and Glu-plasminogen were incor-
porated into the SDS-PAG followed by 1 h incubation
at 37˚C in thrombin solution (1 NIH/ml). After elec-
trophoresis the gel was stained according to the
standard protocol. To detect fibrin-unspecific plasmi-
nogen activators from snake venom incubation in
thrombin solution was substituted for 12 h incubation
in 50 mM Tris-HCl (pH 7.4). To detect fibrinogen-
degrading enzymes fibrinogen-containing gel was
used. Activity of protease/activator was visualized in
the gel as clear bands against the dark background.
These new techniques offer several advantages in-
cluding determination of the quantity and activity of
t-PA and urokinase, however cannot be recommended
for precise quantification of activators; the total pro-
cedure is quite quick and simple; method is conven-
ient tool for detection of novel protein-protein inter-
actions in haemostasis system; the sensitivity of the
method is ≤0.01 IU per track.
Keywords: Substrate-Containing Electrophoresis;
Enzyme Electrophoresis; Haemostasis; Proteins Activity;
Proteins Identification
1. INTRODUCTION
Protein electrophoresis is a convenient approach to
characterize sample composition, protein interactions,
purity, molecular weight, isoelectric point, and to purify
small amounts of protein for further analysis. Different
modifications of this widely used method have been
developed to suit a variety of purposes [1].
Erickson [2] and Brunner [3] offered fibrin autogra-
phy to detect protease activators/inhibitors previously
fractionated by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE). After separation the
proteins and the substrate were transferred electropho-
retically into the fibrin indicator gel. The positions of
activators/inhibitors were revealed by the formation of
contrast fibrinolytic or lysis-resistant zones.
Hanspal et al. [4] described a technique to detect the
activity of protease inhibitors present in enzyme sub-
strate-containing sodium dodecyl sulfate polyacrylamide
gel (SDS-PAG). The method involved 1) incorporation of
substrate (gelatin or casein) into the SDS-PAG at the
time of casting; 2) electrophoresis of the protease in-
hibitors in the presence of SDS; 3) removal of SDS by
washing the gel in 2.5% (w/v) Triton X-100; 4) incuba-
tion of the gels in a solution containing the proteolytic
enzyme at 37 degrees C for 16 h; and 5) staining undi-
gested substrate with amido black.
Sensitive methods for detecting proteases/protease in-
hibitors by using fluorescent protease substrates in gels
are reported [5,6]. Wilkesman and Schroder [7] used 2-D
zymography, a technique that combines IEF (isoelectric
focusing) and zymography. Procedures including high-
molecular-mass substrates within the gel, such as starch
for identification of amylase activity, and protein sub-
strates, such as gelatin, casein, and collagen, for reveal-
ing protease/protease inhibitors activity, have been de-
scribed [8-10].
There are several features of enzyme activity deter-
mination including protection of protein functional ac-
tivity. This provides the possibility of enzyme identifica-
tion after all biochemical manipulations. Although, it is
not possible to save 100% of functional activity, how-
ever most of researchers succeeded to save nativity of
the protein (including its activity) at the level which is
sufficient authentic and adequate identification.
In the present study we report 1D SDS-PAG enzyme
electrophoresis method. The technique is optimized for
identification of proteases/protease activators of haemo-
stasis system in the blood plasma or other tissue samples.

Ostapchenko L. I. et al. / Advances in Bioscience and Biotechnology 2 (2011) 20-26 21
The major advantage of the method is the possibility to
detect active plasminogen activators – tissue-type plas-
minogen activator (t-PA) and urokinase which simplifies
analytical work [11-13]. Enzyme electrophoresis can be
used as a rapid diagnostic method as gives information
not only about the amount and MW of proteins but also
reveals active forms of t-PA and urokinase.
Fibrinogen or (fibrinogen and plasminogen) was in-
corporated into the gel as a substrate for proteases. The
convertion of fibrinogen to fibrin under thrombin treat-
ment provides conditions for fibrin-dependent activators
to generate plasmin causing the background substrate
degradation [14].
2. MATERIALS AND METHODS
Chemicals and proteins. Tris, glycine, SDS, acrylamide,
bisacrylamide, ammonium persulfate (APS), N,N,N’,N’-
tetramethylethylenediamine (TEMED), sucrose, all of
analytical grade, were purchased from Amersham Bio-
sciences AB (Sweden). t-PA, urokinase and elastase
were obtained from Sigma, Germany. Streptokinase was
supplied by Kabi Pharmacia AG, Sweden.
Sample preparation. Mouse blood plasma from Lewis
carcinoma line С57В1/6 was received from Kavetskiy
Institute of experimental pathology, oncology and radio-
biology of the National Academy of Siences of Ukraine.
Subretinal fluid isolated by surgical operation from
patients with regmatogenic retina exfoliation was re-
ceived from Filatov Institute of eye diseases and tissue
therapy of the Academy of Medical Sciences of Ukraine.
The crystalline snake venom of Agkistrodon halys
halys was received from serpentarium of Tripolskiy
biochemical plant, Ukraine.
All samples were mixed with treatment buffer in the
ratio 1:1 (v/v) and stored before electrophoresis at 4˚C.
The treatment buffer was made ready according to
Amersham Biosciences protocol [15] with modifications:
1) glycerine was replaced by sucrose to the final
concentration 5% and 2) DTT was not added to prevent
the loss of enzyme activity. The obligatory condition for
enzyme electrophoresis samples preparation was non-
thermal treatment of the samples before separation to
avoid the loss of enzyme activity.
3. ENZYME ELECTROPHORESIS
We have developed a technique on the basis of the
method described by Heusen C. and Dowdle E., [16]
with following modifications: fibrinogen (1 mg/mL) or
(fibrinogen (1 mg/mL) and Glu-plasminogen (10 mkg/
mL) was incorporated into separating PAG. Fibrinogen
was used to detect proteases capable of fibrinogen
cleavage (in this study plasminogen and mini plasmino-
gen, see Example 4, Figures 4(a) and 5). Fibrinogen
and plasminogen were incorporated into SDS-PAG for
plasminogen activators detection. After separation the
gel was incubated in thrombin solution (1 NIH/mL) for 1
h at 37˚C. Fibrin formation was required for develop-
ment of the fibrin-dependent plasminogen activators (t-
PA and urokinase) activity [17]. t-PA or urokinase ap-
peared as the clear bands, corresponding to the area
where plasmin has degraded fibrin. The separation gel
concentration can vary from 11% to 15% to prevent mi-
gration of incorporated proteins during electrophoresis.
The technique involved: 1) incorporation of fibrino-
gen or (fibrinogen and plasminogen) into the SDS-PAG
of required concentration; 2) electrophoretic separation
under usual conditions [15]; 3) gel washing in 2.5% Tri-
ton Х-100 with shaking for 1 h at 25˚C for SDS removal;
4) for (fibrinogen and plasminogen)-incorporated gel
—treatment with thrombin solution with shaking for 1 h
at 37˚C; 5) proteins visualization according to standard
protocol [15].
For electoforesis performing and gel staining proce-
dures Hoefer Mighty Small system and Hoefer Processor
Plus (Amersham Biosciences AB, Sweden) were used.
The sensitivity of the method was ≤0.01 IU of activa-
tor or protease per track.
4. WESTERN BLOTTING
Procedure was performed according to the protocol [17].
Proteins were transferred to a nitrocellulose membrane
for 1 h at 4˚C and 60 MА in 15 mМ Tris-HCl, рН 8.4
with 120 mМ glycine and 20% methanol. The mem-
brane was stained with 0.1% Ponso “Sigma” in 5% ace-
tic acid with shaking for 30 min followed by overnight
incubation in 5% BSA at 4˚C. Proteins were probed
using monoclonal antibody (MAb) directed against
plasminogen (Merck KGaA, Germany) in dilution
1:1000 and secondary antibody (1:3000) labeled with
alkaline phosphatase. Each procedure was followed by
rinsing step with 3 buffer substitutions. The washing
buffer consisted of 50 mM Tris-HCl with 0.1% Twin-20.
The blot was developed using 5-bromo-4-chloro-3-
indolyl phosphate.
5. PROTEINS PURIFICATION
Human Glu-plasminogen was purified from citrate-
anticoagulated blood plasma by affinity chromatography
on Lys-Sepharose [18]. The citrate-anticoagulated blood
plasma was diluted 1:1 with 50 mM sodium-phosphate,
pH 7.4 containing aprotinin (1000 IU/L), filtered and
loaded onto Lys-Sepharose column previously equili-
brated in the same buffer lacking aprotinin. After loading
the column was washed with 50 mM sodium-phosphate,
pH 7.4 with 200 mМ NaCl, and eluted with 150 mМ
6-aminohexacapronic acid in the same buffer. By adding

Ostapchenko L. I. et al. / Advances in Bioscience and Biotechnology 2 (2011) 20-26 22
ammonium sulfate (41 mg/ml), crude plasminogen was
precipitated from the eluate and gel-filtrated using
Sephacryl S-200 column. Purified plasminogen was
stored frozen at –20˚C until used.
Activation of plasminogen was performed on a
column loaded with insolubilized urokinase. [19]. Glu-
plasminogen was incubated within the column for 1 h at
37˚C in 50 mМ Tris-НCl, pH 7.4 with 150 mM NaCl
and 25% glycerol. The purity of plasmin was controlled
by SDS-PAGE and found to be homogenous. The pro-
tein was stored frozen at –20˚C in 50% glycerol.
Mini-plasmin was obtained according to the method
described by March, Parikh and Cuatrecasas [20]. Glu-
plasminogen (10 mg per 1 ml) was hydrolyzed with
pancreas elastase 1:50 (v/v) in 50 mМ Tris-HCl, pH 8.5
with 100 mМ NaCl for 5 h at 25˚C. The reaction was
stopped by adding of pNFGB to final concentration 10
mМ. The hydrolyzate was loaded onto Superdex 75 for
mini-plasminogen separation from cryngls 4 and 1-3.
The purified mini-plasminogen was lyophilized and
stored at –20˚C. The purity of mini-plasminogen was
controlled by SDS-PAGE and found to be homogenous.
6. RESULTS AND DISCUSSION
Human, rabbit, bovine, porcine and mouse haemostasis
proteases/protease activators were visualized through
this method. To provide optimal results fibrinogen and
plasminogen of target mammal should be used for in-
corporation. However, authors examined usage of hu-
man plasminogen and fibrinogen as a background sub-
strate for haemostasis proteases of mammalian species
listed above. This significantly simplifies the analysis
[21-25].
Advantages and possibilities of enzyme-electro-
phoresis method are shown in four different cases. In the
present study authors did not bring out data of alterna-
tive techniques of haemostasis system analysis. Previous
reports have confirmed results obtained by enzyme-
electrophoresis, references to original studies are indi-
cated in the text and included to the list.
Example 1: The increased risk of acute myocardial
infarction (AMI) is associated with reduced fibrinolytic
activity. Increased levels of thrombin-antithrombin III
complex, fibrinopeptide A, prothrombin fragment F1 + 2
and D-dimer are detectable in patients affected by
thrombosis. [26-30]. The level of t-PA antigen is increased
but associated with decreased t-PA activity [31-33].
For patients who had AMI, we documented an in-
creased fibrinolytic potential after streptokinase (Sk)
administration [21]. To determine the reason of plasmin
formation we used rabbit and porcine models of throm-
bolytic therapy. Whereas Sk activates human and rabbit
plasminogen, it fails to activate porcine. This allowed us
to distinguish plasmin and Sk effects. The total plasmi-
nogen activators were determined using enzyme-
electrophoresis. (Figures 1(a), (b)). PAG was prepared
in the presence of both fibrinogen and Glu-plasminogen.
After separation the gel was treated with thrombin. Ac-
tivity of plasminogen activators was visualized as bands
cleared from fibrin by the activated proenzyme included
to the gel.
Figure 1(a) demonstrates that rabbit blood plasma
contains proteins with fibrinolytic activity and molecular
weights of 82, 70 and 54 kDa, which correspond to
plasmin, t-PA and two-chain urokinase-type plasmino-
gen activator (tcu-PA). Increment of plasmin and t-PA
activities was visible 1 hour after streptokinase admini-
stration. Porcine plasma contained the same proteins, but
54 kDa band was barely visible indicating the trace
amount of tcu-PA in the sample (Figure 1(b)). t-PA
activity was significantly increased 1 hour after throm-
bolytic agent administration. This indicates that t-PA
activity is associated with Sk independently of plasmin
formation.
(a)
(b)
Figure 1. Enzyme electrophoregram of rabbit (a)
and porcine (b) blood plasma: 1) control (before
streptokinase administration); 2) 1 h after strep-
tokinase administration; 3) 4 h after strepto-
kinase administration; 4) 1 day after strepto-
kinase administration; 5) 3 days after strepto-
kinase administration; 6) 7 days after strepto-
kinase administration; 7) t-PA standard (MW 70
kDa); 8) urokinase standard (MW 56, 33 kDa); 9)
plasmin standard (MW 82 kDa).

Ostapchenko L. I. et al. / Advances in Bioscience and Biotechnology 2 (2011) 20-26 23
Example 2: Snake venoms are complex mixtures
containing many different biologically active proteins
and peptides. A number of these proteins affect the
blood coagulation pathway, endothelial cells, and plate-
lets [34]. Several venom enzymes have been used as
anticoagulants and other are under examination of their
possible therapeutic potential. We used enzyme-
electrophoresis to detect potential plasminogen activator
in Agkistrodon halys halys venom. For this purpose fi-
brinogen and plasminogen were incorporated into the
PAGE. The mixture of previously purified activator and
plasminogen 1:1 (q/q) was used as a sample. The gel
was not treated with thrombin but after SDS on Triton
X-100 substitution was incubated for 12 hours in 50 mM
Tris-HCl, рН 7.4 for development of plasmin activity
due to action of potential activator. In the analyzed sam-
ple (Figure 2) two bands were found. One of them cor-
responded to 82 kDa plasmin, which had been formed
under activator action. The second band corresponded to
MW of activator itself (32 kDa), its appearance was pro-
vided by plasminogen incorporation. These results sug-
gest that induction of plasmin formation by the snake
venom activator was specific and involved a bond
cleavage at a specific site in the plasminogen molecule
without formation of active fragments.
These results were completely confirmed using spe-
cific chromogenic substrate for plasmin [22].
Example 3: To detect components of plasminogen ac-
tivation system in the subretinal fluid of patients with
regmatogenic retina exfoliation enzyme-electrophoresis
was performed. This substance is accumulated in the
Figure 2. Enzyme electrophoregram
of plasminogen and plasminogen ac-
tivator from snake venom mixture: 1)
plasminogen and plasminogen acti-
vator mixture; 2) plasmin standard
(MW 82 kDa); 3) plasminogen stan-
dard used for incubation and incorpo-
ration.
cavity formed by pathologic retina exfoliation. It was
shown that subretinal fluid possessed fibrinolytic activity
and included an activator capable of plasminogen trans
formation. To determine the nature of this activator (or
activators) fibrinogen and plasminogen-incorporated gel
was used. After separation and SDS removal the gel was
incubated in thrombin for fibrin formation. This step was
necessary due to fibrin-dependent nature of t-PA and
urokinase activities. The 54 kDa band was revealed at
the electrophoregram (Figure 3(a)) and confirmed the
existence of tcu-PA in the sample.
Previous reports have confirmed results obtained by
substrate-incorporated electrophoresis [23,35].
Example 4: Production of elastase is significantly
enhanced in tumor cells leading to formation of plasmi-
nogen internal fragments (angiostatine that consists of
kringles 1 to 4 and approximately 85% of kringle 5) [36-
39]. Unbound plasminogen/plasmin cringles are biologi-
cally active molecules that act on distant sites affecting
fibrinolytic efficiency [39-42].
Using enzyme-electrophoresis method we analyzed
proteases and plasminogen activators composition in the
blood plasma of Lewis carcinoma mice (Figure 4(a)).
The analysis of haemostasis system during Lewis carci-
noma growth is reflected in the study [24].
Fibrinogen and Glu-plasminogen were incorporated
into the PAG. After separation of the samples and SDS
removal the gel was incubated in thrombin solution for
fibrinogen transformation. Appearance of active bands
revealed plasminogen activators and proteases capable
of fibrin cleavage. As migration pattern of the size (Mr)
standards elastase (27, 29 and 31 kDa), urokinase (33
and 56 kDa), tPA (70 kDa) and mini-plasmin (36 kDa)
were used. The resulting electropherogram for this case
Figure 3. Enzyme electropho-
regram of subretinal fluid: 1)
subretinal fluid; 2) t-PA stan-
dard (MW 70 kDa); 3) uroki-
nase standard (MW 56, 33 kDa).

Ostapchenko L. I. et al. / Advances in Bioscience and Biotechnology 2 (2011) 20-26 24
(a)
(b)
Figure 4. (a) Enzyme electrophoregram of plasminogen
activators and active proteases from Lewis carcinoma
mice blood plasma: 1) elastase standard (MW 31, 29, 27
kDa); 2) urokinase standard (MW 56, 33 kDa); 3) t-PA
standard (MW 70 kDa); 4-11) blood plasma samples; 12)
miniplasmin standard (MW 36 kDa); (b) A Enzyme
electrophoregram of active proteases capable of fibrino-
gen cleavage from Lewis carcinoma mice blood plasma:
1) control blood plasma; 2-9) Lewis carcinoma mice
blood plasma samples; 10) standards: A-plasmin (MW
82 kDa), B-miniplasmin (MW 36 kDa).
is shown on the Figure 4. All samples displayed an ac-
tive zones with MW of 31 kDa, which corresponded to
elastase, 36 kDa (mini-plasmin), 70 kDa (t-PA) and 33
kDa (urokinase). Some samples contained high molecu-
lar weigh urokinase (band with MW of 56 kDa).
In order to detect active proteases capable of fibrino-
gen cleavage, incorporation of plasminogen and fibrino-
gen into fibrin transformation steps were excluded. This
analysis of Lewis carcinoma mice blood plasma revealed
active zones with MW corresponding to plasmin (82
kDa) and mini-plasmin (36 kDa). To confirm our results
western-blot with MAb directed against plasminogen [43]
was performed (Figure 5). Data obtained by western
blotting testified that enzyme electrophoresis could be
used for plasmin and mini-plasmin detection.
Figure 5. Western-blot of Lewis carcinoma
mice blood plasma with MAb directed against
plasminogen: 1) plasmin standard (MW 82
kDa); 2) miniplasmin standard (MW 36 kDa);
3) blood plasma sample.
7. CONCLUDING REMARKS
Our findings indicate that enzyme-electrophoresis meth-
od shows reliable results for identification of active t-PA
and urokinase. The technique can be used for studying of
protease composition and protein-protein interactions in
haemostasis system. The total procedure is quite quick
and simple and can be recommended as alternative
medical diagnostic method used for the rapid assessment
of plasma fybrinolytic potencial.
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