Advanced Materials Research Online: 2012-09-28

ISSN: 1662-8985, Vol. 569, pp 789-794

doi:10.4028/www.scientific.net/AMR.569.789
© 2012 Trans Tech Publications Ltd, Switzerland

A Study of Fibrin Zymography Method for the Assay of Plasminogen

Activators
Ming Xing Huang1*, Xiao Qian Yu2, Yun Ye1
1
Department of Biological Engineering, Faculty of Chemical Engineering and Light Industry,
Guangdong University of Technology, Guangzhou 510006, China
2
Haikou Qili Pharmaceutical Co. Ltd., Haikou, 570100, China
*Corresponding author. Fax: +86-20-3932-2428
E-mail address: mingxing19828282@gmail.com, jobjobye@126.com

Keywords: Fibrin zymography, Plasminogen activators, Fibrinogen, Urokinase, Protein molecular

weight marker

Abstract. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is the most
important and widely used technology which is mainly used to analyze the protein molecular weight.
Fibrin zymography based on the SDS-PAGE is the best method for qualitative analysis of unknown
plasminogen activators (PAs), especially for the analysis of molecular weight. In electrophoresis
technique, molecular weight marker is the most important factor. However, it is difficult to detect
protein molecular weight markers in fibrin zymography. In this study, some important factors, such
as concentrations of fibrinogen and plasminogen, are discussed. Our results provide an efficient and
convenient method which can clearly exhibit the dark blue bands of protein molecular weight (MW)
markers and the transparent bands of PAs against the light blue background on one gel at the same
time, and show high sensitivity.

Introduction
PAs are serine proteases of tryptic specificity which can convert the proenzyme, plasminogen, into
an active enzyme plasmin [1,2]. For the assay of PAs activity, four techniques have been established.
Fibrin plate method [3,4] and colorimetric assay using chromogenic substrates [5,6] are two early
methods which can be used for semi-quantitative analysis. Reverse fibrin autography [7] and fibrin
zymography [8,9] are two new qualitative techniques which can be used to detect the molecular
weight or isoelectric point of unknown PAs. The major drawback to the reverse fibrin autography is
that it is time-consuming and has low sensitivity for enzymatic activity as well [8]. The fibrin
zymography based on the SDS-PAGE, in which the polyacrylamide gel is copolymerized with the
fibrin substrate which can be degraded by the plasmin, is more convenient and sensitive than the
reverse fibrin autography. However, the original researchers could not detect protein MW markers
on gel. So this method just can be used to analyze how many kinds of PAs are contained in gross
sample. The molecular weights of PAs cannot be estimated. In this report, modifications have been
described in order that the dark blue bands of protein MW markers and the transparent bands of PAs
can be exhibited against the light blue background on one gel at the same time.

Materials and methods

Materials. Powder of Thrombin, fibrinogen and urokinase (Sigma) was separately dissolved in 50
mM PBS (pH 7.2) with 0.15 M NaCl according to the concentration of Table 1, and then preserved
at -80℃.

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790 Advanced Materials Design and Mechanics

Preparation of fibrin zymography separating gel. Components of fibrin zymography

separating gel are given in Table 1. Separating gel solution (12%) containing fibrinogen was
prepared in a total volume of 10 ml. Every component should be added in proper order. Firstly,
Bisacrylamide-Acrylamide, tris-Cl buffer, bovine thrombin and distilled water were mixed.
Secondly, bovine fibrinogen and SDS were added into the above mixture. Finally, Ammonium
persulfate and tetramethylethylenediamine (TEMED) were added to the gel solution in final
concentrations of 1% and 0.04% (v/v) respectively.
Electrophoresis and staining. Electrophoresis followed the method of Laemmli [10] using the
electrophoresis cell of DYCZ-24DN (Beijing WoDelife Sciences Instrument Company, China). The
separating and stacking gel were 1.5 mm. Electric voltage were 30 V and 120 V for stacking gel and
separating gel respectively.
After electrophoresis, the separating gel was cut down and soaked into washing buffer (50 mM
Tris, pH 8.0; 2.5% Triton X-100) for 60 min. Subsequently, the separating gel was incubated in
reaction buffer (50 mM PBS, pH 7.2; 0.15 M NaCl) at 37℃ for 18 hours and stained by Coomassie
brilliant blue solution (0.25% Coomassie brilliant blue R250, 45% methanol, 5% acetic acid) for 8
hours. The dyed gel was destained by destaining solution (45% methanol, 10% acetic acid) until the
dark blue bands of protein molecular weight markers and the transparent bands of PAs against the
light blue background were clearly exhibited. The gel was scanned by GS-800™ Calibrated
Densitometer (BIO-RAD).

Table 1 Components of SDS-fibrin zymography separating gel (12%)

Concentration of
fibrinogen (w/v) 0.025% 0.05% 0.075% 0.1%
Volume [ml]
Bisacrylamide-
Acrylamide(1%:29%) 4 4 4 4
1.5 M Tris-Cl buffer
(pH 8.8) 2.5 2.5 2.5 2.5
Bovine thrombin
(1 NIH unit/ml) 1 1 1 1
Bovine fibrinogen
(0.01 g/ml) 0.25 0.5 0. 75 1
SDS (10%) 0.1 0.1 0.1 0.1
Ammonium persulfate
(10%) 0.1 0.1 0.1 0.1
TEMED 0.004 0.004 0.004 0.004
Distilled water 2.05 1.8 1.55 1.3
Total volume 10 [ml]

Results and discussion

The relationship between concentration of fibrinogen and sensitivity. Four dose levels of
fibrinogen were prepared in order to test the relationship between concentration of fibrinogen and
sensitivity. Under each dose level of fibrinogen, six dose levels of urokinase with a two-fold
interval were tested. 0.39µU to 12.5µU (about 0.78 ng to 25 ng) urokinase in sample loading buffer
 Advanced Materials Research Vol. 569 791

without reducing agent was electrophoresed following the method of Laemmli, which was followed
by soaking the separating gel into washing buffer (50 mM Tris, pH 8.0; 2.5% Triton X-100) for 60
min. Subsequently, the separating gel was incubated in reaction buffer (50 mM PBS, pH 7.2; 0.15
M NaCl) at 37℃ for 18 hours and dyed by Coomassie brilliant blue. The result shows that the
sensitivity has little change as the decreasing of the fibrinogen concentration. The breadth of the
transparent bands can keep coincidence under each dose level of fibrinogen, but the bands under
low fibrinogen concentration cannot be seen clearly because of the low contrast of background
color. In order to have a great visual effect, we recommend that the concentration of fibrinogen
should not be less than 0.075%. Figure 1 shows the result of 0.075% fibrinogen. The detectable
amount of urokinase is as low as 0.39μU (about 0.78 ng). The sensitivity of detecting urokinase is
as about ten folds as detecting plasmin (detectable amount is 6.3 ng) by the fibrin zymography
method [8], mainly due to the cascade amplification of enzymatic reaction. When the amount of
urokinase is greater than 50 ng, the transparent bands would become dispersed, which is similar to
streptokinase described by choi et al. [9].

Fig. 1 SDS-fibrin zymography of urokinase in 12%

polyacrylamide gel with 0.075% fibrinogen. Lane 1-6,
12.5µU (about 25 ng) to 0.39µU (about 0.78 ng)
urokinase with a two-fold interval.

SDS-fibrin zymography of protein molecular weight markers. One main aim of our study is
to exhibit the bands of protein MW markers and the transparent bands of PAs on one gel at the same
time, in order that the molecular weight of unknown PAs contained in total protein can be
determined precisely. It can be achieved by decreasing the background color and increasing the
amount of protein MW markers. Under each dose level of fibrinogen, five dose levels of protein
MW markers with a half-nanogram interval for each band were tested. The protein MW markers
(TaKaRa) contain phosphatase b from rabbit muscle (97 kDa), bovine serum albumin (66 kDa), egg
albumin (44 kDa), bovine carbonate glycosidase (29 kDa), trypsin inhibitor from soybean (20 kDa)
and lysozyme from egg white (14 kDa). 0.5µg to 2.5µg protein MW markers for each band in
sample loading buffer without reducing agent were electrophoresed as described above.
Subsequently, the separating gel was dyed by Coomassie brilliant blue without undergoing the
washing and the reaction steps. Figure 2 shows that the sensitivity obviously decreased as the
increasing of the fibrinogen concentration. In order to have great sensitivity, we recommend that the
concentration of fibrinogen should not be more than 0.075%. Under low fibrinogen concentration,
transparent bands of urokinase cannot be clearly seen because of the low contrast of background
color. On the contrary, the sensitivity of detecting protein MW marker is low under high fibrinogen
concentration. When the concentration of fibrinogen is greater than 1%, the bands of protein MW
markers can hardly be shown, though faintly perceptible by eyes just as description by Kim et al. [8].
On the other hand, the mass of protein MW marker has effect on the result. When the protein MW
marker is less than 1μg for each band, it is hard to see bands on gels under any dose level of
fibrinogen.
792 Advanced Materials Design and Mechanics

Fig. 2 SDS-fibrin zymography of protein MW markers in

12% polyacrylamide gel under four dose levels of fibrinogen
After non-denaturing electrophoresis, the gels were dyed
directly without any other procedures. (A), 0.025% fibrinogen;
(B), 0.05% fibrinogen; (C), 0.075% fibrinogen; (D), 0.1%
fibrinogen; Lane 1-5, 0.5μg to 2.5μg protein MW marker
with a half-nanogram interval for each band.

SDS-fibrin zymography of urokinase and protein molecular weight markers on one gel.
When the urokinase and protein MW markers in sample loading buffer without reducing agent were
electrophoresed on one gel, followed by soaking the separating gel into the washing buffer and
reaction buffer sequentially as described above, the result was not as expected. Some bands of
protein MW marker (bovine serum albumin, egg albumin and trypsin inhibitor from soybean) were
fuzzy, or even disappeared (Fig. 3). Because the sample loading buffer was not added with reducing
agent, the disulfide bonds of protein were not broken. When the SDS was removed from the gel by
the washing buffer, protein refolded in the reaction buffer. These active protein MW markers may
interact with the components of gel. Because the trypsin inhibitor can interact with the serine
protease, such as plasminogen/plasmin, the 20 kDa band of trypsin inhibitor from soybean
disappeared. The reason why the two bands of bovine serum albumin and egg albumin were so
fuzzy is not clear.

Fig. 3 SDS-fibrin zymography of urokinase and protein MW

markers in 12% polyacrylamide gel with 0.075% fibrinogen.
Lane 1, 12.5μU (about 25 ng) urokinase; Lane 2-6, 0.5μg to
2.5μg protein MW marker with a half-nanogram interval for
each band.

In order to prevent protein MW markers from reacting with some components of gel, protein
MW markers were denatured by adding 0.02 M dithiothreitol into the sample loading buffer before
electrophoresis. Urokinase without denaturing and denatured protein MW markers were
electrophoresed on one gel, followed by soaking the separating gel into the washing buffer and
reaction buffer sequentially as described above. Figure 4 shows that most of the bands of protein
MW markers and the transparent bands of urokinase can be exhibited clearly on the gel. One
problem is that the crosslinked fibrin on the top of the separating gel can be broken by reducing
agent. Because these fragments of fibrin can migrate downwards under the electric field, a part of
blue background disappears and several extra blue bands appear on the top of the separating gel.
The band of bovine serum albumin can not be distinguished under the influence of these extra blue
bands.
Based on the above analysis, the method of denaturing protein MW markers is not recommended.
We can not get perfect background and bands, but the electrophoretic mobility of protein MW
markers under denaturing condition will change to some extent. The method that both PAs and
protein MW markers are electrophoresed under non-denaturing condition may be much better, but
the prerequisite is that the protein MW markers must be screened finely. Phosphatase b from rabbit
muscle, bovine carbonate glycosidase and lysozyme from egg white are some good choices, but
 Advanced Materials Research Vol. 569 793

bovine serum albumin, egg albumin and trypsin inhibitor from soybean are not suitable. Another
good strategy is cutting down the lane of protein MW markers from the gel that contains some lanes
of PAs after non-denaturing electrophoresis, and subsequently dyed directly without undergoing the
washing and the reaction steps.

Fig. 4 SDS-fibrin zymography of urokinase and denatured

protein MW markers in 12% polyacrylamide gel with 0.075%
fibrinogen. Lane 1, 1.56μU (about 3.125 ng) urokinase; Lane
2-6, 0.5μg to 2.5μg protein MW marker with a half-nanogram
interval for each band.

For some unknown PAs which will be purified from total protein, the fibrin zymography is the
best method for preliminary qualitative analysis, especially for molecular weight analysis. Although
the plasminogen concentration may be important to quantitative analysis of PAs by fibrin plate
method [11], it has little influence on the fibrin zymography. The plasminogen is from the
contaminant of the fibrinogen [12], but it is enough for this analysis. Walton [13] took the same
strategy for the study of fibrin plate method.

Conclusions
In this study, some important factors, such as concentrations of fibrinogen and plasminogen, are
discussed. Our results provide an efficient and convenient method which can clearly exhibit the
dark blue bands of protein molecular weight (MW) markers and the transparent bands of PAs
against the light blue background on one gel at the same time, and show high sensitivity. The
detectable amount of urokinase is as low as 0.39μU (about 0.78 ng). In order to have great
sensitivity, we recommend that the concentration of fibrinogen should be 0.05%. The method of
denaturing protein MW markers is not recommended, and the method that both PAs and protein
MW markers are electrophoresed under non-denaturing condition is much better. Phosphatase b
from rabbit muscle, bovine carbonate glycosidase and lysozyme from egg white are some good
choices as protein MW markers, but bovine serum albumin, egg albumin and trypsin inhibitor from
soybean are not suitable. Comparing with the method of Choi et al. [9], our method has the
following modifications: 1) None additional plasminogen was added into the separating gel; 2) The
final concentration of thrombin was increased to 0.1 NIH unit/ml instead of 0.01 NIH unit/ml; 3)
The final concentration of fibrinogen was decreased to some extent (0.025%-0.1%).

Acknowledgement
This work was supported by the key science and technology projects of Haikou, “novel agent for
cardiovascular disease——The construction of engineering strain with fibrinolytic enzyme of
yellow mealworm and the activity analysis of expressed product” (NO. 2011-0044).
794 Advanced Materials Design and Mechanics

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