Abstract
Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new fibrinogenolytic agents requires the separation of each component first and then checking their fibrinogenolytic activities. Currently, polyacrylamide gel electrophoresis (PAGE) and chromatography are mostly used in the separating stage. Meanwhile, the fibrinogen plate assay and reaction products based PAGE are usually adopted to display their fibrinogenolytic activities. However, because of the spatiotemporal separation of those two stages, it is impossible to separate and display the active fibrinogenolytic agents with the same gel. To simplify the separating and displaying processes of fibrinogenolytic agent identification, we constructed a new fibrinogen-PAGE method to rapidly separate and display the fibrinogenolytic agents of peanut worms (Sipunculus nudus) in this study. This method includes fibrinogen-PAGE preparation, electrophoresis, renaturation, incubation, staining, and decolorization. The fibrinogenolytic activity and molecular weight of the protein can be detected simultaneously. According to this method, we successfully detected more than one active fibrinogenolytic agent of peanut wormhomogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.
Introduction
In recent years, due to the continued rise of thrombotic diseases, thrombotic diseases have become a new major global health problem1. At present, antithrombotic drugs are classified into three groups: anti-platelet aggregation drugs, anticoagulants, and thrombolytic drugs. Among them, thrombolytic drugs, such as urokinase (UK), tissue plasminogen activator (tPA), etc., are the only clinically used drugs that can hydrolyze thrombus2. Meanwhile, more safe and effective thrombolytic drugs are being developed by the identification of novel thrombolytic agents3.
However, the identification of novel thrombolytic agents is time-consuming and labor-intensive, which mainly involves the separation/purification and characterization/checking stages. The former is to separate each component, and the latter is to display their fibrino(geno)lytic activities4,5. In previous studies, despite the fact we have successfully isolated a novel fibrino(geno)lytic enzyme (sFE) from the peanut worm (Sipunculus nudus) by affinity chromatography and fibrin(ogen) plate assay6,7,8, these processes are such time and labor-consuming. First, it is necessary to determine whether the peanut worm homogenates have fibrino(geno)lytic activity by the fibrin(ogen) plate method and reaction products based on PAGE9. Then, a series of chromatography, such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, and other methods, have to be carried out for separation and purification10,11. Subsequently, the fibrin plate assay is performed again to check the fibrinogenolytic activity of each separated component. Finally, native-PAGE and sodium dodecyl sulfate (SDS)-PAGE are performed to determine the molecular weight of the active fibrinogenolytic agents12. Therefore, it is critical to rapidly separate and display the active fibrinogenolytic agents.
To rapidly separate and display the active fibrinogenolytic agents in the peanut worm homogenates, a new fibrinogen-PAGE method was developed by combining the PAGE and fibrinogen plate methods, i.e., the substrates of fibrinogenolytic agents fibrinogen were added to the native-PAGE gel. After native-PAGE, the components were separated by their molecular weight. Meanwhile, each active fibrinogenolytic component can be displayed by staining. By this method, we successfully detected more than one active fibrinogenolytic agent of peanut worm homogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.
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Protocol
1. Peanut worm homogenate preparation
- Add 50 g of peanut worms and 150 mL of saline solution into the homogenizer.
- Homogenize at 24000 rpm for 60 s.
NOTE: Repeat 3 times. - Centrifuge the homogenate at 9710 x g for 30 min at 4 °C.
- Collect the supernatant as the peanut worm homogenate for further study.
2. Fibrinogen-PAGE gel preparation
- Weigh 0.01 g of fibrinogen into a 50 mL glass beaker.
- Add 1.9 mL of DDH2O, 1.3 mL of Tris-HCl (1.5 M, pH 8.8), and 0.05 mL of SDS (10%, w/v) into the beaker; mix well by pipetting.
- Place the beaker at 37 °C for 30 min.
NOTE: This step is to dissolve the fibrinogen. - Add 1.7 mL of acrylamide (30%, w/v), 0.05 mL of ammonium persulfate (APS; 10%, w/v), 0.002 mL of TEMED; mix well by pipetting.
NOTE: This is separating gel. - Pour the separating gel into a 10-well gel mold.
- Add 2 mL of DDH2O into the gel mold; wait for 30 min.
- Remove the water thoroughly by turning the mold upside down.
- Add 1.4 mL of DDH2O, 0.25 mL of Tris-HCl (1.0 M, pH 6.8), 0.02 mL of SDS (10%, w/v), 0.33 mL of acrylamide (30%, w/v), 0.02 mL of APS (10%, w/v), 0.002 mL of tetramethylethylenediamine (TEMED) into the beaker; mix well by pipetting.
NOTE: This is loading gel. - Pour the loading gel into the same gel mold as step 2.5; insert the comb immediately.
NOTE: The fibrinogen-PAGE gel used here was composed of the fibrinogen (0.2%) containing separating gel and loading gel. The concentration of fibrinogen can be adjusted as needed.
3. Electrophoresis
- Place the prepared fibrinogen-PAGE gel together with the glue mold into the electrophoresis tank; pour 1300 mL of electrophoresis solution (1.963 g of Tris, 12.22 g of glycine, 0.65 g of SDS, and 1300 mL of DDH2O) into the tank; take out the comb.
- Add 5 µL of 5x non-denaturing loading buffer to the 20 µL of sFE and 20 µL of peanut worm homogenate; mix well by pipetting; load them into the prepared fibrinogen-PAGE gel.
NOTE: Do not boil the mixture. sFE is a fibrino(geno)lytic enzyme identified previously6,7,8 and is used as a positive control in this study. Peanut worm homogenate is the sample to be detected. Non-denaturing loading buffer without bromophenol blue, β-mercaptoethanol, and SDS compared with the SDS-PAGE loading buffer. Each sample lane is separated with the 1x SDS-PAGE loading buffer to clearly detect the movement of the protein. - Perform electrophoresis at 80 V for 30 min, or 120 V for 20 min.
- Remove the fibrinogen-PAGE gel from the glue mold.
4. Renaturation
- Transfer the fibrinogen-PAGE gel to a plastic box.
- Add 100 mL of Tris-HCl (0.05 M, pH 7.4) and 2 mL of Triton X-100 into the box. Place it on a shaker at 60-70 rpm for 10 min.
- Remove the wash buffer by pipetting.
NOTE: Repeat three times.
5. Incubation
- Add 50 mL of Tris-HCl (0.05 M, pH 7.4) into the plastic box.
- Incubate at 37 °C for 4 h.
NOTE: Incubation time can be adjusted according to the strength of fibrinogenolytic activity.
6. Staining
- Remove the incubation buffer by pipetting.
- Add 50 mL of Coomassie Brillant Blue Staining solution into the box and place the box on a shaker at 60-70 rpm for 30 min.
7. Decolorization
- Remove the staining solution by pipetting.
- Add 50 mL of DDH2O into the box.
- Place the box on a shaker at 60-70 rpm for 30 min.
NOTE: DD H2O was used as the destaining solution here. The shaking time can be adjusted according to the strength of fibrinogenolytic activity.
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Representative Results
After electrophoresis, all the bands of the marker were clearly displayed. The 1x SDS-PAGE loading lanes only showed 10 kDa bands (bromophenol blue). The sFE and peanut worm samples did not show any observable bands (Figure 1). Although the bands of the samples are not visible, the performance of the marker and bromophenol blue indicated that the electrophoresis was successful, and the samples were separated according to their molecular weight.
Although the whole gel turned blue due to the staining, the decolorization made the area where fibrinogen was degraded by fibrinogenolytic agents transparent. Compared with the marker, the sFE indicated an active fibrinogenolytic band near 25 kDa. The peanut worm homogenate displayed 25 kDa, 33 kDa, 43 kDa, and a series of bands bigger than 70 kDa (Figure 2). The results indicated that sFE only contained a 25 kDa fibrinogenolytic component, but the peanut worm homogenate contained sFE components and other fibrinogenolytic agents. Supplementary Figure 1 is an example of a gel image showing the marker in deep blue and the target samples as bright bands.
Figure 1: Gel after electrophoresis. M: Marker; Lane 1: 1x SDS-PAGE loading buffer; Lane 2: sFE; Lane 3: 1x SDS-PAGE loading buffer; Lane 4: peanut worm homogenate; Lane 5: 1x SDS-PAGE loading buffer. Please click here to view a larger version of this figure.
Figure 2: Gel after decolorization. M: Marker; Lane 1: 1x SDS-PAGE loading buffer; Lane 2: sFE; Lane 3: 1x SDS-PAGE loading buffer; Lane 4: peanut worm homogenate; Lane 5: 1x SDS-PAGE loading buffer. Please click here to view a larger version of this figure.
Supplementary Figure 1: Gel image showing the marker in deep blue and the target samples as bright bands. M: Marker; Lane 1: 1x SDS-PAGE loading buffer; Lane 2: sFE; Lane 3: 1x SDS-PAGE loading buffer; Lane 4: peanut worm homogenate; Lane 5: 1x SDS-PAGE loading buffer. Please click here to download this File.
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Discussion
sFE is a novel fibrino(geno)lytic enzyme isolated from peanut worms by our team previously3,6,8,13. However, the identification processes of sFE were time- and labor-consuming, involving the fibrinogenolytic activity detection, protein components separation, and activity confirmation stages. As a simple method, fibrinogen plate assay is mostly used in the preliminary screening stage to check the fibrinogenolytic activity of the samples. However, the fibrinogen plate assay method cannot separate which component is active. Therefore, chromatography and native/SDS-PAGE have to be used to separate their components after activity detection. Then, reaction products are resolved again by the SDS-PAGE to confirm the degradation of substrate fibrinogen. In this work, the substrate fibrinogen was added to PAGE gel to detect the fibrinogenolytic agent. After electrophoresis, renaturation, incubation, staining, and decolorization, the molecular weight and fibrinogenolytic activity of the active fibrinogenolytic agents were clearly displayed with the same gel. By this method, we successfully detected more than one active fibrinogenolytic agent from peanut worm homogenate within 6 h.
To achieve the optimum results, more attention should be paid to the fibrinogen-PAGE gel preparation, renaturation, and incubation. Generally speaking, the lower the concentration of fibrinogen, the more colorless the fibrinogenolytic zone, which indicates fibrinogenolytic activity of the agents. However, the lower the concentration of fibrinogen, the more colorless the background, and the more difficult it is to distinguish the fibrinogenolytic zone from the background. The renaturation time will affect the fibrinogenolytic activity of components significantly. If this time is too short to wash off the SDS in the PAGE gel, the fibrinogenolytic activity of components will be inhibited by remanent SDS. Similarly, If the incubation time is too short, the fibrinogenolytic reaction is not thorough. While the incubation time is too long, the marker used to indicate the molecular weight will be washed away. In this study, although those conditions have been optimized, these conditions were set to separate and display the fibrinogenolytic agents from peanut worms. So, personalized conditions of those critical steps should be set according to specific fibrinogenolytic agents.
It is important to mention that this fibrinogen-PAGE is only suitable for screening the fibrinogenolytic agents with fibrinogen lytic activity but not for the agents with fibrinogen lytic activity or plasminogen activation activity. If we want to display those activities, the method can be optimized by replacing the fibrinogen with fibrin or plasminogen. Another point worth noting is that the active fibrinogenolytic agents tested in this study were monomers, which were not affected under the SDS electrophoresis condition. Therefore, if the tested agents are polymers, this method should be adjusted to a native-PAGE condition that can not display the molecular weight. Therefore, the fibrinogen-PAGE method introduced here was only a general basic version, which can be optimized to screen the other fibrinogenolytic agents quickly.
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Disclosures
The authors have no conflicts of interest to disclose.
Acknowledgments
This research was funded by the Science and Technology Bureau of Xiamen City (No. 3502Z20227197), the Science and Technology Bureau of Fujian Province (No. 2019J01070; No. 2022J01311), and High-level Talents Innovation and Entrepreneurship Project of Quanzhou Science and Technology Plan (No. 2022C015R). We thank Fucai Wang (Huaqiao University) and Lei Tong (Huaqiao University) for their technical assistance.
Materials
| Name | Company | Catalog Number | Comments |
| 1 M Tris-HCl (pH 6.8) | Solarbio | T1020 | |
| 1.5 M Tris-HCl (pH 8.8) | Solarbio | T1010 | |
| 30% Acrylamide/Bis-acrylamide | Biosharp | BL513B | |
| Ammonium persulfate | XiLONG SCIENTIFIC | 7727-54-0 | |
| Beaker | PYREX | 2-9425-02 | |
| Centrifuge Tube (1.5 mL) | Biosharp | BS-15-M | |
| Constant Temperature Incubator | JINGHONG | JHS-400 | |
| Coomas Brillant Blue Stainning solution | Beyotime | P0017F | |
| Electronic Analytical Balance | DENVER | TP-213 | |
| Fibrinogen | Solarbio | F8051 | |
| Gel loading pipette tips, Bulk | Biosharp | BS-200-GTB | |
| Homogenizer | AHS | ATS-1500 | |
| Horizontal rotation oscillator | NuoMi | NMSP-600 | |
| Milli-Q Reference | Millipore | Z00QSV0CN | |
| Mini-PROTEAN Tetra Cell | BIO-RAD | 165-8000~165-8007 | |
| N,N,N',N'-Tetramethylethylenediamine | Sigma | T9281 | |
| Pipette Tip (1 mL) | Axygene | T-1000XT-C | |
| Pipette Tip (10 µL) | Axygene | T-10XT-C | |
| Pipette Tip (200 µL) | Axygene | T-200XT-C | |
| Pipettor (1 mL) | Thermo Fisher Scientific | ZY18723 | |
| Pipettor (10µL) | Thermo Fisher Scientific | ZX98775 | |
| Pipettor (200 µL) | Thermo Fisher Scientific | ZY20280 | |
| Pipettor (50 µL) | Thermo Fisher Scientific | ZY15331 | |
| Refrigerated Centrifuge | cence | H1650R | |
| Sodium dodecyl sulfate | Sigma-Aldrich | V900859 | |
| Tris | Solarbio | T8060 | |
| Tris-HCl | Solarbio | T8230 | |
| Triton X-100 | Solarbio | T8200 |
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