Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis

Here, we present a fibrinogen-polyacrylamide gel electrophoresis (PAGE) protocol to rapidly separate and display the fibrinogenolytic agents of Sipunculus nudus.

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.

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, th....

1. Peanut worm homogenate preparation

1. Add 50 g of peanut worms and 150 mL of saline solution into the homogenizer.
2. Homogenize at 24000 rpm for 60 s.
   NOTE: Repeat 3 times.
3. Centrifuge the homogenate at 9710 x g for 30 min at 4 °C.
4. Collect the supernatant as the peanut worm homogenate for further study.

2. Fibrinogen-PAGE gel preparation

1. Weigh 0.01 g of fibrinogen into a 50 mL glass.......

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 .......

sFE is a novel fibrino(geno)lytic enzyme isolated from peanut worms by our team previously^3,6,8,13. 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 th.......

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.