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

Summary

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

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 activities^4,5. 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 assay^6,7,8, 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 purification^10,11. 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.

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…

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 …

Discussion

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…

Materials

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