fibrinolytic activity of the fibrinolytic enzyme purified from sipunculus nudus

Purificação de uma enzima fibrinolítica de Sipunculus nudus via cromatografia de afinidade

Thrombosis is a major threat to public health, especially following the Covid-19 global pandemic1,2. Clinically, many plasminogen activators (PAs), such as tissue-type plasminogen activator (tPA) and urokinase (UK), have been widely used as thrombolytic drugs. PAs can activate patients&#39; plasminogen into active plasmin to degrade fibrin. Thus, their thrombolytic efficiency is heavily restricted by the patients&#39; plasminogen status3,4. Fibrinolytic agents, such as metalloproteinase plasmin and serine plasmin, are another type of clinical thrombolytic drug that also include fibrinolytic enzymes (FE) such as plasmin, which can dissolve clots directly but are quickly inactivated by various plasmin inhibitors5. Subsequently, a novel type of fibrinolytic agent has been reported that can dissolve the thrombus by not only activating the plasminogen into plasmin but also degrading the fibrin directly6-the fibrinolytic enzyme from the ancient peanut worm Sipunculus nudus (sFE)6. This bifunction endows sFE other advantages over traditional thrombolytic drugs, especially in terms of abnormal plasminogen status. Compared with other bifunctional fibrinolytic agents7,8,9, sFE displays several advantages, including safety, over non-food derived agents for drug development, especially for oral drugs. This is because the biosafety and biocompatibility of Sipunculus nudus have been well-established10.
Similar to the other natural fibrinolytic agents isolated from microorganisms, earthworms, and mushrooms, the purification of sFE from S. nudus is very complicated and includes multiple stages, such as tissue homogenization, ammonium sulphate precipitation, desalination, anion-exchange chromatography, hydrophobic interaction chromatography, and molecular sieving10,11,12. Such a purification system not only depends on proficient skills and expensive materials, but also requires several days to complete the whole procedure. Therefore, a simple purification program of sFE is of great significance for the further development of sFE. Fortunately, two crystals of sFE (PDB: 8HZP; PDB: 8HZO) have been successfully obtained (see Supplemental File 1 and Supplemental File 2). Through structural analysis and molecular docking experiments, we found that the catalytic core of sFE could specifically bind to targets containing arginine or lysine residues.
Herein, an affinity purification system was proposed for the first time, based on the crystal structure of sFE. By following this protocol, highly pure and highly active sFE could be purified from the crude extracts in a single affinity purification stage. The protocol developed here is not only important for the large scale preparation of sFE, but also may be applied for the purification of other fibrinolytic agents.

Autor em destaque: Purificação de afinidade de uma enzima fibrinolítica de Sipunculus nudus  

Purificação por Afinidade de uma Enzima Fibrinolítica de Sipunculus nudus

Our research is mainly about the purification of fibrinolytic enzyme. This work tries to establish a simple purification method for fibrinolytic enzyme. In this work, we report an arginine or lysine best-affinity purification system of fibrinolytic enzyme for the first time.Compared with the conventional purification methods, this is a simple, inexpensive, and efficient approach for the purification of fibrinolytic enzymes from Sipunculus nudus. The development of this protocol is of greater significance for the further utilization of fibrinolytic enzymes from Sipunculus nudus and other similar agents.

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Fibrinolytic Activity of the Fibrinolytic Enzyme Purified from  Sipunculus nudus  

Atividade Fibrinolítica da Enzima Fibrinolítica Purificada de Sipunculus nudus

Comece a preparação da placa de fibrina misturando 25 miligramas de fibrinogênio com 1,25 mililitros de soro fisiológico. Em seguida, prepare a solução de trombina misturando cuidadosamente 100 unidades de trombina com 1,05 mililitros de soro fisiológico. Em seguida, prepare a solução de agarose adicionando 0,5 gramas de agarose a 22,5 mililitros de tampão ácido de Tris-cloridrato de 0,02 molar.Aqueça a solução de agarose a 100 graus Celsius até dissolver completamente. Arrefecer a solução de agarose a cerca de 50 graus Celsius antes de lhe adicionar a solução de fibrinogénio. Adicione imediatamente a solução de trombina, misture rapidamente e despeje a mistura em uma placa de cultura de 60 milímetros.Para carregar a amostra, perfure poços de três milímetros nas placas de fibrina usando um Boer estéril e preencha-as com 10 microlitros de amostras. Incubar as placas a 37 graus Celsius por 18 horas antes de medir o tamanho de suas zonas de degradação e calcular a atividade fibrinolítica. A avaliação da atividade fibrinolítica mostrou zona de lise de 1,3 centímetro no controle uroquinase, zona de lise no tampão fisiológico, 2,1 centímetros da zona de lise no poço de proteína bruta e 1,8 centímetros de diâmetro da zona de lise no poço de enzima fibrinolítica purificada.

Research

JoVE Journal

Biochemistry

Fibrinolytic Activity of the Fibrinolytic Enzyme Purified from Sipunculus nudus

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin ...

Bioquímica

Protein Isolation from Sipunculus nudus

Protein Isolation from Sipunculus nudus  

Isolamento proteico de Sipunculus nudus

Affinity Chromatography Mediated Fibrinolytic Enzymes Purification from Crude Protein Extracts 

Purificação de enzimas fibrinolíticas mediadas por cromatografia de afinidade a partir de extratos de proteína bruta 