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Published on: May 25, 2018
Purification and characterization of a serine protease with fibrinolytic activity from Tenodera sinensis (praying
1Natural Products Research Institute, Seoul National University, 28 Yeonkun-Dong, Jongno-Ku, Seoul 110-460, South Korea.
Abstract:
Mantis egg fibrolase (MEF) was purified from the egg cases of Tenodera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60 and affinity chromatography on DEAE Affi-Gel blue gel. The protease was assessed homogeneous by SDS-polyacrylamide gel electrophoresis and has a molecular mass of 31500 Da. An isoelectric point of 6.1 was determined by isoelectric focusing. Amino acid sequencing of the N-terminal region established a primary structure composed of Ala-Asp-Val-Val-Gln-Gly-Asp-Ala-Pro-Ser. MEF readily digested the Aalpha- and Bbeta-chains of fibrinogen and more slowly the gamma-chain. The nonspecific action of the enzyme results in extensive hydrolysis of fibrinogen and fibrin releasing a variety of fibrinopeptide. The enzyme is inactivated by Cu2+ and Zn2+ and inhibited by PMSF and chymostatin, yet elastinal, aprotinin, TLCK, TPCK, EDTA, EGTA, cysteine, beta-mercaptoethanol, iodoacetate, E64, benzamidine and soybean trypsin inhibitor do not affect activity. Antiplasmin was not sensitive to MEF but antithrombin III inhibited the enzymatic activity of MEF. Among chromogenic protease substrates, the most sensitive to MEF hydrolysis was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at pH 7.0 and 30 degrees C. MEF preferentially cleaved the oxidized B-chain of insulin between Leu15 and Tyr16. D-Dimer concentrations increased on incubation of cross-linked fibrin with MEF, indicating the enzyme has a strong fibrinolytic activity.
Insights
Mantis egg fibrolase (MEF), a novel protease from Tenodera sinensis egg cases, demonstrates potent fibrinolytic activity. This enzyme effectively degrades fibrinogen and fibrin, showing potential for therapeutic applications.
Area of Science:
- Biochemistry
- Enzymology
- Proteomics
Background:
- Fibrinogen and fibrin are key components of the blood coagulation cascade.
- The identification and characterization of novel proteases with fibrinolytic activity are crucial for understanding hemostasis and developing new therapeutic agents.
Purpose of the Study:
- To purify and characterize Mantis egg fibrolase (MEF) from Tenodera sinensis egg cases.
- To investigate the enzymatic activity and substrate specificity of MEF, particularly its fibrinolytic potential.
Main Methods:
- Purification using ammonium sulfate fractionation, gel filtration, and affinity chromatography.
- Characterization by SDS-PAGE, isoelectric focusing, and N-terminal amino acid sequencing.
- Enzyme activity assays using fibrinogen, fibrin, insulin, and chromogenic substrates; inhibition studies.
Main Results:
- MEF was purified to homogeneity with a molecular mass of 31,500 Da and an isoelectric point of 6.1.
- MEF efficiently digested Aalpha- and Bbeta-chains of fibrinogen, and the gamma-chain more slowly, releasing fibrinopeptides.
- The enzyme exhibited strong fibrinolytic activity, evidenced by increased D-dimer concentrations upon incubation with cross-linked fibrin.
- MEF activity was inhibited by Cu2+, Zn2+, PMSF, chymostatin, and antithrombin III, but not by various other protease inhibitors or chelators.
- Optimal activity was observed at pH 7.0 and 30°C, with benzoyl-Phe-Val-Arg-p-nitroanilide as a sensitive chromogenic substrate.
Conclusions:
- Mantis egg fibrolase (MEF) is a serine protease with significant fibrinolytic properties.
- MEF's ability to degrade fibrin suggests potential applications in thrombolytic therapy.
- Further research is warranted to explore MEF's therapeutic potential and mechanism of action.
