Related Experiment Video
Updated: Jul 12, 2026
Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography
Published on: March 30, 2022
Preparation and purification of microplasmin
1Biochemistry Department, Medical College, National Cheng-Kung University, Taiwan, Republic of China.
Abstract:
A catalytically active, human microplasmin was produced by incubation of [Lys]plasmin in buffer at pH 11.0 for up to 12 hr. The microplasmin was purified by affinity chromatography that used lysine-Sepharose and soybean trypsin inhibitor-Sepharose columns. It is homogeneous and pure by electrophoretic analysis in NaDodSO4/polyacrylamide gels and by gel filtration on a Superose 12 column. The molecular weight of the microplasmin determined by NaDodSO4 gel electrophoresis is 29,000 and 26,500 under reducing condition, whereas the molecular weight of native plasmin is 76,500. Microplasmin consists mainly of the ligh (B) chain of native human plasmin and possesses one active site per protein molecule when titrated with p-nitrophenyl p'-guanidinobenzoate. Microplasmin hydrolyzes the peptide substrate NH2-D-Val-Leu-Lys-p-nitroanilide (S-2251) with a Km of 0.361 +/- 0.017 mM and a kcat of 40.3 +/- 3.3 s-1 at pH 7.4 and 37 degrees C, whereas native plasmin has a Km of 0.355 +/- 0.002 mM and a kcat of 27.9 +/- 0.3 s-1 under the same conditions.
Insights
Researchers created active human microplasmin from plasmin. This smaller enzyme form, purified and characterized, shows distinct kinetic properties, offering potential for targeted applications in fibrinolysis research.
Area of Science:
- Biochemistry
- Enzymology
- Proteolysis
Background:
- Human plasmin is a key enzyme in fibrinolysis, responsible for breaking down blood clots.
- Understanding plasmin variants and their catalytic properties is crucial for developing targeted therapies.
Purpose of the Study:
- To produce and characterize a catalytically active human microplasmin.
- To compare the enzymatic activity of microplasmin with native plasmin.
Main Methods:
- Production of microplasmin via incubation of Lys-plasmin at pH 11.0.
- Purification using affinity chromatography (lysine-Sepharose, soybean trypsin inhibitor-Sepharose).
- Characterization by SDS-PAGE, gel filtration, and kinetic assays (S-2251 substrate).
Main Results:
- Homogeneous microplasmin (MW 29,000/26,500 Da) was successfully produced and purified.
- Microplasmin, mainly the light (B) chain, exhibited one active site per molecule.
- Microplasmin showed a higher catalytic rate (kcat) than native plasmin for S-2251 hydrolysis.
Conclusions:
- A catalytically active human microplasmin was generated and characterized.
- Microplasmin demonstrates altered kinetic properties compared to native plasmin.
- This microplasmin variant may offer advantages for specific fibrinolytic applications.
