Preparation and Purification of Microplasmin

• Published in: Proceedings of the National Academy of Sciences - PNAS 1987-12, Vol.84 (23), p.8292-8295
• Main Authors: Wu, Hua-Lin, Shi, Guey-Yueh, Bender, Myron L.
• Format: Article
• Language: English
• Subjects: Active sites; Analytical, structural and metabolic biochemistry; Biochemistry; Biological and medical sciences; Biotechnology; Catalytic activity; Chemical synthesis for preparing modified enzymes, enzyme fragments and enzyme analogs; Chromatography, Affinity; Electrophoresis; Enzyme engineering; Enzymes; Enzymes and enzyme inhibitors; Fibrinolysin - analysis; Fundamental and applied biological sciences. Psychology; Gels; Humans; Hydrolases; Methods. Procedures. Technologies; Molecular Weight; Peptide Fragments - isolation & purification; Phosphates; Production of selected enzymes; Sodium; Soybeans
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.84.23.8292

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 light (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.