Partial Purification and Characterization of Fibrinolytic Enzymes from Yellow Mealworm

• Published in: International journal of peptide research and therapeutics Vol. 18; no. 2; pp. 153 - 161
• Main Authors: Huang, Ming Xing, Ye, Yun, Chen, Ya Xiong, Han, Ya Li
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.06.2012; Springer Nature B.V
• Subjects: Animal Anatomy; Biochemistry; Biomedical and Life Sciences; Enzymes; Histology; Life Sciences; Molecular Medicine; Morphology; Pharmaceutical Sciences/Technology; Pharmacology/Toxicology; Polymer Sciences; Precipitation; Proteases; Proteins; Worms; Serine protease; Fibrinolytic enzyme; Thrombolytic agent; Metal chelate
• ISSN: 1573-3149; 1573-3904
• DOI: 10.1007/s10989-012-9288-x

Two proteins with fibrinolytic activity were partially purified from yellow mealworm ( Tenebrio molitor ) by ammonium sulfate precipitation between 30 and 70% saturation, gel filtration on Sephacryl-S200-HR, ion exchange chromatography on DEAE-Sepharose-FF and metal chelate on Cu–HiTrap–IMAC–FF, but the enzymes had not been completely separated from each other. The two partially purified fibrinolytic enzymes were designated as TMFE-I and TMFE-II ( Tenebrio molitor fibrinolytic enzyme) with molecular weights of 27.5 and 24.9 kDa by SDS-PAGE individually. The partially purified solution of TMFE-I and TMFE-II was considerably stable in the range of pH 5–10 and characterized by pH optimum of the enzymatic activity at 8.0. Thermal stability of TMFE was excellent at 45°C and below. The K M value was 0.26 mM for amidolysis of Bz–Arg–pNA. According to inhibitor analysis by fibrin plate method, phenylmethylsulfonyl fluoride and tosyl-lysine chloromethyl ketone inactivated TMFE almost completely, but trans -(epoxysuccinyl)- l -leucylamino-4-guanidinobutane (E-64) and EDTA had little effect on their fibrinolytic activity. According to metal ion analysis by fibrin plate method, the effect of metal ions on activity of TMFE showed a great difference. Na + , K + and Zn 2+ had little effect on the activity of TMFE. Mg 2+ and Cu 2+ showed inhibition effect on the fibrinolytic activity of TMFE, but Ca 2+ increased the fibrinolytic activity of TMFE at final concentration varying from 0 to 30 mM.