Purification and physicochemical characterization of a serine protease with fibrinolytic activity from latex of a medicinal herb Euphorbia hirta.
A 34 kDa serine protease, designated as hirtin, with fibrinolytic activity was purified to homogeneity from the latex of Euphorbia hirta by the combination of ion exchange and gel filtration chromatography. The N-terminal sequence of hirtin was found to be YAVYIGLILETAA/NNE. Hirtin exhibited esterase and amidase activities along with azocaseinolytic, gelatinolytic, fibrinogenolytic and fibrinolytic activities. It preferentially hydrolyzed Aα and α-chains, followed by Bβ and β, and γ and γ-γ chains of fibrinogen and fibrin clot respectively. The optimum pH and temperature for enzyme activity was found to be pH 7.2 and 50 °C respectively. Enzymatic activity of hirtin was significantly inhibited by PMSF and AEBSF. It showed higher specificity for synthetic substrate p-tos-GPRNA for thrombin. The CD spectra of hirtin showed a high content of β-sheets as compared to α-helix. The results indicate that hirtin is a thrombin-like serine protease and may have potential industrial and therapeutic applications.
Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667, India.
Girijesh Kumar
GK
Patel
Ashish Ashok
AA
Kawale
Ashwani Kumar
AK
Sharma
D000595:Amino Acid Sequence; D028481:Euphorbia / Q000737:chemistry / Q000201:enzymology*; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007840:Latex / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010946:Plants, Medicinal / Q000737:chemistry / Q000201:enzymology; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014315:Triterpenes / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
D005343:Fibrinolytic Agents; D007840:Latex; D014315:Triterpenes; C437025:hirtin; D005337:Fibrin; D005340:Fibrinogen; D057057:Serine Proteases