title: Biochemical characterization of a new glycosylated protease from Euphorbia cf. lactea latex.
abstract: A dimeric protease designated as EuP-82 was purified from Euphorbia cf. lactea latex. Since its proteolytic activity was inhibited by a serine protease specific inhibitor (PMSF), EuP-82 was classified as a serine protease. N-glycan deglycosylation tests revealed that EuP-82 was a glycosylated protein. MALDI-TOF MS showed that EuP-82 was a homodimer, which was its active form. The optimal conditions for fibrinogenolytic activity were at pH 11 and 35 °C. EuP-82 enzyme had broad range of pH stability from 4 to 12. Moreover, the enzyme was still active in the presence of reducing agent (β-mercaptoethanol). EuP-82 was a proline-rich enzyme (about 20.69 mol%). Increased proline production can be found in higher plants in response to both biotic and abiotic stresses, high proline in the molecule of EuP-82 might stabilize its activity, structure and folding. Based on the N-terminal amino acid sequences and peptide mass fingerprint (PMF) of EuP-82, the enzyme was identified as a new serine protease. The digested products from EuP-82 cleavage of human fibrinogen were analyzed by SDS-PAGE and PMF. The results confirmed that EuP-82 could digest all subunits of human fibrinogen. EuP-82 cleaved fibrinogen with a Michaelis constant (Km) of 3.30 ± 0.26 μM; a maximal velocity (Vmax) of 400.9 ± 0.85 ng min(-1); and a catalytic efficiency (Vmax/Km) of 121.5 ± 9.25 ng μM(-1) min(-1). EuP-82 has potential for use in medicinal treatment, for example thrombosis, since the enzyme had fibrinogenolytic activity and high stability.
authors.affiliation: School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand; Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima, Thailand. Electronic address: jaruwan_siritape@yahoo.com.
authors.forename: Jaruwan
authors.initials: J
authors.lastname: Siritapetawee
authors.affiliation: Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Punchapat
authors.initials: P
authors.lastname: Sojikul
authors.affiliation: Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Sompong
authors.initials: S
authors.lastname: Klaynongsruang
mesh_terms: D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D028481:Euphorbia / Q000737:chemistry*; D005340:Fibrinogen / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007840:Latex / Q000737:chemistry*; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D011392:Proline / Q000032:analysis; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013312:Stress, Physiological; D013696:Temperature; D013927:Thrombosis / Q000188:drug therapy
chemical_list: D005343:Fibrinolytic Agents; D007840:Latex; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D008623:Mercaptoethanol; D005340:Fibrinogen; D011392:Proline; D057057:Serine Proteases
keywords: Euphorbia cf. lactea; Fibrinogenolytic; Plant latex; Serine protease