title: Purification and biochemical characterization of a novel fibrinolytic enzyme, PSLTro01, from a medicinal animal Porcellio scaber Latreille.
abstract: A novel protease, named PSLTro01, with fibrinolytic and anticoagulant activity was isolated from Porcellio scaber Latreille and was purified by a combination of hollow fibre membrane molecular weight cut-off (MWCO), ammonium sulfate fractionation, gel filtration and ion-exchange chromatography. PSLTro01 is a single-chain protein with a molecular mass of 38,497 Da as estimated by non-reduced SDS-PAGE and MALDI-TOF MS spectrometry, and its N-terminal 15 amino acid sequence was determined as DINGGGATLPQPLYQ. PSLTro01 is stable in the range of 20-40 °C and pH 6.0-10.0, with a maximum fibrinolytic activity at 40 °C and pH 7.0. The PSLTro01-induced fibrinolytic activity was not influenced by K(+) or Na(+) but was slightly increased by Mg(2+) and completely inhibited by aprotinin and pepstatin A. Fibrin plate assays revealed that PSLTro01 could not directly degrade fibrin but was a plasminogen activator. PSLTro01 exhibited high specificity for the substrate S-2251 for plasmin, followed by S-2238 for thrombin and S-2444 for urokinase. Moreover, the fibrinogenolysis pattern of PSLTro01 was Aα-chains>Bβ-chains>γ-chain. Tail-thrombus of the enzyme treated group was significantly shorter than the physiological saline treated group and the thrombus decrement was correlated with the enzyme dose. PSLTro01 prolongs both thrombin time (TT) and activated partial thromboplastin time (APTT). These results indicate that PSLTro01 may have potential applications in the prevention and treatment of thrombosis.
authors.affiliation: Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing 210023, China; Key Laboratory of Separation Engineering for Chinese Medicine Compound, Nanjing University of Chinese Medicine, Nanjing 210029, China.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Tian
authors.affiliation: Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing 210023, China; Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor, Nanjing University of Chinese Medicine, Nanjing 210023, China; The First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing 210046, China. Electronic address: zodlee@163.com.
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.affiliation: Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing 210023, China; Key Laboratory of Separation Engineering for Chinese Medicine Compound, Nanjing University of Chinese Medicine, Nanjing 210029, China.
authors.forename: Liwei
authors.initials: L
authors.lastname: Guo
authors.affiliation: Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor, Nanjing University of Chinese Medicine, Nanjing 210023, China; The First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing 210046, China. Electronic address: sannychb@126.com.
authors.forename: Mianhua
authors.initials: M
authors.lastname: Wu
authors.affiliation: Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing 210023, China; Key Laboratory of Separation Engineering for Chinese Medicine Compound, Nanjing University of Chinese Medicine, Nanjing 210029, China.
authors.forename: Tingming
authors.initials: T
authors.lastname: Fu
authors.affiliation: Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor, Nanjing University of Chinese Medicine, Nanjing 210023, China; The First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing 210046, China.
authors.forename: Haibo
authors.initials: H
authors.lastname: Cheng
authors.affiliation: Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing 210023, China; Key Laboratory of Separation Engineering for Chinese Medicine Compound, Nanjing University of Chinese Medicine, Nanjing 210029, China.
authors.forename: Huaxu
authors.initials: H
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D003445:Crustacea / Q000201:enzymology*; D004195:Disease Models, Animal; D004789:Enzyme Activation; D004795:Enzyme Stability; D005342:Fibrinolysis; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008670:Metals; D051379:Mice; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D013927:Thrombosis / Q000139:chemically induced / Q000188:drug therapy
chemical_list: D000925:Anticoagulants; D005343:Fibrinolytic Agents; D007477:Ions; D008670:Metals; D011480:Protease Inhibitors; D010447:Peptide Hydrolases
keywords: Anticoagulation; Fibrinolytic enzyme; Porcellio scaber Latreille; Purification; Wet ultra-file grinding