Pan; E mail: [email protected] Author to whom correspondence must be addressed; Email: [email protected]; Tel.: 81528392732; Fax: 81528348090. Received: 18 July 2014 / Accepted: 18 August 2014 / Published: 25 AugustAbstract: A novel hemorrhagic metalloproteinase, okinalysin, was isolated in the venom of Ovophis okinavensis. It possessed caseinolytic and hemorrhagic activities, as well as hydrolyzed fibrinogen and collagen. These activities had been inhibited by ethylenediaminetetraacetic acid (EDTA) but not by pamidinophenyl methanesulfonyl fluoride hydrochloride (APMSF). The molecular mass of okinalysin was 22,202 Da measured by MALDI/TOF mass spectrometry. The principal structure of okinalysin was partially determined by Edman sequencing, plus the putative zincbinding domain HEXXHXXGXXH was discovered to be present in its structure. From these information, okinalysin is defined as a metalloproteinase belonging to a PI class. The partial amino acid sequence of okinalysin was homologous towards the Cterminus of MP 10, a putative metalloproteinase induced from transcriptome of the venom gland cDNA sequencing of O. okinavensis. Okinalysin possessed cytotoxic activity on cultured endothelial cells, as well as the EC50 on human pulmonary artery endothelial cells was determined to be 0.six g/mL. The histopathological study also showed that okinalysin causes the leakage of red blood cells and neutrophil infiltration. These final results indicate that destruction of blood vessels by okinalysin is one of the main causes of hemorrhage.Toxins 2014, 6 Search phrases: Ovophis okinavensis venom; vascular endothelial cell; cytotoxicity hemorrhagic toxin; metalloproteinase;1. Introduction Amongst the different kinds of enzyme and protein current in snake venoms, metalloproteinase (SVMP: snake venom metalloproteinase) is amongst the most important elements. The role of SVMPs in the pathologies associated with Viperidae envenomation has extended been specially studied. Varieties of SVMPs were reported which bring about symptoms for instance hemorrhage, fibrinogenolysis, necrosis and apoptosis [10]. Fox and Serrano described the protein structural classification of SVMPs [11]; Class PI has only a metalloproteinase domain, Class PII consists of metalloproteinase and disintegrin domains, Class PIII is synthesized with metalloproteinase, disintegrinlike and cysteinerich domains, and Class PIV has the PIII domain structure and lectinlike domains. Venom gland cDNA sequencing studies indicated that these SVMPs were biosynthesized as latent precursor proproteinases [12,13]. Generally, the hemorrhagic activity of SVMPs of Class PI is much less active than PIII SVMPs, because disintegrinlike domains and cysteinerich domains are regarded as to possess functions in interacting with cell surface or cell matrix [14].Price of 2-Bromo-6-hydroxybenzaldehyde In the southern islands of Japan, most snake envenomation is due to Okinawa habu (Protobothrops flavoviridis).(2-Cyanopyridin-3-yl)boronic acid uses The frequency of envenomation by Himehabu (O.PMID:23892407 okinavensis) is low because of the short venomous fangs and modest content of venom. Because the typical number of victims of Himehabu envenomation within a year is about 10, this venom has not been studied in detail. Aird et al. [15] analyzed the venom gland cDNA transcripts of O. okinavensis and showed that 95 venomrelated proteins are integrated. The key venom constituents were serineproteinases (93.1 ) and the percentage of metalloproteinases was only four.2 . In contrast, the dominant constituents of P. flavoviridis venom glands are phospholipase A2 (32.1 ).