Pan; E mail: [email protected] Author to whom correspondence really should 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 from the venom of Ovophis okinavensis. It possessed caseinolytic and hemorrhagic activities, and also hydrolyzed fibrinogen and collagen. These activities have 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 primary structure of okinalysin was partially determined by Edman sequencing, as well as the putative zincbinding domain HEXXHXXGXXH was found to be present in its structure. From these data, 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 on the venom gland cDNA sequencing of O.6-Bromo-8-fluoroisoquinolin-1(2h)-one web okinavensis.Formula of 1370633-67-2 Okinalysin possessed cytotoxic activity on cultured endothelial cells, and the EC50 on human pulmonary artery endothelial cells was determined to be 0.PMID:33533528 6 g/mL. The histopathological study also showed that okinalysin causes the leakage of red blood cells and neutrophil infiltration. These benefits indicate that destruction of blood vessels by okinalysin is among the key causes of hemorrhage.Toxins 2014, 6 Keywords and phrases: Ovophis okinavensis venom; vascular endothelial cell; cytotoxicity hemorrhagic toxin; metalloproteinase;1. Introduction Among the different kinds of enzyme and protein existing in snake venoms, metalloproteinase (SVMP: snake venom metalloproteinase) is one of the most significant elements. The function of SVMPs in the pathologies connected with Viperidae envenomation has extended been especially studied. Varieties of SVMPs had been reported which result in symptoms which include 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 have been biosynthesized as latent precursor proproteinases [12,13]. Generally, the hemorrhagic activity of SVMPs of Class PI is significantly less active than PIII SVMPs, simply because disintegrinlike domains and cysteinerich domains are viewed as to have functions in interacting with cell surface or cell matrix [14]. Within the southern islands of Japan, most snake envenomation is resulting from Okinawa habu (Protobothrops flavoviridis). The frequency of envenomation by Himehabu (O. okinavensis) is low as a result of the short venomous fangs and little content of venom. Since 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 important venom constituents have been serineproteinases (93.1 ) along with the percentage of metalloproteinases was only four.two . In contrast, the dominant constituents of P. flavoviridis venom glands are phospholipase A2 (32.1 ).