Determination of Primary Structures of Fibrinogen-specific proteases from Trimeresurus flavoviridis Venom

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1. Amino Acid Sequence of a Coagulant Enzyme, Flavoxobin, from Trimeresurus Flavoviridis venom we previously isolated a proteses (named flavoxobin) from the T. Flavoviridis venom which acts on fibrinogen to produce a fibrin clot upon release of fibrinopeptide A. The molecular weight was estimated to be about 26,000 daltons. The protein was S-pyridylethylated and cleaved with cyanogen bromide and clostripain. The cyanogen bromide peptides were further fragmented with Staphylococcus Aureus V8 protease, Achromobacter protease I, and hydroxylamine. Sequence analyses of S-pyridylethylated protein and its fragments by means of Edman degradation enabled us to determine the primary structure of flavoxobin which consisted of the 236 amino acid residues. No carbohydrate was detected. Flavoxobin was found to be highly (69%) homologous in sequence to batroxobin, a coagulant enzyme from Bothrops atrox but 27-39% homologous to bovine thrombin, bovine trypsin, and human Kallikrein.2. Amino Acid Sequences of Basic Proteins I and II from Trimeresurus flavoviridis Venom These enzymes were purified as protease which specifically cleave fibrinogen at an intermediate locus of A -chain. The S-pyridylethylated basic protein i was fragmented with cyanogen bromide and formic acid. The cyanogen bromide fragments were further digested with chymotrypsin, Achromobacter protease I, and Staphylococcus aureus V8 protease. Analyses of amino acid sequences of S-pyridylethylated protein and its fragments established the primary structure of basic protein I composed of the 122 amino acid residues. The sequence of basic protein II was determined analogously but with a minor modification. The basic proteins I and II were found to be homologous in sequence to phospholipases A_2 so far reported and were characterized as LYS-49-phospholipase A_2. the fibrinogenolytic activity detected previously was ascribed to metal protease which contaminates the enzyme preparations although in a very minor quantity.