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

1.竹叶青蛇毒中一种凝血酶的氨基酸序列分析我们从竹叶青蛇毒中分离到一种蛋白酶，命名为flavoxobin。黄绿病毒属毒液，作用于纤维蛋白原，在释放纤维蛋白肽A时产生纤维蛋白凝块。分子量估计为约26，000道尔顿。将蛋白质S-吡啶基乙基化并用溴化氰和梭菌蛋白酶切割。溴化氰肽进一步用金黄色葡萄球菌V8蛋白酶、无色杆菌蛋白酶I和羟胺片段化。通过Edman降解法对S-吡啶基乙基化蛋白及其片段进行序列分析，确定了由236个氨基酸残基组成的flavoxobin的一级结构。未检测到碳水化合物。发现Flavoxobin在序列上与来自Bothrops atrox的凝血酶巴曲酶高度同源（69%），但与牛凝血酶、牛胰蛋白酶和人激肽释放酶具有27-39%的同源性。竹叶青蛇毒碱性蛋白I和II的氨基酸序列这些酶被纯化为蛋白酶，其特异性切割A链中间位点的纤维蛋白原。用溴化氰和甲酸将S-吡啶基乙基化的碱性蛋白i片段化。用胰凝乳蛋白酶、无色杆菌蛋白酶I和金黄色葡萄球菌V8蛋白酶进一步消化溴化氰片段。对S-吡啶基乙基化蛋白及其片段的氨基酸序列分析确定了由122个氨基酸残基组成的碱性蛋白I的一级结构。类似地测定碱性蛋白II的序列，但有微小的修改。碱性蛋白I和II与已报道的磷脂酶A_2具有同源性，命名为LYS-49-磷脂酶A_2。先前检测到的纤维蛋白原溶解活性归因于金属蛋白酶，该金属蛋白酶污染酶制剂，尽管其量非常小。

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.