Department of Biochemistry, Institute of Biology (IB), State University of Campinas (UNICAMP), P.O. Box 6109, Zip code 13083-970, Campinas, SP, Brazil.
Toxicon. 2012 Feb;59(2):231-40. doi: 10.1016/j.toxicon.2011.11.018. Epub 2011 Dec 6.
A new serine protease with thrombin-like activity (TLBan) from Bothrops andianus (Andean Lancehead) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. TLBan is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with Mr ∼29 kDa under reducing conditions and non-reducing ∼25 kDa conditions and confirmed by MALDI-TOF mass spectrometry (25,835.65 Da) and exhibited high specificity for BAρNA, Michaelis-Menten behavior with Km 5.4 × 10(-1) M and the V(max) 7.9 × 10(-1) nmoles ρ-NA/L/min for this substrate and high stability when was analyzed at different temperatures (25 to 60 °C), pHs (4.0 to 8.0), was inhibited by soybean trypsin inhibitor, EDTA and phenylmethylsulfonyl fluoride (PMSF). The total amino acid sequence was obtained through sequencing of selected tryptic peptides and by inference obtained using SwissProt database http://br.expasy.org/ with the search restricted to serine proteases from Crotalinae snakes and show high amino acid sequence identity with other serine proteases from snake venom. TLBan showed the presence of His(44), Asp(91) residues and Ser was deduced (187) position, in the corresponding positions to the catalytic triad established in the serine proteases and Ser(187) are inhibited by phenylmethylsulfonyl fluoride (PMSF). In this work, we investigated the ability of TLBan to degrade fibrinogen and we observed that it is able to cause α- and β-chain cleavage. Enzymatic activities as well as the platelet aggregation were strongly inhibited when were incubated with PMSF, a specific inhibitor of serine protease. TLBan showed a potential medical-scientific interest to understand the pathophysiological mechanism of the snake venom action and identification of new blood coagulation cascade acting enzymes of natural sources.
一种新型丝氨酸蛋白酶具有凝血酶样活性 (TLBan) 从 Bothrops andianus (安第斯矛头蝮) 在 LC 分子排阻和反相-HPLC 的两个色谱步骤中分离。TLBan 是一种糖蛋白，其结构中既含有 N-连接的碳水化合物，也含有唾液酸，在还原条件下 Mr 约为 29 kDa，在非还原条件下约为 25 kDa，并通过 MALDI-TOF 质谱 (25,835.65 Da) 证实，并对 BAρNA 表现出高度特异性，米氏常数 Km 为 5.4×10(-1) M，最大反应速度 V(max) 为 7.9×10(-1) nmoles ρ-NA/L/min，对该底物具有米氏酶动力学行为，在不同温度 (25 至 60°C)、pH 值 (4.0 至 8.0) 下分析时具有高稳定性，被大豆胰蛋白酶抑制剂、EDTA 和苯甲基磺酰氟 (PMSF) 抑制。通过对选定的胰蛋白酶肽进行测序，以及通过对瑞士Prot 数据库 http://br.expasy.org/的推断，获得了全长氨基酸序列，搜索限制在来自 Crotalinae 蛇的丝氨酸蛋白酶，与来自蛇毒的其他丝氨酸蛋白酶具有高度的氨基酸序列同一性。TLBan 显示存在 His(44)、Asp(91)残基和 Ser(187)位置，在丝氨酸蛋白酶中建立的催化三联体的相应位置，Ser(187)被苯甲基磺酰氟 (PMSF) 抑制。在这项工作中，我们研究了 TLABan 降解纤维蛋白原的能力，观察到它能够引起 α-和 β-链的切割。当与丝氨酸蛋白酶的特异性抑制剂 PMSF 孵育时，酶活性以及血小板聚集强烈抑制。TLBan 具有潜在的医学科学意义，可用于了解蛇毒作用的病理生理学机制以及鉴定天然来源的新凝血级联作用酶。
