Purification, characterization and biological activities of the L-amino acid oxidase from Bungarus fasciatus snake venom
Purification, characterization and biological activities of the L-amino acid oxidase from Bungarus fasciatus snake venom
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金环蛇蛇毒L-氨基酸氧化酶的纯化、表征及生物活性。
DOI：
10.1016/j.toxicon.2009.04.017
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发表时间：
2009-09-01
期刊：
影响因子：
2.8
通讯作者：
He, Shao-Heng
中科院分区：
文献类型：
--
作者：
Wei, Ji-Fu;Yang, Hai-Wei;He, Shao-Heng
L-氨基酸氧化酶（LAAO）广泛存在于蛇毒中，对蛇毒的毒性起重要作用。银环蛇（Bungarus fasciatus，B. fasciatus）蛇毒之前没有分离过。在本研究中，从B.采用SP-SepharoseHP阴离子交换层析和Heparin-SepharoseFF亲和层析纯化了淡江蝮蛇蛇毒中的一种蛋白酶，命名为BF-LAAO。BF-LAAO在SDS-PAGE中的估计分子量为55 kDa，在尺寸排阻色谱中的表观分子量为70 kDa，表明BF-LAAO是单体蛋白。动力学研究表明，BF-LAAO对L-Tyr、L-Asp、L-Phe、L-Glu、L-Trp、L-His、L-Gln、L-Ile、L-Met、L-Leu的活性很强，对L-Lys、L-Arg、L-Ala、L-Asn的活性中等。BF-LAAO对A549细胞有明显的细胞毒作用，作用12 h后A549细胞凋亡率达41.2%。在小鼠腹膜中，BF-LAAO引起注射后中性粒细胞、淋巴细胞和巨噬细胞数量的显著增加。它还以剂量依赖性方式诱导兔血小板聚集。在注射后3 h，BF-LAAO引起小鼠腓肠肌严重炎症，但未能引起明显的器官损伤。结论：BF-LAAO的细胞毒性和促炎活性可能是引起蛇咬伤局部炎症的主要原因，这有助于我们了解蛇咬伤的发病机制。(C)2009爱思唯尔有限公司保留所有权利。
L-Amino acid oxidases (LAAOs) are widely distributed in snake venoms, which contribute to the toxicity of venoms. However, LAAO from Bungarus fasciatus (B. fasciatus) snake venom has not been isolated previously. In the present study, LAAO from B. fasciatus snake venom was purified by SP-Sepharose HP anion exchange chromatography followed by Heparin-Sepharose FF affinity chromatography procedure and the purified enzyme was named BF-LAAO. BF-LAAO presented an estimated molecular weight of 55 kDa in SDS-PAGE and an apparent molecular weight of 70 kDa in size-exclusion chromatography suggesting that BF-LAAO is a monomeric protein. Kinetics studies showed that BF-LAAO was very active against L-Tyr, L-Asp, L-Phe, L-Glu, L-Trp, L-His, L-Gln, L-Ile, L-Met, L-Leu and moderately active against L-Lys, L-Arg, L-Ala and L-Asn. BF-LAAO exhibited a cytotoxic effect on A549 cells and caused up to 41.2% apoptosis of A549 cells following 12 h incubation period. In the mouse peritoneum, BF-LAAO provoked a marked increase in the number of neutrophils, lymphocytes and macrophages following injection. It also induced rabbit platelet aggregation in a dose-dependent manner. At 3 h following injection, BF-LAAO elicited severe inflammation in the gastrocnemius muscles of mice, but failed to induce significant organ damage. In conclusion, the cytotoxic and proinflammatory activities of BF-LAAO could be the main cause of the local inflammation, which helps us to understand the pathogenesis of snakebite. (C) 2009 Elsevier Ltd. All rights reserved.