Barbaro K C, Sousa M V, Morhy L, Eickstedt V R, Mota I
Laboratório de Imunopatologia, Instituto Butantan, São Paulo, SP, Brazil.
J Protein Chem. 1996 May;15(4):337-43. doi: 10.1007/BF01886859.
Loxosceles spider venom usually causes a typical dermonecrotic lesion in bitten patients, but it may also cause systemic effects that may be lethal. Gel filtration on Sephadex G-100 of Loxosceles gaucho, L. laeta, or L. intermedia spider venoms resulted in three fractions (A, containing higher molecular mass components. B containing intermediate molecular mass components, and C with lower molecular mass components). The dermonecrotic and lethal activities were detected exclusively in fraction A of all three species. Analysis by SDS-PAGE showed that the major protein contained in fraction A has molecular weight approximately 35 kDa in L. gaucho and L. intermedia, but 32 kDa in L. laeta venom. These toxins were isolated from venoms of L. gaucho, L. laeta, and L. intermedia by SDS-PAGE followed by blotting to PVDF membrane and sequencing. A database search showed a high level of identity between each toxin and a fragment of the L. reclusa (North American spider) toxin. A multiple sequence alignment of the Loxosceles toxins showed many common identical residues in their N-terminal sequences. Identities ranged from 50.0% (L. gaucho and L. reclusa) to 61.1% (L. intermedia and L. reclusa). The purified toxins were also submitted to capillary electrophoresis peptide mapping after in situ partial hydrolysis of the blotted samples. The results obtained suggest that L. intermedia protein is more similar to L. laeta toxin than L. gaucho toxin and revealed a smaller homology between L. intermedia and L. gaucho. Altogether these findings suggest that the toxins responsible for most important activities of venoms of Loxosceles species have a molecular mass of 32-35 kDa and are probably homologous proteins.
棕色遁蛛毒液通常会在被叮咬的患者身上引发典型的皮肤坏死性损伤，但也可能导致致命的全身效应。对高乔棕色遁蛛、巴西棕色遁蛛或中间棕色遁蛛的毒液进行葡聚糖凝胶G - 100凝胶过滤，得到三个组分（A，含有较高分子量成分；B，含有中等分子量成分；C，含有较低分子量成分）。在这三种蜘蛛的所有样本中，仅在组分A中检测到皮肤坏死和致死活性。SDS - PAGE分析表明，组分A中含有的主要蛋白质在高乔棕色遁蛛和中间棕色遁蛛毒液中的分子量约为35 kDa，而在巴西棕色遁蛛毒液中为32 kDa。通过SDS - PAGE从高乔棕色遁蛛、巴西棕色遁蛛和中间棕色遁蛛的毒液中分离出这些毒素，随后印迹到PVDF膜上并进行测序。数据库搜索显示，每种毒素与隐居褐蛛（北美蜘蛛）毒素的一个片段具有高度同源性。棕色遁蛛毒素的多序列比对显示，它们的N端序列中有许多相同的残基。同源性范围从50.0%（高乔棕色遁蛛和隐居褐蛛）到61.1%（中间棕色遁蛛和隐居褐蛛）。在对印迹样本进行原位部分水解后，还对纯化的毒素进行了毛细管电泳肽图谱分析。所得结果表明，中间棕色遁蛛的蛋白质与巴西棕色遁蛛毒素的相似性高于高乔棕色遁蛛毒素，且中间棕色遁蛛和高乔棕色遁蛛之间的同源性较小。这些发现共同表明，导致棕色遁蛛毒液最重要活性的毒素分子量为32 - 35 kDa，可能是同源蛋白。
