Zamudio F, Saavedra R, Martin B M, Gurrola-Briones G, Hérion P, Possani L D
Departamento de Bioquímica, Universidad Nacional Autónoma de México, Cuernavaca.
Eur J Biochem. 1992 Feb 15;204(1):281-92. doi: 10.1111/j.1432-1033.1992.tb16635.x.
Two toxins, which we propose to call toxins 2 and 3, were purified to homogeneity from the venom of the scorpion Centruroides noxius Hoffmann. The full primary structures of both peptides (66 amino acid residues each) was determined. Sequence comparison indicates that the two new toxins display 79% identity and present a high similarity to previously characterized Centruroides toxins, the most similar toxins being Centruroides suffusus toxin 2 and Centruroides limpidus tecomanus toxin 1. Six monoclonal antibodies (mAb) directed against purified fraction II-9.2 (which contains toxins 2 and 3) were isolated in order to carry out the immunochemical characterization of these toxins. mAb BCF2, BCF3, BCF7 and BCF9 reacted only with toxin 2, whereas BCF1 and BCF8 reacted with both toxins 2 and 3 with the same affinity. Simultaneous binding of mAb pairs to the toxin and cross-reactivity of the venoms of different scorpions with the mAb were examined. The results of these experiments showed that the mAb define four different epitopes (A-D). Epitope A (BCF8) is topographically unrelated to epitopes B (BCF2 and BCF7), C (BCF3) and D (BCF9) but the latter three appear to be more closely related or in close proximity to each other. Epitope A was found in all Centruroides venoms tested as well as on four different purified toxins of C. noxius, and thus seems to correspond to a highly conserved structure. Based on the cross-reactivity of their venoms with the mAb, Centruroides species could be classified in the following order: Centruroides elegans, Centruroides suffusus suffusus = Centruroides infamatus infamatus, Centruroides limpidus tecomanus, Centruroides limpidus limpidus, and Centruroides limpidus acatlanensis, according to increasing immunochemical relatedness of their toxins to those of Centruroides noxius. All six mAb inhibited the binding of toxin 2 to rat brain synaptosomal membranes, but only mAb BCF2, which belongs to the IgG2a subclass, displayed a clear neutralizing activity in vivo.
从墨西哥毒蝎（Centruroides noxius Hoffmann）毒液中纯化出两种毒素，我们提议将其分别命名为毒素2和毒素3，并将它们纯化至同质。确定了这两种肽（各含66个氨基酸残基）的完整一级结构。序列比较表明，这两种新毒素具有79%的同一性，并且与先前鉴定的墨西哥毒蝎毒素高度相似，最相似的毒素是苏氏墨西哥毒蝎毒素2和特科曼氏墨西哥毒蝎毒素1。为了对这些毒素进行免疫化学表征，分离出了六种针对纯化的II-9.2组分（其中含有毒素2和毒素3）的单克隆抗体（mAb）。单克隆抗体BCF2、BCF3、BCF7和BCF9仅与毒素2反应，而BCF1和BCF8以相同亲和力与毒素2和毒素3都发生反应。检测了单克隆抗体对与毒素的同时结合以及不同蝎子毒液与单克隆抗体的交叉反应性。这些实验结果表明，单克隆抗体确定了四个不同的表位（A-D）。表位A（BCF8）在拓扑结构上与表位B（BCF2和BCF7）、C（BCF3）和D（BCF9）无关，但后三个表位似乎彼此之间关系更密切或位置更接近。在所有测试的墨西哥毒蝎毒液以及四种不同的墨西哥毒蝎纯化毒素上都发现了表位A，因此它似乎对应于一种高度保守的结构。根据其毒液与单克隆抗体的交叉反应性，可将墨西哥毒蝎物种按以下顺序分类：秀丽墨西哥毒蝎、苏氏墨西哥毒蝎=臭名昭著墨西哥毒蝎、特科曼氏墨西哥毒蝎、清澈墨西哥毒蝎、阿卡特兰清澈墨西哥毒蝎，依据其毒素与墨西哥毒蝎毒素免疫化学相关性的增加。所有六种单克隆抗体均抑制毒素2与大鼠脑突触体膜的结合，但只有属于IgG2a亚类的单克隆抗体BCF2在体内表现出明显的中和活性。
