Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography
Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography
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DOI：
10.1007/s00216-013-7453-5
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发表时间：
2014-01-01
影响因子：
4.3
通讯作者：
Halassy, Beata
中科院分区：
文献类型：
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作者：
Brgles, Marija;Kurtovic, Tihana;Halassy, Beata
为了发挥其功能，蛋白质经常与其他蛋白质相互作用。蛋白质相互作用的研究方法很多，亲和层析法就是其中之一。蛇毒主要由蛋白质组成，并且已经发现毒液中的各种蛋白质复合物表现出比单独的各个组分更高的毒性水平。复合物可以调节毒液的毒液蛰入活性和/或增强其作用。我们以前的数据表明，最有毒的成分的蝰蛇ammodytes ammodytes（Vaa）毒液分离到目前为止，ammodytoxins（Atxs），有助于毒液的毒性只有适度的，因此，我们的目的是探索他们是否有一些相互作用的合作伙伴（S）增强毒性。为了筛选可能的相互作用，免疫亲和色谱结合质谱鉴定。使用各种化学物质（环氧、羰基二咪唑、乙二胺）以及蛋白G官能团将抗体固定在整料载体（对流相互作用介质盘）上。已经证明整料更适合大生物分子的分离。使用这种方法，几个蛋白质被指示为潜在的Atx结合蛋白。其中，Atxs与Kunitz型抑制剂的相互作用通过远Western斑点印迹和表面等离子体共振测量证实。结论：整体柱亲和层析结合质谱鉴定是一种成功的蛋白质相互作用筛选方法，并首次检测到Vaa毒中Atx与Kunitz型抑制剂的相互作用。
In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far-ammodytoxins (Atxs)-are contributing to the venom's toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.