Radish phospholipid hydroperoxide glutathione peroxidase (RsPHGPx) was identified as a mitochondrion-targeting PHGPx in previous work. To determine its cleavage site of the targeting peptide, the immunoaffinity chromatography (IAC) purification approach was carried out to isolate the native RsPHGPx protein.Polyclonal antibodies directed against recombinant RsPHGPx were raised in rabbit. Monospecific anti-RsPHGPx antibodies were isolated by means of affinity chromatography using the recombinant RsPHGPx as affinity ligand, and employed in assembling an IAC column. A single-step, highly specific and easy-to-use protocol was developed for purification of the active RsPHGPx protein through the assembled IAC column. Using this approach, a specific protein of the expected molecular size was obtained from the mitochondrial fraction of radish seedlings. Western blot analysis showed that it could be specifically recognized by anti-RsPHGPx antibodies, and an enzyme activity assay indicated that it exhibited significant PHGPx activity, suggesting that the purified protein should be the desired native RsPHGPx. These results will lead to clarification of the targeting peptide and the active mature protein of RsPHGPx and will be helpful to further probe the intracellular localization mechanism and biological fun ction of this plant PHGPx.%萝卜磷脂氢谷胱甘肽过氧化物酶(RsPHGPx)是一个定位于线粒体的蛋白质.为了阐明该蛋白质线粒体定位信号的准确切割位点,采用了免疫亲和层析方法纯化天然的RsPHGPx.用重组RsPHGPx蛋白免疫兔子获得了抗RsPHGPx的多克隆抗血清,以重组RsPHGPx蛋白为配体,采用亲和层析技术对抗血清进行了纯化,得到了单特异性的抗RsPHGPx的抗体.将纯化好的抗体偶联到一个N-羟基琥珀酰亚胺(NHS)预先激活的琼脂糖柱子上,装配成一个以单特异性的抗RsPHGPx抗体为配体的免疫亲和层析柱.经过对纯化条件的摸索和优化,形成了一个简单、特异的一步法纯化方案.按照该方案,从萝卜幼苗线粒体总蛋白质提取物中纯化到一个分子质量与预期值相一致的特异蛋白质.免疫印迹分析表明,该蛋白质被抗RsPHGPx的抗血清特异识别.酶活性分析表明,该蛋白质具有显著的PHGPx活性.这些结果表明,纯化到的特异蛋白质是萝卜的RsPHGPx天然蛋白.这是首个关于定位于植物细胞器的PHGPx蛋白纯化的报道.这一结果为准确测定RsPHGPx信号肽的切割位点奠定了基础,并将有助于对植物PHGPx的亚细胞定位机制及其生理功能的深入研究.
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