Immunochemical method to detect proteins that undergo selective modification by 1,2-naphthoquinone derived from naphthalene through metabolic activation
Immunochemical method to detect proteins that undergo selective modification by 1,2-naphthoquinone derived from naphthalene through metabolic activation
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DOI：
10.2131/jts.35.843
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发表时间：
2010-12-01
影响因子：
2
通讯作者：
Kumagai, Yoshito
中科院分区：
文献类型：
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作者：
Miura, Takashi;Kumagai, Yoshito
萘通过多种酶进行生物转化，产生 1,2-萘醌 (1,2-NQ)，这是一种与蛋白质共价结合的反应性代谢物。由于这种共价修饰被认为是造成萘毒性的原因，因此需要一种检测与大分子结合的 1,2-NQ 的程序。在本研究中，我们制备了针对 1,2-NQ 的多克隆抗体，并检查了该抗体对各种芳香结构和醌功能性区域化学的特异性。 Western blot分析显示，针对1,2-NQ制备的抗体可以识别带有邻二羰基的萘部分，但不能识别对二羰基；此外，具有不同芳香环数（n = 1、3、4、5、6）的邻醌几乎没有交叉反应性。使用多克隆抗体进行的斑点印迹和蛋白质印迹分析能够定量测定萘代谢活化过程中蛋白质结合的 1,2-NQ 的形成。本方法有望应用于细胞和组织中萘衍生的1,2-NQ分子靶标的鉴定。
Naphthalene undergoes biotransformation by a variety of enzymes to yield 1,2-naphthoquinone (1,2-NQ), a reactive metabolite that binds covalently to proteins. Because this covalent modification is thought to account for naphthalene toxicity, a procedure to detect 1,2-NQ bound to macromolecules is required. In this study, we prepared a polyclonal antibody against 1,2-NQ and examined the specificities of the antibody for various aromatic structures and for the regiochemistry of the quinone functionality. Western blot analysis revealed that the antibody prepared against 1,2-NQ recognized the naphthalene moiety with the ortho-dicarbonyl group, but not with the para-dicarbonyl group; in addition, little cross-reactivity of ortho-quinones with different numbers of aromatic rings (n = 1, 3, 4, 5, 6) was seen. Dot blot and Western blot analyses with the polyclonal antibody enabled quantitative determination of the formation of protein-bound 1,2-NQ during the metabolic activation of naphthalene. The present method can be expected to applicable for the identification of the molecular targets of 1,2-NQ derived from naphthalene in cells and tissues.