Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose- (concentration) dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 μCi) of (14)C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2 D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 hr post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5-11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with (14)C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts.

中文翻译：

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C-萘-蛋白质加合物的自由流动电泳分离和 AMS 定量。

萘是一种挥发性芳香烃，人类通过各种来源接触到它，包括移动空气源和香烟烟雾。萘对鼠肺的气道上皮细胞产生剂量（浓度）依赖性损伤，在远低于当前职业接触标准的浓度下观察到这种损伤。毒性取决于细胞色素 P450 介导的母体底物代谢活化为不稳定代谢物，这些代谢物与组织蛋白共价结合。近 70 种蛋白质已被鉴定为使用体外系统与反应性萘代谢物形成加合物，但在体内进行的工作很少，因为必须施用相当大量 (100 μCi) 的 (14)C 标记母体化合物才能产生可​​检测的加合物水平通过 2D 凝胶电泳分离标记的蛋白质后，在存储荧光屏上。这里描述的工作是为了提供概念证明，即通过自由流动电泳分离蛋白质，然后 AMS 检测含有蛋白质结合反应性代谢物的蛋白质级分，将在服用痕量标记萘的动物中提供加合物蛋白质谱。小鼠以 2 DPM/nmol (1 pCi/nmol) 的计算比活腹膜内注射 200 mg/kg 萘，注射后 4 小时通过裂解灌洗获得呼吸上皮组织。自由流动电泳 (FFE) 使用低分子量酸和碱来改变 pH 值，从而在较大的 pH 值范围 (2.5-11.5) 内分离液相中的蛋白质。该装置将组分分离到标准 96 孔板中，可用于其他蛋白质分析技术。然而，馏分的缓冲液具有非常高的碳含量，需要进行透析以产生与 (14) C-AMS 兼容的缓冲液。我们描述了将 FFE 与 AMS 结合以定量蛋白质加合物所需的处理技术。自由流动电泳 (FFE) 使用低分子量酸和碱来改变 pH 值，从而在较大的 pH 值范围 (2.5-11.5) 内分离液相中的蛋白质。该装置将组分分离到标准 96 孔板中，可用于其他蛋白质分析技术。然而，馏分的缓冲液具有非常高的碳含量，需要透析以产生与 (14) C-AMS 兼容的缓冲液。我们描述了将 FFE 与 AMS 耦合以定量蛋白质加合物所需的处理技术。自由流动电泳 (FFE) 使用低分子量酸和碱来改变 pH 值，在大 pH 值范围 (2.5-11.5) 内分离液相中的蛋白质。该装置将组分分离到标准 96 孔板中，可用于其他蛋白质分析技术。然而，馏分的缓冲液具有非常高的碳含量，需要透析以产生与 (14) C-AMS 兼容的缓冲液。我们描述了将 FFE 与 AMS 耦合以定量蛋白质加合物所需的处理技术。并且需要透析以产生与 (14)C-AMS 兼容的缓冲液。我们描述了将 FFE 与 AMS 耦合以定量蛋白质加合物所需的处理技术。并且需要透析以产生与 (14)C-AMS 兼容的缓冲液。我们描述了将 FFE 与 AMS 耦合以定量蛋白质加合物所需的处理技术。