Sun J D, Dent J G
Chem Biol Interact. 1980 Oct;32(1-2):41-61. doi: 10.1016/0009-2797(80)90067-8.
A new method has been developed for measuring the total covalent binding of metabolically activated compounds to cellular macromolecules. This method employs equilibrium dialysis, in the presence of the detergent sodium dodecyl sulfate (SDS), to remove unbound radiolabeled compound and its metabolites from cellular macromolecules. [14C] Bromobenzene (80 microM), [14C]aflatoxin B1 (5 microM) or 3-[14C]methylcholanthrene (100 microM) was incubated (37 degrees C) with primary hepatocytes or liver microsomes isolated from Fischer-344 rats. The covalent binding of 14C-radiolabel to hepatic or microsomal macromolecules was measured by SDS-equilibrium dialysis and compared with that measured by exhaustive extraction. After 1 h of incubation with hepatocytes or microsomes, 2--7 times more covalent binding was detected by SDS-equilibrium dialysis, than by exhaustive extraction. The radioactivity associated with these hepatic or microsomal macromolecules migrated to discrete positions on SDS-polyacrylamide disc gels. The non-dialysable radioactivity from incubations with [14C] bromobenzene could not be extracted with diethyl ether even after treatment of the dialysin with beta-glucuronidase-sulfatase or dilute acid. This was taken to indicate that the radioactivity in the dialysin did not include free bromobenzene or its metabolites, a conclusion supported by thin-layer chromatography analysis of the dialysin. The lower amount of covalent binding detected by exhaustive extraction may be related to the inability of trichloroacetic acid to quantitatively precipitate small molecular weight macromolecules. SDS-equilibrium dialysis is an easy, rapid and non-destructive technique for measuring covalent binding. The macromolecular integrity of the sample is maintained and allows further studies concerning the specificity of the covalent interactions.
已开发出一种新方法，用于测量代谢活化化合物与细胞大分子的总共价结合。该方法在去污剂十二烷基硫酸钠（SDS）存在的情况下采用平衡透析，以从细胞大分子中去除未结合的放射性标记化合物及其代谢物。将[14C]溴苯（80微摩尔）、[14C]黄曲霉毒素B1（5微摩尔）或3-[14C]甲基胆蒽（100微摩尔）与从Fischer-344大鼠分离的原代肝细胞或肝微粒体在37℃下孵育。通过SDS平衡透析测量14C放射性标记与肝或微粒体大分子的共价结合，并与通过彻底提取测量的结果进行比较。与肝细胞或微粒体孵育1小时后，通过SDS平衡透析检测到的共价结合比通过彻底提取检测到的多2至7倍。与这些肝或微粒体大分子相关的放射性在SDS-聚丙烯酰胺圆盘凝胶上迁移到离散位置。即使在用β-葡萄糖醛酸酶-硫酸酯酶或稀酸处理透析物后，用[14C]溴苯孵育产生的不可透析放射性也不能用乙醚提取。这表明透析物中的放射性不包括游离溴苯或其代谢物，这一结论得到了透析物的薄层色谱分析的支持。通过彻底提取检测到的较低共价结合量可能与三氯乙酸无法定量沉淀小分子重量大分子有关。SDS平衡透析是一种用于测量共价结合的简便、快速且非破坏性的技术。样品的大分子完整性得以维持，并允许对共价相互作用的特异性进行进一步研究。
