Zhu M, Spink D C, Yan B, Bank S, DeCaprio A P
Department of Environmental Health and Toxicology, University at Albany, State University of New York, USA.
Chem Res Toxicol. 1995 Jul-Aug;8(5):764-71. doi: 10.1021/tx00047a017.
n-Hexane is metabolized to the gamma-diketone 2,5-hexanedione (2,5-HD), a derivative that covalently binds to lysine residues in neurofilament (NF) protein to yield 2,5-dimethylpyrrole adducts. Studies comparing the pyrrole-forming potential and neurotoxic potency of gamma-diketones have demonstrated that pyrrolylation is an absolute requirement in the neuropathogenesis. Autoxidative cross-linking of pyrrolylated NF proteins occurs and is proposed as a second required event. In the present study, the role of nucleophilic thiols and amines in the pyrrole-mediated cross-linking reaction was investigated. When pyrrolylated ribonuclease was incubated with N-acetyllysine, N-acetylcysteine, or glutathione in physiologic buffer (pH 7.4) under air, pyrrole-to-pyrrole cross-linking was inhibited only by the thiol-containing compounds. Stable thiol--pyrrole conjugates containing a bridge from the pyrrole ring at C-3 to the sulfur atom of the thiol were characterized by thermospray LC/MS and 1H-NMR spectroscopy. In contrast to low-molecular-mass thiols, SDS--PAGE studies indicated that, under the same incubation conditions, free thiols present in proteins did not undergo reaction with pyrrole adducts to form cross-links. Further experiments using a low-molecular-mass pyrrole derivative indicated that glutathione may also able to suppress pyrrole dimerization without conjugate formation, possibly via inhibition of a free radical-dependent mechanism. The results suggest the following: (1) 2,5-HD-induced protein cross-linking is mediated primarily by pyrrole-to-pyrrole bridging under physiologic conditions, and (2) glutathione and other low-molecular-mass thiols may inhibit the pyrrole dimerization reaction by two distinct pathways. These findings have significant implications for the mechanism of gamma-diketone neuropathy.
正己烷代谢生成γ-二酮2,5-己二酮（2,5-HD），该衍生物与神经丝（NF）蛋白中的赖氨酸残基共价结合，生成2,5-二甲基吡咯加合物。比较γ-二酮的吡咯形成潜力和神经毒性效力的研究表明，吡咯化是神经发病机制中的一个绝对必要条件。吡咯化NF蛋白发生自氧化交联，并被认为是第二个必要事件。在本研究中，研究了亲核硫醇和胺在吡咯介导的交联反应中的作用。当吡咯化的核糖核酸酶在空气存在下于生理缓冲液（pH 7.4）中与N-乙酰赖氨酸、N-乙酰半胱氨酸或谷胱甘肽一起孵育时，仅含硫醇的化合物会抑制吡咯与吡咯的交联。通过热喷雾液相色谱/质谱和1H-核磁共振光谱对含有从吡咯环的C-3到硫醇硫原子的桥的稳定硫醇-吡咯共轭物进行了表征。与低分子量硫醇不同，SDS-PAGE研究表明，在相同的孵育条件下，蛋白质中存在的游离硫醇不会与吡咯加合物发生反应形成交联。使用低分子量吡咯衍生物的进一步实验表明，谷胱甘肽也可能能够在不形成共轭物的情况下抑制吡咯二聚化，可能是通过抑制自由基依赖性机制。结果表明：（1）2,5-HD诱导的蛋白质交联在生理条件下主要由吡咯与吡咯的桥接介导，（2）谷胱甘肽和其他低分子量硫醇可能通过两种不同途径抑制吡咯二聚化反应。这些发现对γ-二酮神经病变的机制具有重要意义。
