Valentine W M, Graham D G, Anthony D C
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710.
Toxicol Appl Pharmacol. 1993 Jul;121(1):71-7. doi: 10.1006/taap.1993.1130.
Covalent cross-linking of proteins by CS2 has been demonstrated in vitro and represents a potential mechanism for the toxicity of this compound. In the present investigation the ability of CS2 to cross-link proteins covalently in vivo is demonstrated using denaturing polyacrylamide gel electrophoresis. Intraperitoneal injection of CS2 in rats at 2 or 5 mmol/kg for 21 or 42 days produced several high-molecular-weight (approximately 410 kDa) proteins eluted from erythrocyte membranes which were not present in control animals. Limited proteolysis of the high-molecular-weight protein bands, monomeric alpha spectrin, and monomeric beta spectrin using endoproteinase glu-C, followed by peptide mapping on denaturing polyacrylamide gels, showed the high-molecular-weight proteins to be alpha,beta heterodimers. The production of multiple heterodimers exhibiting different distances of migration was consistent with the existence of several preferred sites for cross-linking. Evidence for the presence of dithiocarbamate ester and thiourea cross-linking structures in spectrin dimers was obtained using selective base hydrolysis. No spectrin dimer was detected in control animals, and dimer formation demonstrated a cumulative dose response in CS2-treated rats. The longevity of red blood cells, the cumulative dose response, and the stability of the cross-linking structures endows spectrin cross-linking with the potential to serve as a biomarker of chronic low-level exposures to CS2 and may provide a means to correlate pathological changes with existing methods of CS2 exposure monitoring. The ability of CS2 to covalently cross-link erythrocyte spectrin suggests that CS2 may also cross-link other proteins in vivo and supports covalent cross-linking of proteins as a possible molecular mechanism through which CS2 manifests toxicity. If so, then spectrin cross-linking may parallel cross-linking reactions in the axon and provide a sensitive, preneurotoxic biomarker of this molecular event.
二硫化碳（CS2）在体外已被证明可使蛋白质发生共价交联，这是该化合物毒性的一种潜在机制。在本研究中，使用变性聚丙烯酰胺凝胶电泳证明了CS2在体内使蛋白质共价交联的能力。以2或5 mmol/kg的剂量给大鼠腹腔注射CS2，持续21或42天，从红细胞膜上洗脱下来的几种高分子量（约410 kDa）蛋白质在对照动物中并不存在。使用内肽酶Glu-C对高分子量蛋白带、单体α血影蛋白和单体β血影蛋白进行有限的蛋白水解，然后在变性聚丙烯酰胺凝胶上进行肽图谱分析，结果表明高分子量蛋白质是α、β异二聚体。多个迁移距离不同的异二聚体的产生与存在几个优先交联位点一致。使用选择性碱水解获得了血影蛋白二聚体中存在二硫代氨基甲酸酯和硫脲交联结构的证据。在对照动物中未检测到血影蛋白二聚体，并且二聚体的形成在CS2处理的大鼠中表现出累积剂量反应。红细胞的寿命、累积剂量反应以及交联结构的稳定性使血影蛋白交联有可能作为慢性低水平接触CS2的生物标志物，并可能提供一种将病理变化与现有的CS2接触监测方法相关联的手段。CS2使红细胞血影蛋白共价交联的能力表明CS2也可能在体内使其他蛋白质交联，并支持蛋白质的共价交联作为CS2表现出毒性的一种可能分子机制。如果是这样，那么血影蛋白交联可能与轴突中的交联反应平行，并提供这种分子事件的敏感的神经毒性前生物标志物。
