Glutathione, glutathione disulfide, and S-glutathionylated proteins in cell cultures
Glutathione, glutathione disulfide, and S-glutathionylated proteins in cell cultures
复制标题
DOI：
10.1016/j.freeradbiomed.2015.10.410
复制
发表时间：
2015-12-01
影响因子：
7.4
通讯作者：
Rossi, Ranieri
中科院分区：
文献类型：
--
作者：
Giustarini, Daniela;Galvagni, Federico;Rossi, Ranieri
分析组织和细胞中的整体硫醇-二硫化物氧化还原状态是一项具有挑战性的任务，因为硫醇和二硫化物在样品操作过程中可能会发生人工氧化还原。因此，测量值（特别是二硫化物的测量值）可能存在显着偏差。尽管这一方法学问题已在红细胞和实体组织样品中得到解决，但以前尚未报道过测量细胞培养物中硫醇和二硫化物的可靠方法。在这里，我们证明，在培养细胞中硫醇和二硫化物分析过程中出现的主要伪影是谷胱甘肽二硫化物 (GSSG) 和 S-谷胱甘肽化蛋白 (PSSG) 过高估计，这是由于样品操作过程中谷胱甘肽 (GSH) 的人工氧化造成的，并且这个方法学问题可以通过在去除培养基后立即添加 N-乙基马来酰亚胺 (NEM) 来解决。当细胞未经NEM处理时，不同系培养细胞中GSSG和PSSG的基础水平分别高出3-5倍和10-20倍。 NEM 预处理还可以防止在细胞暴露于氧化剂刺激的预分析阶段发生的二硫化物的人为还原。事实上，在没有 NEM 的情况下，去除培养基后，由于还原酶的活性和氧化剂的缺乏，GSH、GSSG 和 PSSG 水平在 15-30 分钟内恢复到其初始值。新开发的方案用于测量常规用于生物医学研究的 16 种不同细胞系的硫醇-二硫化物氧化还原状态，无论是在基础条件下还是在用硫醇特异性氧化剂双硫仑（0-200 μM 浓度范围）处理后。我们的数据表明，在大多数细胞系中，双硫仑处理仅在最高浓度时影响 GSH 和 GSSG 的水平。另一方面，PSSG水平在较低药物浓度下也显着增加，并且对于几乎所有细胞系来说，增加是显着的（在200μM浓度下从100倍到1000倍）并且呈剂量依赖性。这些数据支持分析培养细胞中 PSSG 作为氧化应激生物标志物的适用性。 (C) 2015 Elsevier Inc. 保留所有权利。
The analysis of the global thiol-disulfide redox status in tissues and cells is a challenging task since thiols and disulfides can undergo artificial oxido-reductions during sample manipulation. Because of this, the measured values, in particular for disulfides, can have a significant bias. Whereas this methodological problem has already been addressed in samples of red blood cells and solid tissues, a reliable method to measure thiols and disulfides in cell cultures has not been previously reported.Here, we demonstrate that the major artifact occurring during thiol and disulfide analysis in cultured cells is represented by glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG) overestimation, due to artificial oxidation of glutathione (GSH) during sample manipulation, and that this methodological problem can be solved by the addition of N-ethylmaleimide (NEM) immediately after culture medium removal. Basal levels of GSSG and PSSG in different lines of cultured cells were 3-5 and 10-20 folds higher, respectively, when the cells were processed without NEM. NEM pre-treatment also prevented the artificial reduction of disulfides that occurs during the pre-analytical phase when cells are exposed to an oxidant stimulus. In fact, in the absence of NEM, after medium removal, GSH, GSSG and PSSG levels restored their initial values within 15-30 min, due to the activity of reductases and the lack of the oxidant. The newly developed protocol was used to measure the thiol-disulfide redox status in 16 different line cells routinely used for biomedical research both under basal conditions and after treatment with disulfiram, a thiol-specific oxidant (0-200 mu M concentration range).Our data indicate that, in most cell lines, treatment with disulfiram affected the levels of GSH and GSSG only at the highest concentration. On the other hand, PSSG levels increased significantly also at the lower concentrations of the drug, and the rise was remarkable (from 100 to 1000 folds at 200 mu M concentration) and dose-dependent for almost all the cell lines. These data support the suitability of the analysis of PSSG in cultured cells as a biomarker of oxidative stress. (C) 2015 Elsevier Inc. All rights reserved.