Go Young-Mi, Pohl Jan, Jones Dean P
Department of Medicine, Emory University, Atlanta, GA, USA.
Methods Mol Biol. 2009;464:303-17. doi: 10.1007/978-1-60327-461-6_17.
Many nuclear proteins contain thiols, which undergo reversible oxidation and are critical for normal function. These proteins include enzymes, transport machinery, structural proteins, and transcription factors with conserved cysteine in zinc fingers and DNA-binding domains. Uncontrolled oxidation of these thiols causes dysfunction, and two major thiol-dependent antioxidant systems provided protection. The redox states of these systems, including the small redox active protein thioredoxin-1 (Trx1) and the abundant, low molecular weight thiol antioxidant glutathione (GSH), in nuclei provide means to quantify nuclear redox conditions. Redox measurements are obtained under conditions with excess thiol-reactive reagents. Here we describe a suite of methods to measure nuclear redox state, which include a redox Western blot technique to quantify the redox state of Trxl, a biotinylated iodoacetamide (BIAM) method for thioredoxin reductase-1 (TrxR1), GSH redox measurement using total protein S-glutathionylation, and a redox isotope-coded affinity tag (ICAT) method for measuring oxidation of specific cysteines in high-abundance nuclear proteins.
许多核蛋白含有硫醇，它们会发生可逆氧化，对正常功能至关重要。这些蛋白质包括酶、转运机制、结构蛋白以及在锌指和DNA结合域中具有保守半胱氨酸的转录因子。这些硫醇的不受控制的氧化会导致功能障碍，而两个主要的硫醇依赖性抗氧化系统提供了保护。这些系统的氧化还原状态，包括小的氧化还原活性蛋白硫氧还蛋白-1（Trx1）和丰富的低分子量硫醇抗氧化剂谷胱甘肽（GSH），在细胞核中提供了量化核氧化还原条件的方法。氧化还原测量是在存在过量硫醇反应试剂的条件下进行的。在这里，我们描述了一套测量核氧化还原状态的方法，包括用于量化Trxl氧化还原状态的氧化还原蛋白质印迹技术、用于硫氧还蛋白还原酶-1（TrxR1）的生物素化碘乙酰胺（BIAM）方法、使用总蛋白S-谷胱甘肽化进行GSH氧化还原测量，以及用于测量高丰度核蛋白中特定半胱氨酸氧化的氧化还原同位素编码亲和标签（ICAT）方法。
