Rigobello Maria Pia, Bindoli Alberto
Department of Biological Chemistry, University of Padova, Padova, Italy.
Methods Enzymol. 2010;474:109-22. doi: 10.1016/S0076-6879(10)74007-6. Epub 2010 Jun 20.
Mitochondrial thioredoxin reductase (TrxR2) maintains thioredoxin (Trx2) in a reduced state and plays a critical role in mitochondrial and cellular functions. TrxR2 has been identified in many different tissues and can be purified to homogeneity from whole organs and isolated mitochondria. Here we describe the detailed steps required to purify this enzyme. A different initial procedure is needed, according to whether purification starts from whole organs or from isolated and purified mitochondria. In the first case, acid precipitation is a critical preliminary step to separate mitochondrial thioredoxin reductase from the cytosolic isoform. Preparation involves ammonium sulfate fractionation, heating, and freeze/thaw cycles, followed by chromatographic passages involving DEAE-Sephacel, 2',5'-ADP-Sepharose 4B affinity, and omega-Aminohexyl-Sepharose 4B columns. The 2',5'-ADP-Sepharose 4B affinity step can be repeated to remove any contaminating glutathione reductase completely. Although several methods are available to detect the activity of this enzyme, reduction of DTNB is an easy and inexpensive test that can be applied not only to the highly purified enzyme but also to lysed mitochondria, provided non-TrxR2-dependent reaction rates are subtracted. TrxR2, like TrxR1, can be inhibited by several different and chemically unrelated substances, usually acting on the C-terminal containing the cysteine-selenocysteine active site. Many of these inhibitors react preferentially with the reduced form of the C-terminal tail. This condition can be evaluated by estimating enzyme activity after removal of the inhibitor by gel filtration of the enzyme preincubated in oxidizing or reducing conditions. Inhibition of thioredoxin reductase has important consequences for cell viability and can lead to apoptosis. Inhibition of TrxR2 causes large production of hydrogen peroxide, which diffuses from the mitochondrion to the cytosol and is responsible for most of the signaling events observed. Methods to measure hydrogen peroxide in isolated mitochondria or cultured cells are described.
线粒体硫氧还蛋白还原酶（TrxR2）可维持硫氧还蛋白（Trx2）处于还原状态，并在线粒体和细胞功能中发挥关键作用。TrxR2已在许多不同组织中被鉴定出来，并且可以从整个器官和分离的线粒体中纯化至同质状态。在此，我们描述了纯化该酶所需的详细步骤。根据纯化是从整个器官还是从分离纯化的线粒体开始，需要不同的初始程序。在第一种情况下，酸沉淀是将线粒体硫氧还蛋白还原酶与胞质同工型分离的关键初步步骤。制备过程包括硫酸铵分级分离、加热和冻融循环，随后进行涉及DEAE-琼脂糖凝胶、2',5'-二磷酸腺苷-琼脂糖4B亲和柱和ω-氨基己基-琼脂糖4B柱的色谱步骤。2',5'-二磷酸腺苷-琼脂糖4B亲和步骤可以重复进行，以完全去除任何污染的谷胱甘肽还原酶。尽管有几种方法可用于检测该酶的活性，但DTNB的还原是一种简单且廉价的测试，不仅可以应用于高度纯化的酶，也可以应用于裂解的线粒体，前提是减去非TrxR2依赖性反应速率。与TrxR1一样，TrxR2可被几种不同且化学性质无关的物质抑制，这些物质通常作用于含有半胱氨酸-硒代半胱氨酸活性位点的C末端。这些抑制剂中的许多优先与C末端尾巴的还原形式反应。这种情况可以通过在氧化或还原条件下预孵育酶后通过凝胶过滤去除抑制剂来估计酶活性来评估。硫氧还蛋白还原酶的抑制对细胞活力有重要影响，并可导致细胞凋亡。TrxR2的抑制会导致大量过氧化氢产生，过氧化氢从线粒体扩散到细胞质中，并导致观察到的大多数信号事件。本文描述了在分离的线粒体或培养细胞中测量过氧化氢的方法。
