Determination of thioredoxin reductase activity in rat liver supernatant
Determination of thioredoxin reductase activity in rat liver supernatant
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DOI：
10.1006/abio.1997.2373
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发表时间：
1997-11-01
影响因子：
2.9
通讯作者：
Burk, RF
中科院分区：
文献类型：
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作者：
Hill, KE;McCollum, GW;Burk, RF
硫氧还蛋白还原酶最近被证明是一种硒酶(1)。Holmgren和Björnstedt设计了一种测量生物样品中硫氧还蛋白还原酶活性的方法，该方法基于NADPH和大肠杆菌硫氧还蛋白存在时胰岛素的减少(2)。我们修改了他们的方法，通过加入一个55℃的加热步骤来获得更低的背景值(3)。然而，这种检测方法仍然耗时且昂贵。一种基于硫氧还蛋白还原酶直接还原5,5 -二硫比斯（2-硝基苯甲酸）（DTNB） 2的更简单的测定方法已被用于在纯化过程中跟踪酶(2)。如上所述，这种基于DTNB的测定不适合用于活性的定量，因为除了硫氧还蛋白还原酶或内源性硫醇（如谷胱甘肽）外，它没有对DTNB的还原进行校正。因此，当与组织组分一起使用时，该分析具有非常高的背景。我们已经修改了基于DTNB的分析，使非硫氧还蛋白还原酶DTNB还原可以被确定和减去。
Thioredoxin reductase was recently shown to be a selenoenzyme (1). Holmgren and Björnstedt devised a method for measurement of thioredoxin reductase activity in biological samples based on the reduction of insulin in the presence of NADPH and Escherichia coli thioredoxin (2). We modified their method to achieve lower background values by including a 55C heating step (3). However, the assay remains time-consuming and expensive.A simpler assay based on the direct reduction of 5, 5-dithiobis (2-nitrobenzoic acid)(DTNB) 2 by thioredoxin reductase has been used to follow the enzyme during purification (2). As described, this DTNB-based assay was not suitable for quantitation of activity because it had no correction for DTNB reduction by enzymes other than thioredoxin reductase or by endogenous thiols such as glutathione. Thus, this assay has a very high background when used with tissue fractions. We have modified the DTNB-based assay so that non-thioredoxin reductase DTNB reduction can be determined and subtracted.