Gergel' D, Cederbaum A I
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
Arch Biochem Biophys. 1997 Nov 15;347(2):282-8. doi: 10.1006/abbi.1997.0352.
Nitric oxide (NO) in an aerobic environment, reacts with the sulfhydryl groups of proteins to form nitroso thiols. Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid), DTNB, is widely used for the determination of -SH groups. In this procedure, DTNB, a symmetric aryl disulfide, reacts with the free thiol to give a mixed disulfide plus 2-nitro-5-thiobenzoic acid (TNB) which is quantified by its absorbance at 412 nm. We observed that the presence of NO during the determination of SH groups in a reaction system containing glutathione (GSH) or bovine serum albumin (BSA) plus DTNB resulted in an inhibition in the detection of TNB. Addition of NO donors or NO gas after TNB was already formed led to the bleaching of yellow color and loss of absorbance at 412 nm. These interactions did not occur under anaerobic conditions. Decreased formation of TNB therefore appeared to be due not only to destruction of SH groups of BSA or GSH by NO (S-nitrosation) and consequently to lower TNB formation, but also to direct reaction of NO/O2 with TNB. The mechanism(s) of inhibition of accumulation of TNB by NO was evaluated. NO generated by DEA/NO, SNAP, or spermine/NO, as well as gaseous NO or BSA-NO, directly interacted with TNB, followed by decreased absorbance at 412 nm in a concentration- and time-dependent manner. Kinetics of NO/O2 interaction with TNB were dependent on the ability of the NO donors to release NO as the donors with a short half-life bleached the yellow color of TNB faster. The requirement for O2 suggests that nitrogen oxide or higher oxides of NOx are responsible for interaction with TNB. The UV/VIS spectrum of the final product formed during the interaction of NO with TNB was identical to that of DTNB. These results suggest that interaction of NO (NOx) with TNB resulted in the formation of an unstable nitrosothiol, followed by oxidation and dimerization back to the corresponding disulfide, DTNB. Therefore, determination of SH groups in proteins by Ellman's reagent after or in the presence of NO treatment is complicated since the reduced form of DTNB, TNB, can be reoxidized by NO back to DTNB, with subsequent loss of absorbance at 412 nm.
在有氧环境中，一氧化氮（NO）与蛋白质的巯基反应形成亚硝基硫醇。埃尔曼试剂，即5,5'-二硫代双（2-硝基苯甲酸），DTNB，被广泛用于测定巯基。在这个过程中，DTNB，一种对称的芳基二硫化物，与游离硫醇反应生成混合二硫化物和2-硝基-5-硫代苯甲酸（TNB），通过其在412nm处的吸光度进行定量。我们观察到，在含有谷胱甘肽（GSH）或牛血清白蛋白（BSA）加DTNB的反应体系中测定巯基时，NO的存在会导致TNB检测受到抑制。在TNB已经形成后添加NO供体或NO气体，会导致黄色褪色以及在412nm处吸光度丧失。这些相互作用在厌氧条件下不会发生。因此，TNB生成减少似乎不仅是由于NO对BSA或GSH的巯基的破坏（S-亚硝基化），从而导致TNB生成降低，还由于NO/O₂与TNB的直接反应。评估了NO抑制TNB积累的机制。DEA/NO、SNAP或精胺/NO产生的NO，以及气态NO或BSA-NO，直接与TNB相互作用，随后在412nm处的吸光度以浓度和时间依赖性方式降低。NO/O₂与TNB相互作用的动力学取决于NO供体释放NO的能力，因为半衰期短的供体使TNB的黄色褪色更快。对O₂的需求表明氮氧化物或更高价的NOx氧化物是与TNB相互作用的原因。NO与TNB相互作用过程中形成的最终产物的紫外/可见光谱与DTNB的相同。这些结果表明，NO（NOx）与TNB的相互作用导致形成不稳定的亚硝基硫醇，随后氧化并二聚化回到相应的二硫化物DTNB。因此，在NO处理后或存在NO的情况下，用埃尔曼试剂测定蛋白质中的巯基会很复杂，因为DTNB的还原形式TNB会被NO重新氧化回DTNB，随后在412nm处的吸光度丧失。
