title: A specific and rapid colorimetric method to monitor the activity of methionine sulfoxide reductase A.
abstract: Considerable evidence indicates that methionine sulfoxide (MetO) reductase A (MsrA) plays an important role in cytoprotection against oxidative stress and serves as a potential drug target. To screen for MsrA regulators, a rapid and specific assay to monitor MsrA activity is required. Most of current assays for MsrA activity are based on the reduction of radioactive substrates such as [3H]-N-acetyl-MetO or fluorescent derivatives such as dimethylaminoazo-benzenesulfonyl-MetO. However, these assays require extraction procedures and special instruments. Here, we developed a specific colorimetric microplate assay for testing MsrA activity quickly, which was based on the fact that MsrA can catalyze the reduction of methyl sulfoxides and simultaneously oxidize dithiothreitol (DTT), whose color can be produced by reacting with Ellman's reagent (dithio-bis-nitrobenzoic acid, DTNB). The corresponding absorbance change at 412nm was recorded with a microplate reader as the reaction proceeded. This method to monitor MsrA activity is easy to handle. Our findings may serve as a rapid method for the characterization of recombinant enzyme and for the screening of enzyme inhibitors, pharmacological activators, gene expression regulators and novel substrates.
authors: Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, China; The Key Laboratory of Neurological Diseases of the Ministry of Education of China, China; The Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China.
authors: Peng-Fei
authors: PF
authors: Wu
authors: Zui
authors: Z
authors: Zhang
authors: Xin-Lei
authors: XL
authors: Guan
authors: Yuan-Long
authors: YL
authors: Li
authors: Jian-Hua
authors: JH
authors: Zeng
authors: Juan-Juan
authors: JJ
authors: Zhang
authors: Li-Hong
authors: LH
authors: Long
authors: Zhuang-Li
authors: ZL
authors: Hu
authors: Fang
authors: F
authors: Wang
authors: Jian-Guo
authors: JG
authors: Chen
mesh_terms: D000818:Animals; D003124:Colorimetry / Q000379:methods*; D004228:Dithionitrobenzoic Acid; D004229:Dithiothreitol / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018384:Oxidative Stress; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D013454:Sulfoxides / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013454:Sulfoxides; D004228:Dithionitrobenzoic Acid; D010088:Oxidoreductases; C488525:MsrA protein, rat; D004229:Dithiothreitol
keywords: Colorimetric detection; DABS; DMSO; DTNB; DTT; Dithiothreitol; IPTG; LC-ESI-ITMS; MetO; Methionine sulfoxide reductase; MsrA; ROS; RSV; Trx; dimethyl aminoazo-benzenesulfonyl; dimethyl sulphoxide; dithio-bis-nitrobenzoic acid; dithiothreitol; isopropyl-β-d-thiogalactopyranoside; liquid chromatography coupled with electrospray ionization-ion trap mass spectrometry; methionine sulfoxide; methionine sulfoxide reductase A; reactive oxygen species; resveratrol; thioredoxin