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A specific and rapid colorimetric method to monitor the activity of methionine sulfoxide reductase A

Paper ID Volume ID Publish Year Pages File Format Full-Text
17069 42638 2013 7 PDF Available
Title
A specific and rapid colorimetric method to monitor the activity of methionine sulfoxide reductase A
Abstract

•MsrA-catalytic DTT oxidation reflects MsrA activity.•MsrA activity is monitored by Ellman's assay.•A rapid method for screening MsrA regulators.

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 412 nm 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.

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Keywords
DABS, dimethyl aminoazo-benzenesulfonyl; DMSO, dimethyl sulphoxide; DTNB, dithio-bis-nitrobenzoic acid; DTT, dithiothreitol; IPTG, isopropyl-β-d-thiogalactopyranoside; LC-ESI-ITMS, liquid chromatography coupled with electrospray ionization-ion trap mass s
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A specific and rapid colorimetric method to monitor the activity of methionine sulfoxide reductase A
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 53, Issues 6–7, 10 December 2013, Pages 391–397
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us