Stereospecific electrophoretically mediated microanalysis assay for methionine sulfoxide reductase enzymes

• Published in: Analytical and bioanalytical chemistry 2014-02, Vol.406 (6), p.1723-1729
• Main Authors: Zhu, Qingfu, El-Mergawy, Rabab G., Heinemann, Stefan H., Schönherr, Roland, Jáč, Pavel, Scriba, Gerhard K. E.
• Format: Article
• Language: English
• Subjects: Analysis; Analytical Chemistry; Assaying; Biochemistry; Buffers; Characterization and Evaluation of Materials; Chemical properties; Chemistry; Chemistry and Materials Science; Chromatography; Diastereomers; Electric potential; Electrophoresis, Capillary - methods; Enzyme Assays - methods; Enzymes; Food Science; Fractionation; Health aspects; Human; Humans; Kinetics; Laboratory Medicine; Methionine; Methionine - analogs & derivatives; Methionine - chemistry; Methionine - metabolism; Methionine Sulfoxide Reductases - metabolism; Models, Molecular; Monitoring/Environmental Analysis; Oxidation; Polymers; Proteins; Reductases; Research Paper; Sodium; Stereoisomerism; Sulfates; Surface active agents; Transcription Factors - metabolism; Voltage
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-013-7596-4

An electrophoretically mediated microanalysis assay (EMMA) for the determination of the stereoselective reduction of l -methionine sulfoxide diastereomers by methionine sulfoxide reductase enzymes was developed using fluorenylmethyloxycarbonyl (Fmoc)- l -methionine sulfoxide as substrate. The separation of the diastereomers of Fmoc- l -methionine sulfoxide and the product Fmoc- l -methionine was achieved in a successive multiple ionic-polymer layer-coated capillary using a 50 mM Tris buffer, pH 8.0, containing 30 mM sodium dodecyl sulfate as background electrolyte and an applied voltage of 25 kV. 4-Aminobenzoic acid was employed as internal standard. An injection sequence of incubation buffer, enzyme, substrate, enzyme, and incubation buffer was selected. The assay was optimized with regard to mixing time and mixing voltage and subsequently applied for the analysis of stereoselective reduction of Fmoc- l -methionine-( S )-sulfoxide by human methionine sulfoxide reductase A and of the Fmoc- l -methionine-( R )-sulfoxide by human methionine sulfoxide reductase B. The Michaelis–Menten constant, K m , and the maximum velocity, v max , were determined. Essentially identical data were determined by the electrophoretically mediated microanalysis assay and the analysis of the samples by CE upon offline incubation. Furthermore, it was shown for the first time that Fmoc-methionine-( R )-sulfoxide is a substrate of human methionine sulfoxide reductase B. Figure Stereospecific EMMA for methionine sulfoxide reductase enzymes Methionine sulfoxide [Met(O)] which may be generated via oxidation by reactive oxygen species (ROS) is reduced by methionine sulfoxide reductase (Msr) enzymes in a stereospecific manner. The present assay allows the in-capillary incubation of recombinant human Msr enzymes followed by separation and analysis of the Met(O) diastereomers as well as the product methionine.