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Stereospecific electrophoretically mediated microanalysis assay for methionine sulfoxide reductase enzymes

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Abstract

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.

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.

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Abbreviations

BGE:

Background electrolyte

Dabsyl:

4-N,N-dimethylaminoazobenzene-4′-sulfonyl

DS:

Dextran sulfate sodium salt

DTT:

Dithiothreitol

EMMA:

Electrophoretically mediated microanalysis

Fmoc:

9-Fluorenylmethyloxycarbonyl

HDB:

Hexadimethrine bromide

hMsr:

Human methionine sulfoxide reductase

Met(O):

l-Methionine sulfoxide

Met-R-(O):

l-Methionine-(R)-sulfoxide

Met-S-(O):

l-Methionine-(S)-sulfoxide

Msr:

Methionine sulfoxide reductase

SMIL:

Successive multiple ionic-polymer layer

TFA:

Trifluoroacetic acid

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Acknowledgments

This work was supported by the Excellence Graduate School “Jena School for Microbial Communication (JSMC)”. Q. Zhu was supported by a stipend from the China Scholarship Council.

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Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, Friedrich Schiller University Jena, Philosophenweg 14, 07743, Jena, Germany

    Qingfu Zhu, Pavel Jáč & Gerhard K. E. Scriba

  2. Department of Biophysics, Friedrich Schiller University Jena, Hans-Knöll-Straße 2, 07745, Jena, Germany

    Rabab G. El-Mergawy, Stefan H. Heinemann & Roland Schönherr

  3. Jena University Hospital, Hans-Knöll-Straße 2, 07745, Jena, Germany

    Rabab G. El-Mergawy, Stefan H. Heinemann & Roland Schönherr

Authors
  1. Qingfu Zhu
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  2. Rabab G. El-Mergawy
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  3. Stefan H. Heinemann
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  4. Roland Schönherr
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  5. Pavel Jáč
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  6. Gerhard K. E. Scriba
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Correspondence to Gerhard K. E. Scriba.

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Zhu, Q., El-Mergawy, R.G., Heinemann, S.H. et al. Stereospecific electrophoretically mediated microanalysis assay for methionine sulfoxide reductase enzymes. Anal Bioanal Chem 406, 1723–1729 (2014). https://doi.org/10.1007/s00216-013-7596-4

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  • DOI: https://doi.org/10.1007/s00216-013-7596-4

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