title: Stereospecific micellar electrokinetic chromatography assay of methionine sulfoxide reductase activity employing a multiple layer coated capillary.
abstract: A micellar electrokinetic chromatography method for the analysis of the l-methionine sulfoxide diastereomers employing a successive multiple ionic-polymer layer coated fused-silica capillary was developed and validated in order to investigate the stereospecificity of methionine sulfoxide reductases. The capillary coating consisted of a first layer of hexadimethrine and a second layer of dextran sulfate providing a stable strong cathodic EOF and consequently highly repeatable analyte migration times. The methionine sulfoxide diastereomers, methionine as product as well as β-alanine as internal standard were derivatized by dabsyl chloride and separated using a 35 mM sodium phosphate buffer, pH 8.0, containing 25 mM SDS as BGE and a separation voltage of 25 kV. The method was validated in the range of 0.15-2.0 mM with respect to linearity and precision. The LODs of the analytes ranged between 0.04 and 0.10 mM. The assay was subsequently applied to determine the stereospecificity of methionine sulfoxide reductases as well as the enzyme kinetics of human methionine sulfoxide reductase A. Monitoring the decrease of the l-methionine-(S)-sulfoxide Km = 411.8 ± 33.8 μM and Vmax = 307.5 ± 10.8 μM/min were determined.
authors.affiliation: Department of Pharmaceutical Chemistry, Friedrich Schiller University Jena, Jena, Germany.
authors.forename: Qingfu
authors.initials: Q
authors.lastname: Zhu
authors.forename: Rabab G
authors.initials: RG
authors.lastname: El-Mergawy
authors.forename: Stefan H
authors.initials: SH
authors.lastname: Heinemann
authors.forename: Roland
authors.initials: R
authors.lastname: Schönherr
authors.forename: Pavel
authors.initials: P
authors.lastname: Jáč
authors.forename: Gerhard K E
authors.initials: GK
authors.lastname: Scriba
mesh_terms: D020374:Chromatography, Micellar Electrokinetic Capillary / Q000295:instrumentation* / Q000379:methods*; D053841:Electroosmosis; D057075:Enzyme Assays; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D057230:Limit of Detection; D008715:Methionine / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D051150:Methionine Sulfoxide Reductases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D013237:Stereoisomerism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D008715:Methionine; D051150:Methionine Sulfoxide Reductases; C013111:methionine sulfoxide
keywords: Methionine oxidation; Methionine sulfoxide; Methionine sulfoxide reductase; Micellar electrokinetic chromatography; Successive multiple ionic-polymer layer