title: Experimental design-guided development of a stereospecific capillary electrophoresis assay for methionine sulfoxide reductase enzymes using a diastereomeric pentapeptide substrate.
abstract: A capillary electrophoresis method has been developed and validated to evaluate the stereospecific activity of recombinant human methionine sulfoxide reductase enzymes employing the C-terminally dinitrophenyl-labeled N-acetylated pentapeptide ac-KIFM(O)K-Dnp as substrate (M(O)=methionine sulfoxide). The separation of the ac-KIFM(O)K-Dnp diastereomers and the reduced peptide ac-KIFMK-Dnp was optimized using experimental design with regard to the buffer pH, buffer concentration, sulfated β-cyclodextrin and 15-crown-5 concentration as well as capillary temperature and separation voltage. A fractional factorial response IV design was employed for the identification of the significant factors and a five-level circumscribed central composite design for the final method optimization. Resolution of the peptide diastereomers as well as analyte migration time served as responses in both designs. The resulting optimized conditions included 50mM Tris buffer, pH 7.85, containing 5mM 15-crown-5 and 14.3mg/mL sulfated β-cyclodextrin, at an applied voltage of 25kV and a capillary temperature of 21.5°C. The assay was subsequently applied to the determination of the stereospecificity of recombinant human methionine sulfoxide reductases A and B2. The Michaelis-Menten kinetic data were determined. The pentapeptide proved to be a good substrate for both enzymes. Furthermore, the first separation of methionine sulfoxide peptide diastereomers is reported.
authors: Department of Pharmaceutical Chemistry, Friedrich Schiller University Jena, Philosophenweg 14, 07743 Jena, Germany.
authors: Qingfu
authors: Q
authors: Zhu
authors: School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, PR China.
authors: Xingyu
authors: X
authors: Huo
authors: Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, Hans-Knöll-Straße 2, 07745 Jena, Germany.
authors: Stefan H
authors: SH
authors: Heinemann
authors: Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, Hans-Knöll-Straße 2, 07745 Jena, Germany.
authors: Roland
authors: R
authors: Schönherr
authors: Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, Hans-Knöll-Straße 2, 07745 Jena, Germany.
authors: Rabab
authors: R
authors: El-Mergawy
authors: Department of Pharmaceutical Chemistry, Friedrich Schiller University Jena, Philosophenweg 14, 07743 Jena, Germany. Electronic address: gerhard.scriba@uni-jena.de.
authors: Gerhard K E
authors: GK
authors: Scriba
mesh_terms: D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008715:Methionine / Q000031:analogs & derivatives / Q000737:chemistry; D051150:Methionine Sulfoxide Reductases / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D008715:Methionine; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase; C013111:methionine sulfoxide
keywords: Electrokinetic chromatography.; Experimental design; Kinetic analysis; Methionine oxidation; Methionine sulfoxide