Stereospecific micellar electrokinetic chromatography assay of methionine sulfoxide reductase activity employing a multiple layer coated capillary

Zhu, Qingfu; El-Mergawy, Rabab G.; Heinemann, Stefan H.; Schönherr, Roland; Jáč, Pavel; Scriba, Gerhard K. E.

doi:10.1002/elps.201300147

Cited by 8 publications

(9 citation statements)

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“…Consequently, two types of Msr enzymes, termed MsrA and MsrB exist, which reduce the S-and the R-sulfoxide, respectively, in a stereospecific manner [38]. While the diastereomers of Met(O) labeled with dabsyl chloride [29] or fluorenylmethyloxycarbonyl (Fmoc) chloride [37] could be separated by MEKC methods employing SDS as surfactant in sodium phosphate buffer, pH 8.0, as background electrolyte, the resolution of the diastereomeric pentapeptide derivative ac-Lys-Ile-Phe-Met(O)-Lys-Dnp required the presence of the achiral crown ether 15-crown-5 in combination with sulfated β-CD in the background electrolyte [34]. The assays were applied to study the stereospecific reduction of Met(O) either in the free form or in peptide substrates by recombinant human and fungal Msr enzymes including the determination of the Michaelis-Menten kinetic data for the various substrates.…”

Section: Pre-capillary Enzyme Assaysmentioning

confidence: 99%

“…Furthermore, assays for monitoring the stereospecificity of enzymatic reactions have been reported. A series of assays was developed for monitoring the stereospecificity of methionine sulfoxide reductase (Msr) enzymes which are ubiquitous enzymes in living cells reducing free methionine sulfoxide [Met(O)] or Met(O) bound in proteins [28,29,36,37]. Due to the chirality of the sulfoxide moiety, Met(O) exists as a pair of diastereomers.…”

Section: Pre-capillary Enzyme Assaysmentioning

confidence: 99%

“…In-capillary derivatization was accomplished by mixing the plugs of the reagent phthalic anhydride and the samples. While the reagent was injected hydrodynamically Enzyme activity [28] l-Methionine sulfoxide 35 mM sodium phosphate, pH 8.0, 25 mM SDS Derivatization with dabsyl chloride, enzyme kinetic data [29] ac-Lys-Ile-Phe-Met(O)-Lys-Dnp 50 mM Tris, pH 7.85, 5 mM 15-crown-5, 14.3 mg mL −1 sulfated β-CD Enzyme kinetic data [36] Fmoc-l-methionine sulfoxide 50 mM sodium phosphate, pH 8.0, 30 mM SDS Enzyme kinetic data [37] Sirtuin 1 N ε -Acetyl-N α -(4-methyl-7-Methoxycoumarin)lysine amide 200 mM phosphate/Tris, pH 2.85…”

Section: Pre-capillary Enzyme Assaysmentioning

confidence: 99%

“…When using substrates without UV chromophores, derivatization of the products and/or substrates can be performed. This has been realized for amino acid-derived substrates by derivatization with dabsyl chloride [28,29], dansyl chloride [30][31][32], fluorenylmethyloxycarbonyl (Fmoc) chloride [33] or phenylisothiocyanate [34]. Carbohydrate substrates were derivatized by reductive amination with 7-aminonaphthalene-1,3-disulfonic acid [35].…”

Section: Pre-capillary Enzyme Assaysmentioning

confidence: 99%

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Advances in Capillary Electrophoresis-Based Enzyme Assays

Scriba

Belal

2015

Chromatographia

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IntroductionEnzymes catalyze metabolic reactions in cells regulating homeostasis, growth and reproduction of living organisms. Enzymes are also often involved in human disease. The analysis of the human genome has revealed that within the so-called druggable genome a significant portion (in some analyses more than half) of the potential drug targets are enzymes [1][2][3]. Consequently, enzymes are important targets in the development of new drugs. Moreover, enzymes play a role in the clinical diagnosis of diseases. Therefore, effective methods for characterization of enzymes as well as for the determination of their activity are important, for example, for the understanding of enzyme-mediated metabolic reactions or the identification of substrates and inhibitors for the treatment of human diseases.Capillary electrophoresis (CE) has been applied to enzyme analysis for more than 20 years since the first report by Banke et al. on an assay of alkaline protease [4]. Since then, all aspects of enzyme-related analysis have been studied by CE methods including the evaluation of enzymatic activity, enzyme kinetics, identification and characterization of enzyme inhibitors and activators as well as metabolic pathways as, for example, summarized in [5][6][7][8][9][10][11][12].Generally, CE-based enzyme assays can be divided in three groups, pre-capillary (offline) assays, in-capillary (online) assays and post-capillary assays. In the pre-capillary assays, all required components including enzyme, substrate, co-factors, etc., are mixed in a vial and incubated for an intended period of time. Subsequently, the reaction is quenched and an aliquot is subjected to CE analysis for the determination of substrate(s) and/or co-substrate(s) and/ or product(s). Multiple sampling or repeated sampling in combination with sequential runs for the determination of Abstract Capillary electrophoresis (CE) has become a flexible and accurate, high-efficiency analytical separation technique in many areas requiring only minute amounts of sample and chemicals. Thus, CE has also been recognized as a suitable technique to study enzymatic reactions including the determination of Michaelis-Menten kinetic data or the identification and characterization of inhibitors. The most often applied CE-based enzyme assay modes can be divided into two categories: (1) pre-capillary assays where incubations are performed offline followed by CE analysis of substrate(s) and/or product(s) and (2) in-capillary assays in which the enzymatic reaction and analyte separation are performed in the same capillary. In case of the in-capillary assays, the enzyme may be immobilized or in solution. The latter is also referred to as electrophoretically mediated microanalysis (EMMA), while in the case of immobilized enzyme the term immobilized enzyme reactor (IMER) is used. The present review summarizes the literature on CEbased enzyme assays published between January 2010 and April 2015. Immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic a...

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Section: Pre-capillary Enzyme Assaysmentioning

confidence: 99%

Section: Pre-capillary Enzyme Assaysmentioning

confidence: 99%

Section: Pre-capillary Enzyme Assaysmentioning

confidence: 99%

Section: Pre-capillary Enzyme Assaysmentioning

confidence: 99%

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Advances in Capillary Electrophoresis-Based Enzyme Assays

Scriba

Belal

2015

Chromatographia

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“…Mixing time and mixing voltage were optimized and applied to human reductases A and B . An similar method using dansyl (4‐ N , N ‐dimethylaminoazobenzene‐4‐sulfonyl) labeling has also been published .…”

Section: Separation Of Enantiomersmentioning

confidence: 99%

Recent advances in amino acid analysis by capillary electromigration methods, 2013–2015

et al. 2015

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We describe the most important research articles published on amino acid analysis using CE during the period from June 2013 to May 2015, and follows the format of the previous articles published in electrophoresis the new developments in amino acid analysis with CE are mainly describing improvements in detection means and injection methods. Enantiomeric separation developments are still important. Focusing the applications, we describe the neurochemical and clinical works, but also the metabolomic studies for which the publication number increase greatly. Finally, works focused on amino acids in food and agricultural applications are described.

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The discovery of methionine sulfoxide reductase enzymes: An historical account and future perspectives

2015

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L-Methionine (L-Met) is the only sulphur-containing proteinogenic amino acid together with cysteine. Its importance is highlighted by it being the initiator amino acid for protein synthesis in all known living organisms. L-Met, free or inserted into proteins, is sensitive to oxidation of its sulfide moiety, with formation of L-Met sulfoxide. The sulfoxide could not be inserted into proteins, and the oxidation of L-Met in proteins often leads to the loss of biological activity of the affected molecule. Key discoveries revealed the existence, in rats, of a metabolic pathway for the reduction of free L-Met sulfoxide and, later, in Escherichia coli, of the enzymatic reduction of L-Met sulfoxide inserted in proteins. Upon oxidation, the sulphur atom becomes a new stereogenic center, and two stable diastereoisomers of L-Met sulfoxide exist. A fundamental discovery revealed the existence of two unrelated families of enzymes, MsrA and MsrB, whose members display opposite stereospecificity of reduction for the two sulfoxides. The importance of Msrs is additionally emphasized by the discovery that one of the only 25 selenoproteins expressed in humans is a Msr. The milestones on the road that led to the discovery and characterization of this group of antioxidant enzymes are recounted in this review.

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Stereospecific micellar electrokinetic chromatography assay of methionine sulfoxide reductase activity employing a multiple layer coated capillary

Cited by 8 publications

References 42 publications

Advances in Capillary Electrophoresis-Based Enzyme Assays

Advances in Capillary Electrophoresis-Based Enzyme Assays

Recent advances in amino acid analysis by capillary electromigration methods, 2013–2015

The discovery of methionine sulfoxide reductase enzymes: An historical account and future perspectives

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