A chiral ligand exchange CE essay with zinc(II)– l-valine complex for determining enzyme kinetic constant of l-amino acid oxidase

• Published in: Talanta (Oxford) Vol. 81; no. 4; pp. 1554 - 1559
• Main Authors: Qi, Li, Yang, Gengliang, Zhang, Haizhi, Qiao, Juan
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.06.2010; Elsevier
• Subjects: Analytical chemistry; Animals; Biochemistry - methods; Catalysis; Chemistry; Chemistry Techniques, Analytical; Chromatographic methods and physical methods associated with chromatography; Electrophoresis, Capillary - instrumentation; Electrophoresis, Capillary - methods; Enantioseparation; Enzyme kinetics; Exact sciences and technology; Hydrogen-Ion Concentration; Keto Acids - chemistry; Kinetics; l-Amino acid oxidase; L-Amino Acid Oxidase - analysis; Ligand exchange CE; Ligands; Other chromatographic methods; Snake Venoms - enzymology; Snakes; Stereoisomerism; Substrate Specificity; Valine - chemistry; Zinc - chemistry; Zinc complex; LAAO; CLE-CE; AA; l-Amino acid oxidase; β-CD; KIC; Enantioseparation; Zinc complex; Dns-AAs; Enzyme kinetics; Ligand exchange CE; Ammonium; Chemical analysis; Separation; Ligand; Design; Boric acid; Efficiency; Enantiomer; Substrate specificity; pH; Quantitative analysis; Catalytic reaction; Capillary electrophoresis; Enzyme; Zinc II; Use; Acetate; Enzymatic method; Cyclodextrin; Valine; Aminoacid; Strategy; Kinetics; L-Amino acid oxidase
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2010.03.001

A new strategy for the enantioseparation of d, l-amino acids employing the principle of ligand exchange capillary electrophoresis with Zn(II)– l-valine complex as a chiral selecting system in the presence of β-cyclodextrin has been designed. Successful enantioseparation of label free and labeled amino acids have been achieved with a buffer of 100.0 mM boric acid, 5.0 mM ammonium acetate, 4.0 mM β-cyclodextrin, 4.0 mM ZnSO 4 and 8.0 mM l-valine at pH 8.1. This new method was shown to be applicable to the quantitative analysis of label free d- and l-aromatic amino acids. Furthermore, the expanding enzymatic use of l-amino acid oxidase to incubate with different l-amino acids has allowed understanding of the substrate's specificity. An on-column incubation assay has been developed to study the l-amino acid oxidase's catalytic efficiency. It was demonstrated that the enzyme kinetic constant could be determined by using this new method.