Chiral ligand-exchange CE assays for separation of amino acid enantiomers and determination of enzyme kinetic constant.
This paper deals with studies on the use of Zn(II)-L-ornithine complex as a chiral selecting system for the enantioseparation and UV detection of amino acids (AAs) by using the principle of ligand-exchange CE. Successful enantioseparation of three pairs of label-free aromatic AAs and four pairs of labeled AA enantiomers have been achieved with a buffer of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4 and 6.0 mM L-Orn at pH 8.2. This new method was shown to be applicable to the quantitative analysis of D- and L-aromatic AAs, with a linear range between 12.5 and 800.0 microg/mL, and a correlation coefficient above 0.99. Thus this assay, which is facile and relatively rapid, allows us to measure the enzyme catalytic activity in the incubation of D,L-AAs with D-AA oxidase. Using this new method, we can determine the enzyme kinetic constant, lending insight into potential enzyme mechanism.
Qi
Li
L
Beijing National Laboratory of Molecular Science, Laboratory of Analytical Chemistry for Life Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing, P. R. China. qili@iccas.ac.cn
Qiao
Juan
J
Yang
Gengliang
G
Chen
Yi
Y
D000596:Amino Acids / Q000737:chemistry / Q000302:isolation & purification*; D003605:D-Amino-Acid Oxidase / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007700:Kinetics; D050830:L-Amino Acid Oxidase / Q000737:chemistry; D008024:Ligands; D016014:Linear Models; D009942:Organometallic Compounds / Q000737:chemistry; D009952:Ornithine / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D015032:Zinc / Q000737:chemistry
D000596:Amino Acids; D008024:Ligands; D009942:Organometallic Compounds; D009952:Ornithine; D050830:L-Amino Acid Oxidase; D003605:D-Amino-Acid Oxidase; D015032:Zinc