Chiral ligand‐exchange CE assays for separation of amino acid enantiomers and determination of enzyme kinetic constant

Abstract

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.