Online Enzyme Discrimination and Determination of Substrate Enantiomers Based on Electrophoretically Mediated Microanalysis

Abstract

We proposed the first application of an electrophoretically mediated microanalysis (EMMA) method for fast online discrimination and determination of substrate enantiomers, which was achieved by just one EMMA assay. Lactate dehydrogenase (LDH)-catalyzed reaction was studied to evaluate the feasibility and performance of the presented method. The L- and D-LDH chiral enzymatic reactions, which are highly stereoselective to the lactate enantiomers, were initiated successively in one capillary, and the corresponding products, nicotinamide adenine dinucleotide (NADH), were online discriminated and detected by UV absorption. Excellent linear dependence of the two NADH peak intensities on the concentration of the corresponding lactate enantiomers was obtained within a wide range of 0.1-10 mM. The limit of detection was 26 μM for D-lactate and 49 μM for L-lactate (S/N = 3). Good repeatability of online chiral discrimination was demonstrated with relative standard deviation (RSD) < 6.3% for NADH peak height and RSD < 1.5% for migration time (n = 5). K(m) values for L- and D-lactate were measured and were consistent with those of the off-line enzyme assays. The presented method was successfully applied to determine the L-/D-lactate in several yogurt and wine samples. Our study shows a new application of the EMMA method utilizing high stereoselectivity of enzymes for fast online chiral analysis.

Advances in enzyme substrate analysis with capillary electrophoresis

“…Most of the on-line incubations described in the paper either use the same pH [13,18,23,24,34,43,56–62], or a pH close to enzyme reaction [26,32,33,41,55]. In some instances a wide difference of pH existed between the enzyme incubation buffer and the separation buffer [31,63].…”

“…On the other hand, the on-line approach initiates enzymatic reaction through mixing in-capillary. The on-line mixing of enzyme, substrate, and inhibitor has been accomplished through electrophoretically mediated microanalysis [58,59,62,63,66,73], transverse diffusion of laminar flow profiles [21,31,41,74,75], and pressure mediated microanalysis [45]. On-line analysis with the use of immobilized enzyme for inhibitor studies have also been developed and high throughput analysis was achieved [18,22,23,56,68,76–78].…”

Advances in Capillary Electrophoresis-Based Enzyme Assays

“…Consequently, the drug and metabolite enantiomers were separated in the CD-containing background electrolyte. Zhao et al presented an interesting approach for enantiomer analysis without using a chiral selector [93]. They exploited the stereospecificity of lactate dehydrogenase (LDH) enzymes for the determination of the enantiomers of lactic acid via alternating analysis by l-LDH and d-LDH which selectively oxidize l-lactic acid and d-lactic acid, respectively.…”

Human protein kinase inhibitor screening by capillary electrophoresis using transverse diffusion of laminar flow profiles for reactant mixing

“…Electrophoretically mediated microanalysis (EMMA) is the most widely used in-capillary mixing technique for enzymatic studies [24][25][26][27]. However, mixing more than two reactants by EMMA is difficult [8,25,26,28].…”