Capillary electrophoretic assay of human acetyl‐coenzyme A carboxylase 2

Abstract

Human acetyl‐coenzyme A carboxylase 2 catalyzes the carboxylation of acetyl coenzyme A to form malonyl coenzyme A, along with the conversion of magnesium‐adenosine triphosphate complex to magnesium‐adenosine diphosphate complex. A simple off‐column capillary electrophoresis assay for human acetyl‐coenzyme A carboxylase 2 was developed based on the separation of magnesium‐adenosine triphosphate complex, magnesium‐adenosine diphosphate complex, acetyl coenzyme A and malonyl coenzyme A with detection by ultraviolet absorption at 256 nm. When Mg2+ was absent from the separation buffer, the zones due to magnesium‐adenosine triphosphate complex and magnesium‐adenosine diphosphate complex both split and migrated as two separate peaks. With Mg2+ added to the separation buffer, magnesium‐adenosine triphosphate complex and magnesium‐adenosine diphosphate complex produced single peaks, and the reproducibility of peak shape and area improved for human acetyl‐coenzyme A carboxylase 2 assay components. The final separation buffer used was 30.0 mM HEPES, 3.0 mM MgCl2, 2.5 mM KHCO3, and 2.5 mM potassium citrate at pH 7.50. The same buffer was used for the enzyme‐catalyzed reaction (off‐column). Inhibition of human acetyl‐coenzyme A carboxylase 2 by CP‐640186, a known inhibitor, was detected using the capillary electrophoresis assay.

Challenging Bioanalyses with Capillary Electrophoresis

“…CE has been used to compare inhibitors of native and recombinant enzymes . In the period under review, inhibitors of enzymes in free solution have been characterized with UV detection for ecto-5-nucleotidase, thrombin, histone deacetylase, glucosamine-6 phosphate synthase, and human acetyl-CoA carboxylase 2 and with laser-induced fluorescence for d -amino acid oxidase and BCR-ABL . Further, inhibition studies have been done for immobilized enzymes including acetylcholinesterase, , alanine aminotransferase, β-glucosidase, and sulfotransferase 1A1 using UV detection.…”

Application of Capillary Electrophoresis with Laser-Induced Fluorescence to Immunoassays and Enzyme Assays

“…The extensive studies of enzymes as current drug targets have encouraged researchers to seek effective methods to characterize enzyme activities and understand their roles in human diseases. Such activities include enzyme kinetics, enzyme substrate identification, enzyme inhibitor screening, and enzyme-mediated metabolic pathways, and these aspects of enzyme-related analysis have been largely studied by CE methods, for example, those summarized in [96,97,98,99]. A typical CE-based enzyme assay starts with small amounts of the substrate, enzyme, and/or inhibitors.…”