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Capillary electrophoretic assay of human acetyl‐coenzyme A carboxylase 2
Author(s) -
Nguyen Thu H.,
Waldrop Grover L.,
Gilman S. Douglass
Publication year - 2019
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201800514
Subject(s) - chemistry , coenzyme a , pyruvate carboxylase , magnesium , capillary electrophoresis , cofactor , chromatography , adenosine triphosphate , biochemistry , adenosine diphosphate , adenosine , enzyme , biology , reductase , organic chemistry , platelet , platelet aggregation , immunology
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 Mg 2+ 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 Mg 2+ 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 MgCl 2 , 2.5 mM KHCO 3 , 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.