Application of liquid chromatography to a study on 4-coumarate: coenzyme A ligase activity.
This report describes the separation of components from a 4-coumarate:CoA ligase assay by means of liquid chromatography. With the aid of polyamide column chromatography it is possible to enrich and isolate chromatographically and UV spectroscopically pure p-coumaroyl-CoA using as a solvent 0.01% NH4OH in methanol subsequent to water and methanol alone. High performance liquid chromatography on octadecylsilane-bonded silica stationary phase allows a discontinuous determination of ligase activity. All components - ATP, Coenzyme A, p-coumaric acid, and the products AMP and p-coumaroyl-CoA - can be separated and accurately quantified within 20 min using a water-acetonitrile gradient, containing 1% phosphoric acid. The presented HPLC method may be used to affirm the accuracy of optical tests.
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003066:Coenzyme A Ligases / Q000378:metabolism*; D003373:Coumaric Acids / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet
D003373:Coumaric Acids; D003066:Coenzyme A Ligases; C019943:4-coumarate-CoA ligase