Michaelis-Menten analysis of bovine plasma amine oxidase by capillary electrophoresis using electrophoretically mediated microanalysis in a partially filled capillary

Abstract

A method for determining bovine plasma amine oxidase (PAO; EC 1.4.3.6) activity with benzylamine (Bz) as substrate is described. Electrophoretically mediated microanalysis (EMMA) combined with micellar electrokinetic capillary chromatography (MEKC) was used to perform an on-capillary enzymatic reaction and to separate the generated benzaldehyde from the other reaction products. The capillary was only partially filled with the separation solution, since the enzyme was unstable in the presence of the applied surfactant. The initial reaction velocity of the enzyme-catalyzed reaction was estimated from the peak area of the enzyme product, benzaldehyde. An amplification step was introduced by means of an on-capillary incubation of 15 min, in order to accumulate enough reaction product to detect spectrophotometrically at 254 nm. This set-up resulted in a fully automated assay, which can be carried out in less then 35 min. Using the Lineweaver-Burk equation, an average Michaelis constant (K(M)) for PAO was calculated to be 0.74 mM +/- 0.05 mM, which is consistent with previously reported values.

Fully automated electrophoretically mediated microanalysis for CYP1A1 activity monitoring optimized by multivariate approach

“…For the EMMA procedure we performed the partial‐filling mode developed by Van Dyck et al . First, a plug (−50 mbar for 6 s) containing CYP1A1 supersomes was hydrodynamically injected into a fused‐silica capillary, followed by a plug (−50 mbar for 6 s) that contained the cofactor (NADPH) and the substrate (7‐EC), then another plug (−50 mbar for 6 s) of CYP1A1 supersomes was injected (sandwich mode).…”

Screening bioactive compounds from natural product and its preparations using capillary electrophoresis

“…There is a paper reported that main phenolic acids and flavonoids in tomato were separated and determined simultaneously using MEKC. Sigrid Van et al achieved on‐line detection of the activity of bovine plasma amine oxidase. CZE was applied at early stage of the study, which is not able to separate benzylamine from other substrates.…”

Advances in Capillary Electrophoretically Mediated Microanalysis for On‐line Enzymatic and Derivatization Reactions

“…After applying a certain voltage, the plugs will overlap each other and thus the incubation is initialized. If, at this stage, the voltage is turned off, the reaction product is accumulated, which is called ‘zero potential amplification’ .…”

“…When the enzymatic reaction is not compatible with the background electrolyte (BGE), the plugs of enzyme and substrate can be separated from the BGE by injecting plugs of incubation buffer in between. This method is named ‘partial filling’ . In a study by Farcaş et al.…”