Off‐line and in‐line determination of catechol‐ O ‐methyltransferase activity in a capillary electrophoretic system

Abstract

The use of capillary electrophoresis for the determination of catechol-O-methyltransferase (COMT) activity with dihydroxybenzoic acid as a substrate was investigated. Both an off-line and in-line capillary electrophoresis determination of COMT activity was developed and the two approaches are discussed. In the presented methods, substrate and reaction products are monitored at the same time. The initial velocity of the reaction is quantified spectrophotometrically by the corrected peak area of the products at 200 nm. In the off-line setup, capillary zone electrophoresis is used to separate and quantify the different reaction compounds. Each electrophoretic run required only 37 nL of the enzymatic reaction solution. Based on the off-line assay, an in-line determination of COMT activity was developed by a methodology known as electrophoretically mediated microanalysis (EMMA). All the different steps (i.e. mixing, incubation, separation and in-line quantitation) are combined in the capillary, which is used as a microreactor for the enzymatic reaction. Full automation of the assay is achieved with this microscale approach.

Electrophoretically mediated microanalysis

“…In this variant, enzyme and substrate(s) are introduced into the capillary as distinct plugs, the first reactant injected being the one with the lower electrophoretic mobility [5,[26][27][28][29][30][31][32][33][34][35][36][37][38][39]. Upon the application of an electric field, the two zones interpenetrate due to differences in their electrophoretic mobilities.…”

Separation methods applicable to the evaluation of enzyme–inhibitor and enzyme–substrate interactions

“…Enzyme-substrate reactions are model systems for analysis using EMMA [109][110][111][112][113][114][115][116][117][118][119]. For example, the Glatz group has used EMMA to analyze the activity of rhodanese, the enzyme which catalyzes the reaction of thiosulfate with cyanide to form thiocyanate and sulfite [120][121][122].…”

Electrochemical Activity Determination of Catechol‐O‐methyl Transferase by Selective Dopamine Detection

“…[45,46] Another effective separation strategy exploits capillary electrophoresis for the detection of dopamine conversion . [47] Fluorine doped tin oxide (FTO) is one electrode material which can be found in actual sensor developments because of its good conductivity and transparency, thus appearing as a valuable alternative to indium tin oxide (ITO) electrodes. [48,49] First studies on the electrochemical behavior of adrenaline, dopamine and other catecholamines at electrodes using FTO as sensing material have been reported recently.…”

“…Recently, for activity measurement of the COMT substrate and product of the enzyme conversion are separated via high performance liquid chromatography followed by an electrochemical detection of the single compounds [45,46] . Another effective separation strategy exploits capillary electrophoresis for the detection of dopamine conversion [47] …”