Capillary electrophoresis as a tool for a cost‐effective assessment of the activity of plant membrane enzyme chlorophyllase

Abstract

The potential of the capillary electrophoresis-based enzymatic assay has been demonstrated in case of a typical plant membrane enzyme - chlorophyllase. An efficient, automated and rapid semi-quantitative method has been developed, which allowed us to assess the activity of the enzyme via two strategies. Firstly, a reaction conducted in a vial placed directly on the sample tray was combined with the concomitant separation and detection of reagents. The method was used to monitor the reaction progress. Secondly, an on-line approach was applied by using an electrophoretically mediated mixing. The reaction was performed in-capillary, resulting in an extreme reduction of the reagent amounts required for a single run. Both methods were effective in the assessment of the activity of a membrane enzyme, a member of protein class known to pose experimental difficulties.

Capillary coating as an important factor in optimization of the off-line and on-line MEKC assays of the highly hydrophobic enzyme chlorophyllase

“…At basic pH, around 9.0, the micellar phase of LDAO provides an optimal medium for both the solubilization of reactants and separation between the substrate (Chla) and the product (chlorophyllide a , Chlidea) after application of a voltage [23]. Hence, this separation method is an example of micellar electrokinetic chromatography (MEKC).…”

“…The reaction mixture (total volume of 50 μL) was prepared by addition of a 10-μL aliquot of Chla in acetone and a 5-μL aliquot of purified Chlase in LDAO solution to the borate buffer (Na 2 B 4 O 7  · 10H 2 O–HCl) of 20 mM ionic strength, pH 9.0, containing 1.0% (v/v) LDAO. We recently showed that in these conditions Chlase maintains its catalytic activity [23]. The final concentration of the substrate was 100 μM and that of the enzyme was 2.0 μg/mL The electrophoretic analysis was performed by several consecutive injections of the reaction mixture into the capillary, with application of a forward pressure of 3.45 kPa (0.5 psi) for 5 s, and separation of Chla and Chlidea by subsequent application of a 30-kV voltage.…”

“…Equal values of the molecular absorption coefficients for Chla and Chlidea were used, as previously [23]. …”

“…However, because of its very high hydrophobicity, any experimental work with Chlase is still a challenge. Recently, we developed a CE-based assay of recombinant Chlase from Arabidopsis thaliana based on a bare silica capillary as the first such assay of a plant membrane enzyme [23].

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Recent advances in CE mediated microanalysis for enzymatic and derivatization reactions

“…In another study by Nowak et al. , a membrane bound plant enzyme assay was established using MEKC. However, in this assay the zwitterionic lauryldimethylamine N‐oxide (LDAO) was used as a surfactant.…”