| PAPEŽOVÁ, Kateřina, Tomáš NĚMEC, Radka CHALOUPKOVÁ and Zdeněk GLATZ. Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary. Journal of Chromatography A. Amsterdam: Elsevier, 2007, vol. 1150, 1-2, p. 327-331. ISSN 0021-9673. |
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@article{699386,
author = {Papežová, Kateřina and Němec, Tomáš and Chaloupková, Radka and Glatz, Zdeněk},
article_location = {Amsterdam},
article_number = {1-2},
keywords = {CE; EMMA substrate; inhibition; haloalkane dehalogenase},
language = {eng},
issn = {0021-9673},
journal = {Journal of Chromatography A},
title = {Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary.},
volume = {1150},
year = {2007}
}
TY - JOUR
ID - 699386
AU - Papežová, Kateřina - Němec, Tomáš - Chaloupková, Radka - Glatz, Zdeněk
PY - 2007
TI - Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary.
JF - Journal of Chromatography A
VL - 1150
IS - 1-2
SP - 327-331
EP - 327-331
PB - Elsevier
SN - 00219673
KW - CE
KW - EMMA substrate
KW - inhibition
KW - haloalkane dehalogenase
N2 - Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this set-up, the part of capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. In the case of haloalkane dehalogenase, a model enzyme selected for this study, the enzymatic reaction was performed in 20 mM glycine buffer (pH 8.6) whereas 20 mM beta-alanine - hydrochloric acid buffer (pH 3.5) was used as a background electrolyte in combination with direct detection at 200 nm. The whole study was performed on poorly soluble brominated substrate - 1,2-dibromoethane. As a result it was first necessary to find the compromise between the concentrations of the enzyme and the substrate preserving both the adequate sensitivity of the assay and at the same time the attainable substrate solubility. By means of the developed EMMA methodology we were able to determine the Michaelis constant (KM) as well as the substrate inhibition constant (KSI). The value of KM and KSI obtained were 7.7 mM and 1.1 mM respectively. Observation of the substrate inhibition of haloalkane dehalogenase by 1,2-dibromoethane is in accordance with previous literature data.
ER -
PAPEŽOVÁ, Kateřina, Tomáš NĚMEC, Radka CHALOUPKOVÁ and Zdeněk GLATZ. Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary. \textit{Journal of Chromatography A}. Amsterdam: Elsevier, 2007, vol.~1150, 1-2, p.~327-331. ISSN~0021-9673.
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