J 2007

Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary.

PAPEŽOVÁ, Kateřina, Tomáš NĚMEC, Radka CHALOUPKOVÁ and Zdeněk GLATZ

Basic information

Original name

Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary.

Name in Czech

Studium substrátové inhibice metodou EMMA v kombinaci s technikou částečného plnění kapiláry

Authors

PAPEŽOVÁ, Kateřina (203 Czech Republic), Tomáš NĚMEC (203 Czech Republic), Radka CHALOUPKOVÁ (203 Czech Republic) and Zdeněk GLATZ (203 Czech Republic, guarantor)

Edition

Journal of Chromatography A, Amsterdam, Elsevier, 2007, 0021-9673

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10600 1.6 Biological sciences

Country of publisher

Netherlands

Confidentiality degree

není předmětem státního či obchodního tajemství

Impact factor

Impact factor: 3.641

RIV identification code

RIV/00216224:14310/07:00020038

Organization unit

Faculty of Science

UT WoS

000247049200043

Keywords in English

CE; EMMA substrate; inhibition; haloalkane dehalogenase

Tags

Tags

International impact, Reviewed
Změněno: 29/6/2008 05:54, Olga Křížová

Abstract

ORIG CZ

V originále

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.

In Czech

Byla vypracována technika pro studium substrátové inhibice metodou EMMA v kombinaci s technikou částečného plnění kapiláry

Links

GA203/06/0047, research and development project
Name: Využití kapilární elektroforézy pro studium metabolismu léčiv
Investor: Czech Science Foundation, Capillary electrophoresis as a tool for the drug-metabolism studies
LC06023, research and development project
Name: Integrované bioanalytické technologie pro mikroanalýzy a diagnostiku s využitím LIF a hmotnostní spektrometrie
Investor: Ministry of Education, Youth and Sports of the CR
MSM0021622413, plan (intention)
Name: Proteiny v metabolismu a při interakci organismů s prostředím
Investor: Ministry of Education, Youth and Sports of the CR, Proteins in metabolism and interaction of organisms with the environment