Application of Electrophoretically Mediated Microanalysis for
Study of Ezymes

D 2004

Application of Electrophoretically Mediated Microanalysis for Study of Ezymes

VYTISKOVÁ, Soňa, Kateřina PAPEŽOVÁ and Zdeněk GLATZ

Basic information

Original name

Application of Electrophoretically Mediated Microanalysis for Study of Ezymes

Name in Czech

Využití metody EMMA pro studium enzymů.

Authors

VYTISKOVÁ, Soňa (203 Czech Republic), Kateřina PAPEŽOVÁ (203 Czech Republic) and Zdeněk GLATZ (203 Czech Republic, guarantor)

Edition

Rakousko, Proceeding of 17th International Symposium on Microscale Separations and Capillary Electrophoresis, p. 258-9, 2 pp. 2004

Publisher

Tyrol Congress

Other information

Language

English

Type of outcome

Stať ve sborníku

Field of Study

10600 1.6 Biological sciences

Country of publisher

Austria

Confidentiality degree

není předmětem státního či obchodního tajemství

RIV identification code

RIV/00216224:14310/04:00009930

Organization unit

Faculty of Science

Keywords in English

capillary electrophoresis; microscale separations

Abstract

ORIG CZ

V originále

Enzymes are biological catalysts that play an important role in biochemical reactions necessary for normal growth, maturation and reproduction through the whole living world. They catalyze virtually all chemical reactions in living systems and the assay of enzyme activity is probably one of the most frequently encountered procedures in biochemistry and molecular biology. Due to their low concentrations in samples containing a large amount of other proteins, direct measurements of enzymes by mass are impossible. However, enzymes can be measured more easily by their catalytic activities, which are the most relevant properties of enzymes in the biochemical context. The enzyme assay is important procedure in elucidation of enzyme properties and function. Determination of kinetic parameters is usually undertaken to characterize an enzyme, to provide a quantitative evaluation of substrate specificity and to study kinetic mechanisms. Capillary electrophoretic systems have been successfully applied for in-line enzyme-catalyzed reaction by a methodology known as Electrophoretically Mediated MicroAnalysis (EMMA), firstly described by Bao and Regnier. In this method, substrate(s) and enzyme are introduced into the capillary as a distinct plugs, the first analyte injected being the one with the lower electrophoretic mobility. Upon the application of an electric field the two zones interpenetrate due to differences in their electrophoretic mobilities. Enzymatic reaction takes place and the resultant reaction product(s) and the unreacted substrate(s) are electrophoretically transported towards the detector, where they are individually detected. This approach was applied in the complex study of rhodanese, important enzyme catalyzing detoxication of cyanide to less toxic thiocyanate after reaction with a sulfur donor, such as thiosulfate. The purpose of this study was to determine the kinetic parameters of rhodanese with thiosufate and cyanide as substrates, and also the inhibition action of 2-oxoglutarate on this enzymatic reaction. This work shows that the Michaelis constants for both substrates (thiosulfate and cyanide), the effect of teperature on rhodanese reaction, and the inhibition parameters can be easily performed by EMMA methodology combined with a partial filling technique. The method can be used not only to estimate KI but also for the determination of the inhibition type of 2-oxoglutarate action on rhodanese reaction. Compared to spectrophotometric and other discontinuous assays, the method is rapid, can be automated, does not need expensive radiolabeled substrates and requires only small amounts of reagents which is especially important in the case of enzymes (injection volume of analytes is in the order of nanoliter). In consequence the EMMA methodology could serve as a progressive tool of modern enzymology in context of metabolomic research.

In Czech

Jde o shrnutí využití metody EMMA pro studium enzymů.

Links

GA203/03/1125, research and development project

Name: Využití kapilární zónové elektroforézy pro studium enzymů

Investor: Czech Science Foundation, Application of capillary zone electrophoresis for the study of enzymes

MSM 143100005, plan (intention)

Name: Strukturně-funkční vztahy biomolekul a jejich role v metabolismu

Investor: Ministry of Education, Youth and Sports of the CR, Biomolecular Structure-function Relationships and their role in the Metabolism

Citovat

VYTISKOVÁ, Soňa, Kateřina PAPEŽOVÁ and Zdeněk GLATZ. Application of Electrophoretically Mediated Microanalysis for Study of Ezymes. In Proceeding of 17th International Symposium on Microscale Separations and Capillary Electrophoresis. Rakousko: Tyrol Congress, 2004, p. 258-9, 2 pp.

@inproceedings{492940,
   author = {Vytisková, Soňa and Papežová, Kateřina and Glatz, Zdeněk},
   address = {Rakousko},
   booktitle = {Proceeding of 17th International Symposium on Microscale Separations and Capillary Electrophoresis},
   keywords = {capillary electrophoresis; microscale separations},
   language = {eng},
   location = {Rakousko},
   pages = {258-9},
   publisher = {Tyrol Congress},
   title = {Application of Electrophoretically Mediated Microanalysis for Study of Ezymes},
   year = {2004}
}

TY  - JOUR
ID  - 492940
AU  - Vytisková, Soňa - Papežová, Kateřina - Glatz, Zdeněk
PY  - 2004
TI  - Application of Electrophoretically Mediated Microanalysis for Study of Ezymes
PB  - Tyrol Congress
CY  - Rakousko
KW  - capillary electrophoresis
KW  - microscale separations
N2  - Enzymes are biological catalysts that play an important role in biochemical reactions necessary for normal growth, maturation and reproduction through the whole living world. They catalyze virtually all chemical reactions in living systems and the assay of enzyme activity is probably one of the most frequently encountered procedures in biochemistry and molecular biology. Due to their low concentrations in samples containing a large amount of other proteins, direct measurements of enzymes by mass are impossible. However, enzymes can be measured more easily by their catalytic activities, which are the most relevant properties of enzymes in the biochemical context. The enzyme assay is important procedure in elucidation of enzyme properties and function. Determination of kinetic parameters is usually undertaken to characterize an enzyme, to provide a quantitative evaluation of substrate specificity and to study kinetic mechanisms. Capillary electrophoretic systems have been successfully applied for in-line enzyme-catalyzed reaction by a methodology known as Electrophoretically Mediated MicroAnalysis (EMMA), firstly described by Bao and Regnier. In this method, substrate(s) and enzyme are introduced into the capillary as a distinct plugs, the first analyte injected being the one with the lower electrophoretic mobility. Upon the application of an electric field the two zones interpenetrate due to differences in their electrophoretic mobilities. Enzymatic reaction takes place and the resultant reaction product(s) and the unreacted substrate(s) are electrophoretically transported towards the detector, where they are individually detected. This approach was applied in the complex study of rhodanese, important enzyme catalyzing detoxication of cyanide to less toxic thiocyanate after reaction with a sulfur donor, such as thiosulfate. The purpose of this study was to determine the kinetic parameters of rhodanese with thiosufate and cyanide as substrates, and also the inhibition action of 2-oxoglutarate on this enzymatic reaction. This work shows that the Michaelis constants for both substrates (thiosulfate and cyanide), the effect of teperature on rhodanese reaction, and the inhibition parameters can be easily performed by EMMA methodology combined with a partial filling technique. The method can be used not only to estimate KI but also for the determination of the inhibition type of 2-oxoglutarate action on rhodanese reaction. Compared to spectrophotometric and other discontinuous assays, the method is rapid, can be automated, does not need expensive radiolabeled substrates and requires only small amounts of reagents which is especially important in the case of enzymes (injection volume of analytes is in the order of nanoliter). In consequence the EMMA methodology could serve as a progressive tool of modern enzymology in context of metabolomic research.
ER  -

VYTISKOVÁ, Soňa, Kateřina PAPEŽOVÁ and Zdeněk GLATZ. Application of Electrophoretically Mediated Microanalysis for Study of Ezymes. In \textit{Proceeding of 17th International Symposium on Microscale Separations and Capillary Electrophoresis}. Rakousko: Tyrol Congress, 2004, p.~258-9, 2 pp.

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