Study of enzyme kinetics of phenol sulfotransferase by
electrophoretically mediated microanalysis

J 2004

Study of enzyme kinetics of phenol sulfotransferase by electrophoretically mediated microanalysis

VYTISKOVÁ, Soňa, Sigrid VAN DYCK, Ann VAN SCHEPDAEL, Jos HOOGMARTENS, Zdeněk GLATZ et. al.

Basic information

Original name

Study of enzyme kinetics of phenol sulfotransferase by electrophoretically mediated microanalysis

Name in Czech

Studium enzymove kinetiky enzymu fenolsulfotranferasy metodou EMMA

Authors

VYTISKOVÁ, Soňa (203 Czech Republic, guarantor), Sigrid VAN DYCK (56 Belgium), Ann VAN SCHEPDAEL (56 Belgium), Jos HOOGMARTENS (56 Belgium) and Zdeněk GLATZ (203 Czech Republic)

Edition

Journal of Chromatography A, Netherlands, Elsevier, 2004, 0021-9606

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: 3.105

RIV identification code

RIV/00216224:14310/04:00009928

Organization unit

Faculty of Science

UT WoS

000220121200043

Keywords in English

Capillary electrophoresis; enzymes; EMMA; sulfotranferase

Abstract

ORIG CZ

V originále

Electrophoretically mediated microanalysis (EMMA) was applied for the study of the kinetic parameters of the enzymatic reaction of phenol sulfotransferase SULT1A1 isoenzyme with 4-nitrophenol as a substrate. The SULT1A1 activity was determined by the quantitation of the product, 4-nitrophenyl sulfate, at 274 nm by using different injection and separation steps. This new approach solved the problem of the presence of the very strong inhibitor, adenosine 3',5'-bisphosphate (PAP), in the co-substrate solution (adenosine 3'-phosphate 5'-phosphosulfate, PAPS) which is unstable at room temperature. The inhibitor PAP was electrophoretically separated from the co-substrate PAPS before the injection of enzyme and substrate inside the capillary (and thus before their in-capillary encountering). With the developed in-capillary SULT1A1 activity assay an average Michaelis constant (Km) for 4-nitrophenol was calculated to be 0.84 M, a value which is consistent with a previously reported value. Strong substrate inhibition (above a 4-nitrophenol concentration of 2.5 M) was observed, and this is also in accordance with literature values.

In Czech

Metoda EMMA byla využita pro studium enzymove kinetiky enzymu fenolsulfotranferasy.

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, Sigrid VAN DYCK, Ann VAN SCHEPDAEL, Jos HOOGMARTENS and Zdeněk GLATZ. Study of enzyme kinetics of phenol sulfotransferase by electrophoretically mediated microanalysis. Journal of Chromatography A. Netherlands: Elsevier, 2004, vol. 1032, (1+2), p. 319-326. ISSN 0021-9606.

@article{492938,
   author = {Vytisková, Soňa and Van Dyck, Sigrid and Van Schepdael, Ann and Hoogmartens, Jos and Glatz, Zdeněk},
   article_location = {Netherlands},
   article_number = {(1+2)},
   keywords = {Capillary electrophoresis; enzymes; EMMA; sulfotranferase},
   language = {eng},
   issn = {0021-9606},
   journal = {Journal of Chromatography A},
   title = {Study of enzyme kinetics of phenol sulfotransferase by electrophoretically mediated microanalysis},
   volume = {1032},
   year = {2004}
}

TY  - JOUR
ID  - 492938
AU  - Vytisková, Soňa - Van Dyck, Sigrid - Van Schepdael, Ann - Hoogmartens, Jos - Glatz, Zdeněk
PY  - 2004
TI  - Study of enzyme kinetics of phenol sulfotransferase by electrophoretically mediated microanalysis
JF  - Journal of Chromatography A
VL  - 1032
IS  - (1+2)
SP  - 319-326
EP  - 319-326
PB  - Elsevier
SN  - 00219606
KW  - Capillary electrophoresis
KW  - enzymes
KW  - EMMA
KW  - sulfotranferase
N2  - Electrophoretically mediated microanalysis (EMMA) was applied for the study of the kinetic parameters of the enzymatic reaction of phenol sulfotransferase SULT1A1 isoenzyme with 4-nitrophenol as a substrate. The SULT1A1 activity was determined by the quantitation of the product, 4-nitrophenyl sulfate, at 274 nm by using different injection and separation steps. This new approach solved the problem of the presence of the very strong inhibitor, adenosine 3',5'-bisphosphate (PAP), in the co-substrate solution (adenosine 3'-phosphate 5'-phosphosulfate, PAPS) which is unstable at room temperature. The inhibitor PAP was electrophoretically separated from the co-substrate PAPS before the injection of enzyme and substrate inside the capillary (and thus before their in-capillary encountering). With the developed in-capillary SULT1A1 activity assay an average Michaelis constant (Km) for 4-nitrophenol was calculated to be 0.84 M, a value which is consistent with a previously reported value. Strong substrate inhibition (above a 4-nitrophenol concentration of 2.5 M) was observed, and this is also in accordance with literature values.
ER  -

VYTISKOVÁ, Soňa, Sigrid VAN DYCK, Ann VAN SCHEPDAEL, Jos HOOGMARTENS and Zdeněk GLATZ. Study of enzyme kinetics of phenol sulfotransferase by electrophoretically mediated microanalysis. \textit{Journal of Chromatography A}. Netherlands: Elsevier, 2004, vol.~1032, (1+2), p.~319-326. ISSN~0021-9606.

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