| Autor: |
Nováková S; Department of Biochemistry, Faculty of Science, Masaryk University Brno, Kotlárská 2, 61137 Brno, Czech Republic., Van Dyck S, Glatz Z, Van Schepdael A, Hoogmartens J |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of chromatography. A [J Chromatogr A] 2004 Apr 02; Vol. 1032 (1-2), pp. 319-26. |
| DOI: |
10.1016/j.chroma.2003.11.082 |
| Abstrakt: |
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 microM, a value which is consistent with a previously reported value. Strong substrate inhibition (above a 4-nitrophenol concentration of 2.5 microM) was observed, and this is also in accordance with literature values. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
|
Full Text from ScienceDirect