title: Study of enzyme kinetics of phenol sulfotransferase by electrophoretically mediated microanalysis.
abstract: 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.
authors.affiliation: Department of Biochemistry, Faculty of Science, Masaryk University Brno, Kotlárská 2, 61137 Brno, Czech Republic.
authors.forename: Sona
authors.initials: S
authors.lastname: Nováková
authors.forename: Sigrid
authors.initials: S
authors.lastname: Van Dyck
authors.forename: Zdenek
authors.initials: Z
authors.lastname: Glatz
authors.forename: Ann
authors.initials: A
authors.lastname: Van Schepdael
authors.forename: Jos
authors.initials: J
authors.lastname: Hoogmartens
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D015239:Arylsulfotransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics
chemical_list: D004791:Enzyme Inhibitors; D000244:Adenosine Diphosphate; C010840:adenosine 3'-phosphate-5'-phosphate; D015239:Arylsulfotransferase