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 ( K m) for 4-nitrophenol was calculated to be 0.84 μM, a value which is consistent with a previously reported value. Strong substrate inhibition (above a 4-nitrophenol concentration of 2.5 μM) was observed, and this is also in accordance with literature values.