An improved HPLC method for the quantitation of 3′-phosphoadenosine 5′-phosphate (PAP) to assay sulfotransferase enzyme activity in HepG2 cells

Abstract

Sulfotransferase-catalyzed (SULT-catalyzed) sulfation is responsible for hormone regulation and xenobiotic detoxification. In the current study, a sensitive and widely applicable method of reversed-phase HPLC–UV was developed for the determination of 3′-phosphoadenosine 5′-phosphate (PAP), a common product of different subtypes of sulfation reactions, in HepG2 cells. The analyte was separated on a ZORBAX Extend-C18 column with the mobile phase of methanol and water containing 75mM KH2PO4, 100mM NH4Cl, and 1mM 1-octylamine (pH 4.55), at a flow rate of 1.0ml/min. The assay exhibited linearity over the range of 0.1–20μM for PAP with a correlation coefficient of 0.9995. The total time per run was under 10min. The intra- and inter-day precision was less than 7.2%, with accuracy in the range 82.6–102.0%. The method was applied to the determination of kinetic parameters Km, Vm, and Kcat, of three different SULTs (SULT1A1, SULT2A1, and SULT1E1) in HepG2. This universal HPLC–UV method was easy to perform, economically feasible, and suitably efficient for the investigation of the enzyme kinetics of the SULT family using multiplex substrates.