Comparison of 2-aminophenol and 4-nitrophenol as in vitro probe substrates for the major human hepatic sulfotransferase, SULT1A1, demonstrates improved selectivity with 2-aminophenol.

Sulfation, catalysed by members of the cytosolic sulfotransferase (SULT) enzyme family, is important in xenobiotic detoxification and in the biosynthesis and homeostasis of many hormones and neurotransmitters. The major human phenol sulfotransferase SULT1A1 plays a key role in chemical defence, is widely expressed in the body and is subject to a common polymorphism that results in reduced protein levels. Study of these enzymes in vitro requires robust probe substrates, and we have previously shown measurement of activity with the widely used SULT1A1 substrate, 4-nitrophenol, does not accurately reflect protein expression. Additionally, the high degree of substrate inhibition observed with this compound further reduces its value as a probe for SULT1A1. Here we show that 2-aminophenol is a more suitable probe substrate for quantifying SULT1A1 activity in human liver. This compound is sulfated at a high rate (V(max) with purified recombinant SULT1A1=121nmol/(minmg) and shows strong affinity for the enzyme (K(m) with purified recombinant SULT1A1=9microM) and, importantly, is a very poor substrate for the other major SULT1 enzyme expressed in liver, SULT1B1 (with V(max) and K(m) values of 17nmol/(minmg) and 114microM, respectively). Experiments with purified recombinant human SULTs and a panel of 28 human liver cytosols demonstrated that 2-aminophenol shows limited substrate inhibition with SULT1A1, and V(max) values measured in liver cytosols correlated strongly with SULT1A1 enzyme protein levels measured by a quantitative immunoblot method. We therefore suggest that 2-aminophenol is a suitable substrate to use for quantifying SULT1A1 enzyme activity.