Simple colorimetric assay for phenotyping the major human thermostable phenol sulfotransferase (SULT1A1) using platelet cytosols

A thermostable phenol sulfotransferase, SULT1A1, has been implicated in num erous detoxification and bioactivation pathways; however, little is known r egarding its endogenous function or its putative role in mediating risk for human environmental disease. A simple endpoint colorimetric assay is descr ibed that can be used for rapid phenotyping of SULT1A1 activity in human po pulations. The assay utilizes a microtiter-plate format and relatively smal l amounts of platelet cytosol-derived enzyme. The enzyme catalyzes the synt hesis of 2-naphthylsulfate from 2-naphthol and 5'-phosphoadenosine 3'-phosp hosulfate (PAPS), whereas addition of p-nitrophenyl sulfate to the assay co ntributes to an effective PAPS-regenerating system. In contrast to other su lfotransferase assay methods, 3'-phosphoadenosine 5'-phosphate (PAP) does n ot accumulate during the incubation to interfere with enzyme activity, but instead serves as a cofactor to cause the removal of sulfate from p-nitroph enyl sulfate to regenerate PAPS. This reaction concomitantly results in gen eration of p-nitrophenol that can be quantified colorimetrically at 405 nm (epsilon = 18,200 M-1) to give an indirect measure of sulfotransferase acti vity. Using platelet enzyme preparations from adult human subjects, sulfati on rates of two prototypical thermostable phenol sulfotransferase substrate s (2-naphthol and p-nitrophenol) and one thermolabile phenol sulfotransfera se substrate (dopamine) were determined using standard radiochemical protoc ols. These data were then compared with results from the colorimetric assay using 2-naphthol as substrate. There was a good correlation between the ph enotyping assay and radiochemical assays for both 2-naphthol sulfotransfera se and p-nitrophenol sulfotransferase activity (r = 0.85 and 0.69, respecti vely). However, SULT1A1 activity was approximately 10 to 20 times higher wi th the colorimetric determination. As anticipated, there was no correlation between SULT1A1 activity and dopamine sulfotransferase activity (r = 0.07) in these human platelet preparations. This inexpensive and rapid method fo r phenotyping SULT1A1 activity may help investigators assess a role for thi s enzyme in disease susceptibility.