SULT1A1 activity was measured using a colorimetric assay in which the formation of pnitrophenol upon the sulfonation of a substrate would correlate directly to sulfonation activity (Figure ). 17 P-Nitrophenol is generated by the desulfonation of p-nitrophenyl sulfate by SULT1A1 to regenerate PAPS thus allowing for more sulfonation of the substrate. P-Nitrophenol has a stable yellow color in solution that can be read at 405 nm on a plate reader. The colorimetric assay quantifying sulfonation of a substrate was originally devised by To make the assay buffer, 1.0 M MES buffer was diluted to 50 mM using UltraPure water. To a 384-well clear bottom black assay plate (Corning® CellBIND® Ref. 3770) was added 9.25 µL of 50 mM MES assay buffer (pH of 7.5) to each well. Then, 5 µL of 1 mM PAPS, which was diluted from a 6.64 mM stock using assay buffer, was added to the wells. Next, the acceptor substrate (YC-1 and analogs) or the positive control (2-Napthol) at 10 mM in DMSO was diluted 20X in the MES assay buffer for a final concentration of 500 µM. Then, 5 µL of the 500 µM acceptor substrate solution was added to the well. After the substrate was added, 5 µL of 5 mM PNPS in assay buffer was added. Finally, the enzymatic reaction was initiated upon addition of 0.75 µL of SULT1A1 (25.1 µM) to the well to give a final SULT1A1 concentration of 0.75 µM and final well volume of 25 µL. The well was pipetted up and down and mixed with the pipette tip. As a negative control (blank), 25 µL of assay buffer was added to a well. The plate was immediately taken to the Tecan Spark® Microplate Reader and absorbance at 405 nm was read for 120 minutes with measurements taken every 5 minutes. Data was graphed and analyzed using the GraphPad Prism software (version 10.2.23).