*2.4. Characterization of Human SULTs Involved in Catalposide Sulfation*

To screen for SULT enzymes involved in catalposide sulfation, each 100 µL of reaction mixture contained 20 µM PAPS; 10 mM dithiothreitol; 5 mM magnesium chloride; 50 mM phosphate buffer (pH 7.4); 150 µM catalposide; and the human liver S9 fraction (40 µg protein) or human cDNA-expressed SULT 1A1\*1, 1A1\*2, 1A2, 1A3, 2A1, 1B1, 1C2, 1C4, or 1E1 supersomes [1A1\*1 (0.25), 1A1\*2 (0.5), 1A2 (0.25), 1A3 (0.2), 2A1 (1.25), 1B1 (0.5), 1C2 (2.5), 1C4 (0.1), 1E1 (0.2 µg protein)]. Incubation proceeded at 37 ◦C for 5 min. The reactions were stopped by adding 100 µL of methanol containing 30 ng/mL 4-methylumbelliferone (an internal standard). After vortex-mixing and centrifugation, 50 µL of each supernatant was diluted with 50 µL deionized water. The mixture was transferred to an injection vial, and an aliquot (5 µL) was injected into the LC-HRMS system.

To explore the kinetics of the metabolism of catalposide to catalposide sulfate, various concentrations of catalposide (10 to 2000 µM) were incubated in duplicate with pooled human liver S9 fractions (40 µg protein) or human cDNA-expressed SULT1A1\*1 (0.5 µg protein), SULT1A1\*2 (0.5 µg protein), SULT1C4 (0.1 µg protein), or SULT1E1 (0.2 µg protein) in the presence of 20 µM PAPS, 10 mM dithiothreitol, and 5 mM magnesium chloride at 37 ◦C for 5 min to obtain *K*<sup>m</sup> and *V*max values.
