Figure 3. Microfluidic sulfotransferase assay to measure inhibition of HS2ST activity in vitro.

Assays were performed using 20 nM HS2ST and the extent of substrate sulfation was determined after 15 min incubation at room temperature, as described in Figure 2. Dose–response curves for inhibition of HS2ST activity by (A) modified heparin derivatives containing different sulfation patterns (assayed in the presence of 0.5 mM MgCl₂) or (B) nucleotides (assayed in the absence of MgCl₂). Assays contained HS2ST and 10 μM PAPS and the indicated concentration of inhibitory ligand or buffer. (C) Inhibition of HS2ST activity by fixed 10 μM PAP, 0.5 mM CoA or 0.5 mM dephospho-CoA in the presence of increasing concentration of PAPS. Inhibition is calculated as a function of no inhibitor for each concentration of PAPS in the absence of MgCl₂. (D) Evaluation of small-molecule HS2ST inhibitory profiles in the presence of 10 μM PAPS. (E) Inhibition of HS2ST activity by 20 μM rottlerin in the presence of varied concentrations of PAPS, suggesting a competitive mode of inhibition. Similar results were seen in multiple experiments.