rhENPP1 activity assay

An enzymatic endpoint assay for ENPP1 activity was developed based its ability to hydrolyze thymidine 5′-monophosphate p-nitrophenyl ester sodium salt (T4510; Sigma-Aldrich, Allentown, PA, USA). A prior lot of rhENPP1 served as the stock ENPP1 used for generating all standard curves in this assay. Test samples and standards were diluted in assay buffer (0.11 M Tris pH 9.0, 0.11 M NaCl, 15 mM MgCl₂). All standards and samples (10 µl) were incubated with 1 mM substrate (90 µl) and incubated in the dark for 30 min at 37°C. Enzymatic activity was determined spectrophotometrically at 450 nm using SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA), with 1 unit of enzyme defined as 1 µmol/min.