Bis p nitrophenyl phosphate

Purified wild type Sebox3 and the mutated strains (Sebox5, Sebox6 and Sebox7) were assayed for in vitro phosphodiesterase activity. The protein concentration was kept uniform after performing Bradford assay. PDE activity was performed using chemically synthesized substrate bis(p-nitrophenyl) phosphate (obtained from Sigma Aldrich) using standard protocol (Liu et al. 2010 (link)) with some minor changes. 50 μl of reaction mixture was prepared consisting of 50 mM Tris–HCl (pH 7.4), 10 mM MnCl₂, 5 mM bis-pNPP to which was added 50 μl of the protein sample. The reaction mixture was mixed well and incubated for 8 h at 37 °C. The amount of released p-nitrophenol was detected and quantified by a Shimadzu UV–Vis spectrophotometer at an OD of 410 nm. Controls were set up without the addition of the protein and enzyme activity was compared in triplicate individual assays to ascertain the reproducibility of the reaction. Benziman had reported that Ca²⁺ strongly inhibited the phosphodiesterase activity in Gluconacetobacter xylinus (Ross et al. 1986 (link), 1990 (link)). Therefore, we added 10 mM CaCl₂ to the reaction mixture to check for Ca²⁺ induced inhibition in the wild type as well as mutant samples.

All reactants, including p-nitrophenylphosphate (pNPP), bis p-nitrophenylphosphate (bis-pNPP), Thymidine 5′-monophosphate p-nitrophenylester sodium salt (TpNPP), calcium glycerophosphate (CaGP) and sodium glycerophosphate (NaGP) were purchased from Sigma-Aldrich. pNPP and glycerophosphate (GP) are phosphomonoesters while bis-pNPP and TpNPP are phosphodiesters.