Phenol nitroprusside

Protein extracts of WT and AsFmd were used to measure formamidase and urease enzymatic activity. Formamidase and urease activity were measured by monitoring ammonia release, as described in [20 (link),22 (link)], with modifications. Briefly, 500 ng of protein extracts were added to 50 μL of 100 mM formamide (for formamidase activity) or 50 mM urea (for urease activity) substrate solution in 100 mM phosphate buffer, pH 7.4, and 10 mM EDTA. The reaction mixture was incubated at 37 °C for 30 min. Next, 80 μL of phenol-nitroprusside and 80 μL alkaline hypochlorite (Sigma Aldrich) were added to the reaction and samples were incubated at 50 °C for 6 min. Absorbance was read at 625 nm and the amount of ammonia released was calculated compared with a standard curve. One unit (U) of formamidase and urease activity was defined as the amount of enzyme required to hydrolyze 1 μmol of formamide and urea, respectively, per minute per mg of total protein. Three experimental replicates were performed.

They were monitored by the release of ammonia from urea [22 (link)]. Cells collected by centrifugation (5,000 rpm, 10 min, 20°C) were resuspended in 400–1000 μL of buffer (PBS 1X pH 7.5, EDTA 10 mM, Protease Inhibitor Cocktail, Roche) and disrupted in a pre-cooled Eaton press, as described [14 (link)]. 10 μg of total proteins (measured with the Bradford protein assay, Biorad) were mixed with 90 μL of PBS EDTA buffer lacking (reaction blank) or containing (samples) 50 mM urea and then incubated for 10 min at 30°C. Tubes were transferred on ice before the addition of 150 μL of phenol nitroprusside (Sigma-Aldrich), 150 μL of alkaline hypochlorite (Sigma-Aldrich) and 700 μL H₂O. Chromophore formation was achieved at 37°C for 10 min before measuring absorbance at 570 nm. After substracting the A₅₇₀ values of blank reactions, the ammonia concentration of the samples was determined from a standard curve constructed with varying NH₄Cl concentrations.

First the protein extracts were incubated with different concentrations of TSC (1 mM, 125 μM, 32 μM and 15 μM) or TSC-C (1 mM, 62 μM, 18 μM and 9 μM).Then, formamidase activity was measured by monitoring the production of ammonia, as previously described [31 (link)]. Protein samples (500 ng) were added to 100 mM formamide, 100 mM phosphate buffer, pH 7.4, and 10 mM EDTA. The reaction mixture was incubated at 37°C for 30 min. Subsequently, 400 mL phenol-nitroprusside and 400 mL alkaline hypochlorite solution (Sigma Aldrich) were added. The samples were incubated at 50°C for 6 min and the enzymatic activity was monitored at 625 nm. The amount of ammonia released was determined through comparison with a standard curve. The specific activity was calculated as the measured absorbance divided by the amount of proteins in μg.