Antipyrine

DDAH activity (μmols/min) was determined by measuring the conversion of ADMA to L-citrulline [22 (link)]. Briefly, the cell lysate (5μL) was incubated with 1mmol/L ADMA (Sigma-Aldrich, St. Louis, MO) and 0.1mol/L phosphate buffer (pH 6.5) at 37°C for 120 minutes. This first reaction was stopped by the addition of equal volume 10% trichloroacetic acid (G-Biosciences, St. Louis, MO). The second step required addition of 0.8% diacetyl monoxime (Thermo Fisher) and 0.5% antipyrine (Thermo Fisher). This mixture was then incubated at 60°C for 110 minutes. 200μL of the supernatant was then transferred into a plate and analyzed by a spectrophotometer at 466nm. An L-citrulline standard curve was created using varying concentrations of L-citrulline (Sigma-Aldrich, St. Louis, MO) DDAH activity (V) was then calculated using the following equation:
$$V=\frac{(A_{cit}-A_0)\times 100\times {10}^{-4}L}{B\times t}$$
Where A_Cit is the measured absorption A₀ is the blank (determined from the L-citrulline standard), B is the slope of the L-citrulline standard curve and t is the time of the enzymatic reaction [23 (link)]. Specific DDAH activity (μmols/mg/min) was calculated after normalization to protein concentration.