Reacción #422227

Ecuación de reacción

fMLP

Dianisidine

citrate

hydrogen peroxide

dianisidine

Dianisidine

o-dianisidine

→

Plumbagin

Condiciones de reacción

Tratamiento posterior

1. 1
   workup.ADDITIONTo the tubes were added 0.5 mL of cells at 37° C.
2. 2
   Otrowas stored at −20° C. for later assay
3. 3
   Temperaturaincrease in absorbance at 450 nm that

Procedimiento

Eppendorf tubes containing either 25 mg of CPPD or 1 uM fMLP (with 0.5 uM cytochalasin B) were maintained at 37° C. To the tubes were added 0.5 mL of cells at 37° C. followed by shaking to initiate the reactions. At appropriate times, tubes were centrifuged at 10,000× g for 10 s and 0.4 mL of supernatant was stored at −20° C. for later assay. Myeloperoxidase (MPO) activity was measured by the increase in absorbance at 450 nm that accompanies the oxidation of o-dianisidine. Dianisidine (7.8 mg) was dissolved in 100 mL of 0.1 M citrate buffer, pH 5.5 and to a 1 mL cuvette were added 0.89 mL of the dianisidine solution, followed by 50 mL of 1% Triton X 100, 10 mL of 0.05% hydrogen peroxide and 50 mL of crystal-cell supernatant. MPO activity was determined from the change in absorbance (450 nm) per minute, (ΔA450), using the following relationship: Dianisidine oxidation (nmol/min)=50 ΔA450

Fuente