Reaction #1091408

Reaction equation

NADPH

L-kynurenine

→

L-3-hydroxykynurenine

Conditions

Workup

1. 1
   Concentrationconcentration), and
2. 2
   Concentration25 μl of different concentrations of inhibitor solutions
3. 3
   workup.ADDITIONThe reaction was stopped by addition of 200 μl of 1 M HClO4 after 60 min incubation

Procedure

The reaction mixture contained: 75 μl of suspended homogenate; 100 μl of substrate solution containing 50 mM K-phosphate buffer pH 7.5, 2 mM MgCl2, 0.4 mM NADPH, 50 μM L-kynurenine (final concentration), and 25 μl of different concentrations of inhibitor solutions. The reaction was stopped by addition of 200 μl of 1 M HClO4 after 60 min incubation. L-3-hydroxykynurenine formed was quantified by HPLC with coulometric detection at a working voltage of +0.2 V. The column was a 10 cm C18 reversed phase (3 μm. The mobile phase consisted of 950 ml distilled water, 20 ml acetonitrile, 9 ml triethylamine, 5.9 ml phosphoric acid, 100 mg sodium EDTA and 1.5 g heptanesulfonic acid. The flow rate was 1 ml/min.

Source