Figure 2. The interaction of NADPH and HRP generates H₂O₂.

Upper panel, the addition of NADPH to reaction mixes containing HRP causes significant AR oxidation leading to the formation of resorufin in the absence of exogenous H₂O₂. Reactions mixes in 140 μL containing potassium phosphate buffer, 50 mM, pH 7.7, 200 mM KCl, 0.5 mM sodium azide, 0.1 mM DETAPAC and AR/HRP (0.01 mM, and 0.5 units/ml, respectively) were preincubated at 37° C in the microplate reader. After 3 min, NADPH, (final concentrations as indicated, closed circles and squares), or control buffer (closed triangles) was added (shown by the arrow). Lower panel, the addition of HRP to reaction mixes causes significant AR oxidation leading to the formation of resorufin in the absence of exogenous H₂O₂. The reaction mixes were the same as above except that 100 μM NADPH was present at the start of the reactions and HRP (0.5 U/ml) was added as indicated by arrows (open circles and triangles). Note that these recordings were done in optical density mode and reactions were carried out in 170 μl volumes in half area transparent flat bottom microplates. Resorufin formation and NADPH degradation were measured simultaneously by changes in absorbance at 570 nm and 340 nm, respectively. Each data point represents the mean ± SD (n = 6). Unless shown, error bars fell within the symbol of each data point.