NADPH oxidase activity in organs

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calucestein's picture
NADPH oxidase activity in organs

Does anyone have a clear protocol which works for the measurement of the NADPH activity in organs with Lucigenin or luminol

qiang wang
qiang wang's picture
I do not know much about

I do not know much about about your tissue, but the ref. attached below was the original one I used to get the NADPH Oxi activity in photosynthetic systems, hope it helps.

Light-Dependent NAD(P)H Oxidation
Quantitation of light-dependent NAD(P)H oxidation was carried out by a modification of the procedure of Mi et al. (1995). The reaction mixture contained (in a total volume of 0.5 mL): 24 mM Tris-HCl (pH 8.6), 48 mM NaCl, 9.6 mM MgCl2, 200 µM NAD(P)H, 100 µM MeV, 10 µM DCMU, and thylakoids corresponding to 4 to 8 µg of Chl. The oxidation of NAD(P)H at 25°C was followed at 340 nm in a spectrophotometer (DW-2000, Aminco, Urbana, IL) with a thermostated cuvette holder. The actinic light was supplied by a halogen lamp (15 V, 150 W; model KL1500, Schott, Cologne, Germany) provided with fiber optics and passed through a 665-nm cut-on filter. The photomultiplier tube was protected from the actinic light with a 340-nm narrow-bandwidth interference filter. The oxidation of NAD(P)H was calculated using an extinction coefficient of 6.2 mM-1 cm-1. Before spectrophotometric analysis, the reagents, except for NAD(P)H, were mixed and the NDH activity was activated by illumination. The sample was surrounded by a water-cooled jacket (25°C) and white light was supplied by a halogen lamp (KL1500 Schott) provided with fiber optics. Details of illumination times are given in ``Results''. Alternatively, activation was accomplished by omitting MgCl2 from the reaction mixture, and incubating the sample for 1 h in the dark in the presence of 12 µM NADPH.

Light-Independent NAD(P)H Oxidation
The rate of light-independent NAD(P)H oxidation was measured spectrophotometrically at 340 to 375 nm (using an extinction coefficient of 4.0 mM-1 cm-1) as the rate of NAD(P)H oxidation at 25°C in an assay mixture containing (in a total volume of 0.5 mL): 20 mM Tris-HCl (pH 7.8), 8 mM MgCl2, 200 µM NAD(P)H, and 120 µM duroquinone. The reaction also contained thylakoids corresponding to 8 µg of Chl or 4 pmol of isolated barley FNR. The reaction was started by the addition of duroquinone.