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Methods for Measuring NADPH-Oxidase

The enzyme NADPH oxidase is identified as a producer of reactive oxygen species during processes including the growth of vascular smooth muscle cells. These reactive species are linked to a range of diseases including hypertension, diabetes, cancer and inflammation; methods for monitoring the activity of this enzyme is critical for possible treatment development.

  1. NADPH Oxidase

    • NADPH oxidase belongs to a family of enzymes that assist with electron transfer between NADPH and oxygen. However, NADPH oxidase has the unique ability of producing reactive oxygen species from this reaction, compared to other family members where reactive oxygen species is generated via other means. A multimeric protein complex, NADPH oxidase consists of the membrane-bound flavocytochrome b558, which is used as a measurement marker.

    Reactive Oxygen Species (ROS) Generation

    • As the chief source of ROS, particularly hydrogen peroxide, NADPH oxidase produces small but consistent quantities via its isoform expressed in blood vessels, which are involved in signaling processes. This consistent production may eventually overwhelm the natural antioxidant system, causing a loss of protective compounds such as nitric oxide, and the generation of even more powerful ROS.

    Chemiluminescence Method

    • Chemiluminescence enables highly sensitive, automated measurement of NADPH oxidase activity over a range of concentrations. During this method, NADPH substrate is added to a tissue sample containing NADPH oxidase. Upon reaction, ROS is generated and reacts with the compound lucigenin to produce a chemilluminescent effect, which is measured. Luminescence is directly proportional to NADPH oxidase activity, as the enzyme determines the rate of the reaction.

    Kinetic Assay via Flow Cytometry

    • In this method, tissue samples are analyzed via flow cytometry. Bone marrow is separated from the sample using Hanks' Balanced Salt Solution. Specific stains and detecting antibodies are added to the sample in polystyrene tubes. The sample is then incubated, washed with Hanks' Balanced Salt Solution, labeled with molecular probes and prepared from flow cytometry. Control tubes are also made for comparison and to calibrate the flow cytometer. The machine is then ran and measurements recorded.

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