采用基于NADPH的荧光寿命成像法选择性探测多核形细胞中NADPH氧化酶
NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalysesthe reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invadingpathogens, for example, the fungus Aspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative andoxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly andfunction of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms bymeans of biexponentialNAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of 3670± 140 picosecondsas compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound tooxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors,we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificityof the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example ofNADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellularfunction.