Journal of Biophysics 
Volume 2008 (2008), Article ID 602639, 13 pages
doi:10.1155/2008/602639
Research Article

Selective Detection of NADPH Oxidase in Polymorphonuclear Cells by Means of NAD(P)H-Based Fluorescence Lifetime Imaging

Raluca Niesner1,2 and Priyanka Narang2,3

 1Institute of Physical and Theoretical Chemistry, University Braunschweig, Hans-Sommer Straße 10, 38106 Braunschweig, Germany
2Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany
3Centre for Immunology and Infection, Department of Biology, University of York, P.O. Box 373, York YO10 5YW, UK
Received 28 May 2008; Accepted 2 September 2008

Recommended by Peter T. C. So

Abstract

NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading pathogens, for example, the fungus Aspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of 3670±140 picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to oxidative 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 specificity of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular function.