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
1Institute of Physical and Theoretical Chemistry, University Braunschweig, Hans-Sommer Straße 10, D-38106 Braunschweig, Germany
2Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany
3Centre for Immunology and Infection, Department of Biology, University of York, P.O. Box 373, York YO10 5YW, UK
4LaVision Biotec GmbH, Meisenstr. 65, D-33607 Bielefeld, Germany
5Institute of Molecular and Clinical Immunology, Otto von Guericke University Magdeburg, Leipzigerstrasse 44, D-39120 Magdeburg, Germany
Received 28 May 2008; Accepted 2 September 2008
Academic Editor: Peter T. C. So
Copyright © 2008 R. Niesner et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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.