Selective Detection of NAD(P)H-Dependent Enzymes during Their Function within Vital Cells by means of Time-Resolved TPLSM

Abstract

The investigation of physiological phenomena at molecular level within vital cells, for instance metabolic processes, represents a major challenge in life sciences. In this context, measuring methods should be non-invasive for the system under study and should provide high sensitivity and specificity. We demonstrate inhere the power of marker-free, spatially and timely resolved intracellular detection of NAD(P)H-dependent enzymes in the study of phenomena of particular biomedical relevance like phagocytosis or autoimmune reactions. The technique is based on biexponential two-photon laser scanning fluorescence lifetime microscopy (TPLSM FLIM) of the physiologic fluorophore NAD(P)H, which exhibits a fluorescence lifetime τ that is strongly dependent on its chemical environment, i.e. on the enzyme to which NAD(P)H is bound to (free NAD(P)H: τ ∼ 400 ps, enzyme-bound NAD(P)H: τ ∼ 2 ns). In order to verify the feasibility of the method, an in-vitro study with different enzymes in solution was performed. The measured fluorescence lifetimes of enzyme-bound NAD(P)H strongly varied from τ = 960±27 ps for malic dehydrogenase to τ = 3640±101 ps for 3α hydroxysteroid dehydrogenase, which made us confident, that an intracellular selective enzyme detection is possible by means of NAD(P)H-FLIM. We indeed succeeded for the first time to specifically and intracellularly detect the NADPH oxidase, a multi-subunit membrane-bound enzyme complex that catalyzes the reduction of free oxygen to its superoxide anion. Thereby, we selectively monitored the NADPH oxidase during its function within differently activated murine polymorphonuclear leucocytes (PMNs). The experiments revealed a specific fluorescence lifetime of 3670±140 ps for NADPH bound to this enzyme both in humorally activated PMNs, i.e. activation with PMA, and in PMNs phagocytosing the fungus Aspergillus fumigatus, confirming the assembly of NADPH oxidase as highly site-specific.