The apolar azide of 5-iodonaphthalene-1-azide (Ina) partitions into the lipid bilayer of biological membranes. Upon photolysis at 314 nm, it is rapidly converted into the reactive nitrene, which efficiently attaches covalently to lipid-embedded domains of proteins and, to a lesser extent, to membrane phospholipids. Above 370 nm, Ina absorption is negligible and photolysis at these wavelengths does not occur. However, on addition of the photosensitizing molecule 3-aminopyrene, trifluoperazine, or 8-anilinonaphthalene-1-sulfonate, followed by irradiation at 380 nm, efficient conversion of Ina to reactive species was observed, as measured by ['251]Ina-labeling of membrane proteins and inactivation of the hormonal response of adenylate cyclase. Irradiation at 480 nm in the presence of a fluorescein derivative of n-undecylamine also resulted in a pattern of [125] Klip and Gitler (1, 2) introduced the use of highly lipid-soluble azides for the labeling of proteins in membranes. Various reagents of this type have been developed and their properties have been reviewed (3-7). 5-Iodonaphthalene-1-azide (Ina) was used frequently and proved to be efficient in labeling hydrophobic domains of membrane proteins (8-12).Ina is highly hydrophobic (partition coefficient >105) (11) and partitions efficiently into the lipid bilayer of various cellular membranes. Upon photolysis (Xmax = 310 nm, E -21,400M-1 cm-1) with the 314-nm line of a mercury lamp, it is rapidly and efficiently converted into the reactive nitrene, which inserts covalently into intrinsic membrane proteins that contain lipid-embedded domains and, to a lesser extent, into phospholipids. When photoactivated in the plasma membrane, Ina blocks transduction of hormonal stimulation of adenylate cyclase by uncoupling of the hormone receptor from the enzyme (13).It was of interest to determine whether energy-transfer processes could be used to photoactivate a discrete fraction of the azide molecules present in a membrane or cell.Resonance energy transfer could not be used because Ina absorbs light down to 260 nm and any donor molecules chosen for its photoactivation would have to have absorption maxima below this wavelength, this clearly being impractical. Aromatic azides, however, are capable of photosensitized activation by donors that absorb at longer wavelengths than those absorbed by the azides themselves (14, 15). Photosensitization occurs by various mechanisms (16, 17) in a process that requires the approach of the photosensitizer and acceptor pair to within the collisional range in order for intermolecular exchange of excitation energy to take place (18). The nature of the reactive intermediates generated by photosensitization of aryl azides in different solvents is not completely understood. However, they seem to be reactive in hydrogen-abstraction, dimerization, and insertion reactions (19,20). Since many sensitizers are aromatic apolar molecules, it is likely that they will parallel the distribution of Ina into lipophilic regions of the membrane. This sh...