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 [125I]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 [125I]Ina-labeled
membrane proteins and
hormone uncoupling indistinguishable from that obtained following direct photolysis at 314 nm.
Photosensitization of the
azide molecules is confined to the vicinity of the
photosensitizer chromophore. This allowed selective labeling of chromophore-bearing
proteins in
solution or in membranes.
Bovine serum albumin-
fluorescein conjugate, in the presence of nonderivatized soluble
proteins, was exclusively labeled by [125I]Ina when irradiated at 480 nm, but random labeling occurred on photolysis at 314 nm. Likewise,
rhodopsin in rod outer segment membranes from frog retina was exclusively labeled by [125I]Ina upon
photosensitization at 380 nm. Random labeling again occurred on direct irradiation at 314 nm. The results suggest that selective labeling in complex
biological systems may be achieved by photosensitized activation of
azides.