Selective labeling of proteins in biological systems by photosensitization of 5-iodonaphthalene-1-azide.
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.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism / Q000528:radiation effects*; D066298:In Vitro Techniques; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000378:metabolism*; D010782:Photolysis; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism
D001386:Azides; D008051:Lipid Bilayers; D008565:Membrane Proteins; C015298:iodonaphthylazide; D012243:Rhodopsin; D000262:Adenylyl Cyclases