Photoaffinity labeling of serotonin-binding proteins

A photosensitive arylazide derivative of serotonin (nitroaryl-azidophenyl serotonin, NAP-serotonin) has been synthesized for use in studying the biochemical nature of serotonin binding sites. ³H]-NAP-serotonin possesses a similar ability to bind to the crude membranes of rat brains does ³H]-serotonin and therefore seems suitable for use as a photoaffinity labeling probe for serotonin binding sites. Upon irradiation with ultraviolet light, ³H]-NAP-serotonin covalently attaches to protein components of the brain homogenate. Several distinct radioactively labeled proteins have been separated by sodium dodecyl sulfate polyacrylamide gel electro-phoresis. Their apparent molecular weights were 80,000, 49,000, and 38,000 (±5%). When 1 μM of unlabeled serotonin or d-lysergic acid diethylamide (d-LSD) was added prior to photolysis, the incorporation of ³H]-NAP-serotonin into these proteins was inhibited significantly. No inhibitory effect was observed when dopamine was used. These observations suggest that the photoaffinity labeled proteins are specific for serotonin binding.