Photoaffinity labeling of rat alpha-fetoprotein.
Two photosensitive estrogen derivatives, 16-diazoestrone and 4-azidoestradiol, have been studied as photoaffinity-labeling agents for the estrogen-binding site of rat alpha-fetoprotein (AFP). 16-Diazoestrone has a high affinity for AFP (121%, relative to 17 beta-estradiol), and photolysis of the 16-diazo[3H]estrone . AFP complex for 30 min at 300 nm results in the covalent attachment of 19% of the ligand bound reversibly to the estradiol site at the time of irradiation. The photocovalent attachment appears to result from both a "chromophore-dependent" process (photoaffinity labeling), whose time course follows the photolytic consumption of the diazoketone chromophore and is not susceptible to scavenging by nucleophiles, and a "chromophore-independent" process (pseudophotoaffinity labeling) that results from covalent attachment of an electrophilic photoproduct and can be intercepted by 20 mM mercaptoethanol. AFP covalently labeled with 16-diazo[3H]estrone has the same electrophoretic mobility as unlabeled AFP on normal and sodium dodecyl sulfate-polyacrylamide gels; labeled AFP has an apparent molecular weight of 69,400 and is distinguishable from albumin (which is also labeled by 16-diazo[3H]estrone, but not in a site-specific manner). While 4-azido[3H]estradiol undergoes extensive photoinduced covalent attachment to AFP, little of this is site-specific.
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D003979:Diazonium Compounds; D004958:Estradiol / Q000031:analogs & derivatives*; D004970:Estrone / Q000031:analogs & derivatives*; D007700:Kinetics; D008027:Light; D010777:Photochemistry; D011485:Protein Binding; D051381:Rats; D000509:alpha-Fetoproteins
D000345:Affinity Labels; D001386:Azides; D003979:Diazonium Compounds; D000509:alpha-Fetoproteins; D004970:Estrone; C027620:16-diazoestrone; C027621:4-azidoestradiol; D004958:Estradiol