Azido auxins : photolysis in solution and covalent binding to soybean

The potential of three auxin analogs, 4-, 5-, and 6-azidoindole-3-acetic (4-N(3)IAA, 5-N(3)IAA, and 6-N(3)IAA), as photoaffinity labeling agents for the detection and isolation of auxin receptors was assessed by irradiating these compounds at 365 nm on TLC plates, in solution, and in contact with soybean (Glycine max L. Merr. var. Wayne) hypocotyl. Photolysis on TLC plates produces immobile spots, indicating extremely polar or covalent binding of the photoproducts to the plates. On irradiation in buffer or in buffer containing sucrose, all three compounds decompose at rates that are first order in N(3)IAA to give fluorescent solutions. Photolysis through a Pyrex filter is slower than that through quartz, but the filter prevents tissue damage and allows a given dose of irradiation to photolyze all three N(3)IAAs to the same extent. The effects of photolysis of these compounds in vivo were evaluated with a straight growth assay using etiolated soybean hypocotyl segments. According to this assay, the photoproducts of the N(3)IAAs possess little auxin activity. Irradiation of soybean hypocotyl tissue after 1-hour exposure to 4-N(3)IAA in the dark causes the tissue to grow during 12 hours as much as tissue that is continuously exposed to 4-N(3)IAA in the dark for this period, suggesting that, on photolysis, this auxin analog binds irreversibly to an auxinsensitive site. Although the fluorescence intensity of the photolyzed N(3)IAAs is weak enough to require another method of detecting the bound analog under physiological conditions, the evidence for covalent binding of the N(3)IAAs on photolysis implies that these compounds will be satisfactory photoaffinity labeling agents.