A fluorescent analog of colcemid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-colcemid, as a probe for the colcemid-binding sites of tubulin and microtubules.
The synthesis and biological testing of the fluorescent analog of colcemid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-colcemid (NBD-colcemid), are here described. NBD-colcemid exhibited a visible absorption maximum at 465 nm and fluoresced in the range of 520-540 nm, highly in environments of low polarity, whereas only slightly in aqueous solution. The addition of NBD-colcemid to bovine brain tubulin was accompanied by a striking enhancement of fluorescence. The fluorescent titration study suggested a stoichiometric binding of NBD-colcemid to tubulin. Assembled microtubules were directly visualized after mixing with NBD-colcemid using a fluorescence microscope. NBD-colcemid reversibly disrupted the metaphase spindles of sea urchin eggs as well as unlabeled colcemid. However, even when the birefringence of spindles was mostly lost, self-quenching properties of the NBD fluorescence allowed tubulin and its oligomers aggregated in higher concentrations in eggs to be visualized under a fluorescence microscope. The results suggest a wide applicability of NBD-colcemid as a fluorescent probe for studying the interactions of colcemid with tubulin and microtubules, as well as for localizing other colcemid-binding structures within cells.
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003703:Demecolcine / Q000031:analogs & derivatives* / Q000378:metabolism*; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D010069:Oxadiazoles / Q000378:metabolism*; D012617:Sea Urchins; D013050:Spectrometry, Fluorescence; D014404:Tubulin / Q000378:metabolism*
D005456:Fluorescent Dyes; D010069:Oxadiazoles; D014404:Tubulin; C052280:NBD-colcemid; D009327:4-Chloro-7-nitrobenzofurazan; D003703:Demecolcine