Fluoro-Gold: composition, and mechanism of uptake.
Determining the mechanism by which fluorescent retrograde neuronal tracers are taken up requires knowledge of their composition. It has been claimed that Fluoro-Gold, a retrogradely transported fluorescent neuronal tracer, is 2-hydroxy-4,4'-diamidinostilbene (hydroxystilbamidine), an amidine antibiotic. However, this appears questionable, since the fluorescence spectrograms reported for Fluoro-Gold differ markedly from the spectrograms previously reported for purified hydroxystilbamidine. To help clarify the mechanism by which Fluoro-Gold might be taken up, it was decided to examine its composition and determine whether hydroxystilbamidine was its active agent. Fluoro-Gold was found by mass spectrometry to contain a component with a molecular weight of 280 Da (identical to that of hydroxystilbamidine), and fluorescence spectroscopy demonstrated the existence of a substance with a fluorescence spectrum similar to that of purified hydroxystilbamidine. Although a major fluorescent impurity was also observed, chromatographic separation of different fluorescent components of Fluoro-Gold suggested that the fraction resembling hydroxystilbamidine was responsible for its retrograde labeling of cells. It is concluded that hydroxystilbamidine is the active constituent of Fluoro-Gold. Chemically, hydroxystilbamidine is a weak base. In this respect it resembles True blue, DAPI, Granular blue, bis-benzimide, Nuclear yellow, and several other retrogradely transported molecules. It is suggested that these agents cross cell membranes in their uncharged form and are trapped in lysosomes and endosomes by a favorable pH gradient. Thus, the uptake of this type of retrograde tracer may be an example of a well-understood process occurring widely throughout biological systems: the trapping of weak bases in acidic cellular compartments.
Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis 55455.
M W
MW
Wessendorf
D000818:Animals; D001933:Brain Stem / Q000033:anatomy & histology; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000493:pharmacokinetics; D006651:Histocytochemistry; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013054:Spectrophotometry, Atomic; D013266:Stilbamidines / Q000737:chemistry
C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; D005456:Fluorescent Dyes; D013266:Stilbamidines; C100274:hydroxystilbamidine