Fluorescence histochemical demonstration of dopa thioethers by condensation with gaseous formaldehyde

Abstract

The usefulness of the formaldehyde (FA) and glyoxylic acid (GA) methods for the fluorescence histochemical demonstration of dopa thioethers has been tested using protein droplet models. Similar fluorescence intensities were recorded from these compounds after either FA or GA treatment. Cysteinyldopa gave a high fluorescence yield similar to that obtained from dopamine and dopa in the FA reaction, whereas gluatitodopa showed a lower, although clearly visible fluorescence. Since the FA method seemed to be the most useful one for demonstration of catechol thioethers, the FA-induced fluorophores of these compounds were further characterized by microspectrofluorometry. The spectral characteristics of the thioether fluorophores (excitation maxima at 420 nm and emission maxima at 480-485 nm) distinguish these substances from dopa and other compounds fluorogenic in the Falck-Hillarp method. Dopa thioethers are proposed to form fluorophores with FA in a manner analogous to that of the primary catecholamines i.e. via low-fluorescent tetrahydroisoquinolines, along two different pathways, to strongly fluorescent 3,4-dihydroisoquinolines and 2-methyl-dihydroisoquinolinium compounds. These dihydroisoquinolines are in a pH-dependent tautomeric equilbrium with their quinoidal forms as reflected by a characteristic spectral shift upon acidification. The results of this study provide the guide-lines for the characterization of fluorogenic compounds in pigment-forming cells.