IN-VIVO SPECTROSCOPIC STUDY OF PHOTORECEPTOR PIGMENTS OF BLEPHARISMA-JAPONICUM RED AND BLUE CELLS

In the coloured ciliate Blepharisma japonicum, step-up photophobic responses are triggered by the endogenous pigment blepharismin. Blepharismin, red in dark-grown cells, is intracellularly photooxidized into a blue form (oxyblepharismin), still acting as photosensing pigment. With the aim of correlating the spectroscopic properties of blepharismin and oxyblepharismin in vivo with their photophysiological features, optical absorption, steady-state and time-resolved fluorescence spectra have been measured on cell suspensions. Both in blepharismin and oxyblepharismin in their physiological molecular environment, three fluorescent species have been observed, with virtually the same lifetimes (similar to 0.2 ns, similar to 1.0 ns, similar to 3.5 ns), but significantly different relative amplitudes. In red cells the long-living component has a very low relative amplitude(similar to 4%) and the short-living one is largely predominant(> 78%), whereas in blue cells the slowly decaying species has a slightly higher relative amplitude (similar to 40%) than the intermediately (similar to 31%) and the fast decaying species (similar to 29%). Together with the spectral width of time-gated spectra, these data are discussed in connection with current hypotheses on the structures of the chromophores. No meaningful difference in the above-mentioned spectroscopic parameters was observed after 30 min of UV-B irradiation, showing that no significant difference exists between red and blue blepharismin as far as UV-B lability is concerned.