Time-resolved fluorescence spectroscopy and photolysis of the photoreceptor blepharismin.

Blepharismin is the photoreceptor for the photophobic response in the ciliate Blepharisma japonicum (Scevoli, P., Bisi, F., Colombetti, G., Ghetti, F., Lenci, F., and Passarelli, V. (1987) J. Photochem. Photobiol.: B. Biol. 1, 75-84; Lenci, F., Ghetti, F., Gioffre, D., Heelis, P.F., Thomas, B., Phillips, G.O., and Song, P.-S. (1989) J. Photochem. Photobiol.: B. Biol. 3, 449-453). Blepharismin was solubilized from the red cells with 2% n-octylglucopyranoside. A crude pigment-protein preparation was then successively subjected to Bio-Gel A1.5 filtration, FPLC/hydroxyapatite and FPLC/DEAE ion-exchange chromatography. At least two spectrally distinct forms of blepharismin, with the respective absorbance maxima at 597 +/- 1 and 601 +/- 1 nm, were resolved. The steady state fluorescence emission maxima were at 602.5 and 617.5 nm, respectively. The fluorescence decay curves for these pigments were non-exponential. The major component possesses relatively short fluorescence lifetime (200-500 ps) for the former, according to a global analysis. This analysis suggests that the excited state of the shorter wavelength-absorbing form of blepharismin undergoes primary photoprocess faster than that of the free parental chromophore hypericin. Photolysis of blepharismin in solution yielded a irreversible product, accompanied by a 10-12 nm bathochromic shift of the absorbance maximum. However, the mechanistic nature of the time-resolved fluorescence and the photochemistry of blepharismin remains to be elucidated.