Sub-picosecond fluorescence spectroscopy of the M intermediate in the photocycle of bacteriorhodopsin by using up-conversion fluorometry

A fluorescence up-conversion method was applied to the measurement of the fluorescence lifetime of the M intermediate with a 13-cis retinal Schiff base. M formed under cw visible light (440–800 nm) irradiation of the purple membrane suspension of Halobacterium salinarum containing diethyl ether or guanidine hydrochloride at 24°C. An excitation source was a frequency-doubled Ti:Sapphire laser (408 nm, 240±10 fs). The 500 nm fluorescence of M was mixed with the fundamental of the laser and converted to UV for detection. The measured lifetimes (0.6 and 0.3 ps in ether and in guanidine, respectively) were shorter than those of protonated all-trans retinal Schiff bases in solution and in apoprotein.