Absolute absorption spectra of batho- and photorhodopsins at room temperature. Picosecond laser photolysis of rhodopsin in polyacrylamide

Abstract

Picosecond laser photolysis of rhodopsin in 15% polyacrylamide gel was performed for estimating absolute absorption spectra of the primary intermediates of cattle rhodopsin (bathorhodopsin and photorhodopsin). Using a rhodopsin digitonin extract embedded in 15% polyacrylamide gel, a precise percentage of bleaching of rhodopsin after excitation of a picosecond laser pulse was measured. Using this value, the absolute absorption spectrum of bathorhodopsin was calculated from the spectral change before and 1 ns after the picosecond laser excitation (corresponding to the difference spectrum between rhodopsin and bathorhodopsin). The absorption spectrum of bathorhodopsin thus obtained displayed a lambda max at 535 nm, which was shorter than that at low temperature (543 nm) and a half band-width broader than that measured at low temperature. The oscillator strength of bathorhodopsin at room temperature was smaller than that at low temperature. The absolute absorption spectrum of photorhodopsin was also estimated from the difference spectrum measured at 15 ps after the excitation of rhodopsin (Shichida, Y., S. Matuoka, and T. Yoshizawa. 1984. Photobiochem. Photobiophys. 7:221-228), assuming a sequential conversion of photorhodopsin to bathorhodopsin. Its lambda max was located at approximately 570 nm, and the oscillator strength was smaller than those of rhodopsin and bathorhodopsin.

Bathoiodopsin, a primary intermediate of iodopsin at physiological temperature.

“…The absorption maximum of bathoiodopsin thus obtained was located at 625 nm, which is a slightly shorter wavelength than that (about 640 nm) obtained by low-temperature spectrophotometry at 78 K (9). The molar extinction coefficient of bathoiodopsin at room temperature is comparable to that of iodopsin at the same temperature but 1.5 times lower than the extinction coefficient at 78 K. Similar observations (Emax of the bathointermediate at room temperature is lower than that at low temperature) have been reported in bacteriorhodopsin (22) and cattle rhodopsin (21).…”

“…Thus, diffusion of the excited molecule in the sample cell makes it difficult to accurately estimate the percentage of bleaching. We have developed a method for accurately estimating the percentage of bleaching of rhodopsin after laser excitation, in which a polyacrylamide gel was used to prevent diffusion of the excited molecules (21 (Fig. 6) using the difference spectrum between bathoiodopsin and iodopsin (Fig.…”

Photoisomerization mechanism of the rhodopsin chromophore: picosecond photolysis of pigment containing 11-cis-locked eight-membered ring retinal.

“…About 10 years ago Shichida et al (5) observed that excitation of cattle rhodopsin with a weak laser pulse by which only the one-photon reaction of rhodopsin took place yielded an intermediate earlier than bathorhodopsin. This intermediate was named photorhodopsin, which has an absorption maximum at a wavelength longer than that of bathorhodopsin (5,6). Recently, Schoenlein et al (7) reported that this primary intermediate is formed from the excited state of rhodopsin within 200 fs on the basis of a femtosecond laser photolytic experiment.…”

“…A 1-kW projector lamp (Rikagaku, Tokyo) was used for irradiation of the samples and wavelengths were selected by using optical filters [blue light at 436 nm, interference filter (Nihonshinku, Tokyo; half-bandwidth = 2 nm); yellow light at wavelengths >490 nm, cutoff filter (Toshiba; VO51)]. A glass optical cell filled with water (light path, 6 cm) was placed as a heat shield between the filters and the light source.…”

Bathorhodopsin structure in the room-temperature rhodopsin photosequence: picosecond time-resolved coherent anti-Stokes Raman scattering.

“…After photon absorption, a series of at least seven intermediates making up the RhRT photosequence appears. These room-temperature intermediates have been identified primarily via transient absorption spectroscopy (3)(4)(5)(6)(7)(8)(9).…”