Bathorhodopsin-rhodopsin difference spectra of native 11-cis-rhodopsin and regenerated 9-cis-rhodopsin were measured at room temperature with a double-beam laser spectrophotometer after excitation at 532 nm. A detailed analysis of data obtained at 85 psec after excitation suggests that the bathorhodopsins generated from 1 1-cis-and 9-cis-rhodopsin differ in their extinction coefficients and that their absorption maxima are shifted in wavelength by about 10 nm from one another. The ratio of quantum yields for photochemical production of the 11-cis-bathorhodopsin and the 9-cis-bathorhodopsin approximates 1. Implications that the early photochemical processes in vision are more complex than previously considered are explored.An improved double-beam laser spectrophotometer was developed to perform a comparative study of the bathorhodopsin photoproducts of native 11-cis-rhodopsin and regenerated 9-cis-rhodopsin. The instrument measures difference spectra of the initial photoproduct (bathorhodopsin) minus the rhodopsin converted for each of these visual pigments under identical experimental conditions. Each spectrum, covering the range of 400-650 nm, was recorded with a single monitoring pulse after excitation at 532 nm. Particular attention was given to optimizing the signal-to-noise ratio of measured absorbance changes in order to achieve data collection with excitation pulse energies that were low enough to avoid multiphoton events. Our goal was to establish, as best possible, characteristic absorption spectra of the transient bathophotoproducts arising from 11-cis-and 9-cis-rhodopsin at room temperature and to compare these with the published spectra obtained by photostationary studies carried out with aqueous glycerol/rhodopsin glasses at low temperature.METHODS AND MATERIALS Visual Pigment Samples. Buffer is defined throughout this work as 0.01 M Hepes, pH 7/0.1 mM EDTA/1.0 mM dithiothreitol. Rhodopsin was prepared from frozen bovine retinae (G. Hormel), solubilized in Ammonyx-LO detergent, and purified by hydroxyapatite chromatography (1, 2). The relative purity of the samples used is indicated by the ratio A278/ A498 = 1.9 + 0.1. For preparation of regenerated 9-cis-rhodopsin, the 9-cis-retinaldehyde isomerwas made byphotolysis of all-trans-retinal (Fluka, Buchs, Switzerland) dissolved in trifluoroethane, with appropriately filtered light (Schott 06455 and 06745). 9-cis-Retinal was isolated by preparative HPLC and was found to be >99% pure by analytical HPLC. The retinal was stored at -700C under N2 until incorporated into opsin. 9-cis-Rhodopsin was prepared from opsin, made by washing bleached disk membranes with buffered NH2OH, and purified 9-cis-retinal. The regenerated pigment was solubilized and purified by hydroxyapatite chromatography (1, 2). The relative purity is indicated by the ratio A278/A485 = 1.8 ± 0.1. The purified 9-cis-rhodopsin was shown to contain the 9-cis-retinal isomer by HPLC. The molar extinction coefficients used throughout this work were eri (498 nm) = 4.06 X 104 M'1cm-1 for ...

The formation of Metarhodopsin II, measured spectroscopically in the excised bovine retina at 37 degrees C, can be described by the sum of two exponentials. The R2 response of a simultaneously recorded early receptor potential can be matched by the difference of two weighted exponentials, whose time constants are identical to those obtained for the spectroscopic signal.

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