Formation of 9-cis- and 11-cis-retinal pigments from bacteriorhodopsin by irradiating purple membrane in acid

Abstract

Both light-adapted and dark-adapted forms of bacteriorhodopsin in purple membrane in 67% glycerol solution were allowed to stand in acidic conditions by the addition of HCl to final concentrations from 4 X 10(-4) to 2 X 10(-2) M for 24 h at 3 degrees C. Over this concentration range, the acid-induced products from both species showed a maximum absorbance around 600 nm and high-performance liquid chromatography of extracted retinal isomers revealed that the acid-induced form of bacteriorhodopsin has 13-cis- and all-trans-retinals in a molar ratio of 4:6, which is intermediate between those of the dark-adapted and the light-adapted forms at neutral pH values. Exposure of the acid-induced form of bacteriorhodopsin to light at wavelengths longer than 670 nm at 3 degrees C caused a decrease of the absorbance around 600 nm with a concomitant rise of the absorbance around 500 nm. The extract from the irradiated products of bacteriorhodopsin in acid contained 9-cis- and 11-cis-retinals in addition to 13-cis- and all-trans-retinals. The absorbance maximum estimated from the analysis of the absorption spectra and the composition of the isomers was found at 495 nm for the 9-cis-retinal pigment and around 560 nm for the 11-cis-retinal pigment. On irradiation with 438-nm light, the 9-cis-retinal pigment disappeared with a concomitant increase of both the 13-cis- and all-trans-retinal pigments as judged by chromophore analysis and the absorption spectrum. The 9-cis-retinal pigment brought to pH 9 exhibited a maximum absorbance at 450 nm; this could be decomposed by the action of hydroxylamine or converted to a form resembling normal bacteriorhidopsin by 438-nm irradiation.