Reversible Dissociation of the Purple Complex in Bacteriorhodopsin and Identification of 13‐<i>cis</i> and all‐<i>trans</i>‐Retinal as its Chromophores

1 Suspensions of the purple membrane from Halobacterium halobium in water-dimethyl-sulfoxide mixtures change their absorption maximum dependent on Me2SO concentration. Between 30 and 60% Me2SO the purple complex equilibrates with a second complex form called the 460-nm complex. The apparent thermodynamic parameters of this reaction have been determined. 2 The equilibrium is influenced by pH and the nature of the buffering ion species in such a way that low pH and triethanolamine or histidine favour the purple complex. 3 The kinetics of the reaction indicate the participation of more than two species and thin-layer chromatography reveals two retinal isomers to be present in both complexes, i.e. 13-cis and all-trans-retinal. Light causes isomerization of the 13-cis to the all-trans form, in the dark a mixture of both isomers is found. 4 Only the 460-nm complex reacts with hydroxylamine to form retinaloxime. Retinaloximes derived from authentic retinal isomers show two spots on thin-layer plates, presumably due to syn and anti forms of the oxime. The reaction product from detergent-treated purple membrane shows the four spots corresponding to the 13-cis and all-trans-retinaloximes. The product from the 460-nm complex forms mainly two spots corresponding to 13-cis and all-trans-retinaloxime in only one form, either the syn or the anti form. 5 The reaction with hydroxylamine has a fast second-order and a slow zero-order component. The products formed at any time are the same. After the end of the fast initial phase only the purple complex is present in the sample and has first to be isomerized before it is dissociated by Me2SO and can react with hydroxylamine. This rate-limiting step does not involve C°C double bond isomerization but interconversion of two unknown complex configurations. 6 Light accelerates the slow part of the reaction with hydroxylamine. The product then consists mainly of syn and anti all-trans-retinaloxime but also syn and anti 13-cis-retinaloxime. This is interpreted as intermediate formation of the hydroxylamine sensitive 412-nm complex by reversible photolysis of the purple complex as described in [16]. In this case apparently no preference for the formation of the syn or the anti form exists.