Bleaching of Purple Membrane With O‐substituted Hydroxylamines

The retinal Schiff base of bacteriorhodopsin, in the purple membrane from Halobacterium halobium, can be cleaved by hydroxylamine in the presence of light. We have further investigated this reaction with a series of O‐substituted hydroxylamines, RONH2, where R = ‐H (HA), ‐CH3 (MHA), ‐SO3− (HAS), benzyl‐ (BHA), p‐nitrobenzyl‐ (NBHA), and pentafluorobenzyl‐ (FBHA). All except MHA caused light‐induced bleaching of the purple membrane and the chromophore could be regenerated from apomembrane and all‐trans retinal. Relative bleaching rate constants were obtained from V=QIak0X/(kr+k0X), where V= bleaching rate, Q= quantum yield, Ia = absorbed light intensity, X= hydroxylamine concentration, k0= rate constant for bleaching and kr= rate constant for return of photoexcited bacteriorhodopsin to the initial state. This equation fits the time‐, concentration‐ and intensity‐dependences of the bleaching reactions in 0.02 M phosphate, pH 7.0. The rate constants k0 relative to HA were: MHA: 0; HAS: 0.3; HA: 1.0; BHA: 1.8; FBHA: 10.1; NBHA: 10.8. The relative rate constants do not correlate with the basicity of the derivatives. Instead, the results suggest that the retinal Schiff base is near a non‐polar cavity into which an aromatic group can be inserted.