Determination of total proton release in purple membrane suspension by umbelliferone fluorescence quenching technique.

A technique for determining total proton release from purple membrane suspension under steady illumination has been described. Illuminated purple membrane is found to quench the fluorescence life-time of umbelliferone indicating the release of protons in the medium. Besides the "stoichiometric" release of protons from bacteriorhodopsin, there seems to be release of protons from sources other than protonated retinylidene Schiff base moiety also.

Bioorganic chemistry of the purple membrane of Halobacterium halobium—Chromophore and apoprotein modified bacteriorhodopsins.

Iodophenyl and anthryl retinal analogues have been synthesized. The transisomers have been isolated and purified by high pressure liquid chromatography. The purified isomers have been further characterized by nuclear magnetic resonance and ultraviolet-visible spectroscopy. Incubation of these retinal analogues with apoprotein (bacterioopsin), isolated from the purple membrane of Halobacterium halobium gave new bacteriorhodopsin analogues. These analogues have been investigated for their absorption properties and stability. The iodophenyl analogue has been found to bind to bacterioopsin rapidly. The pigment obtained from this analogue showed a dramatically altered opsin shift of 1343 cm-1. The anthryl analogue based bacteriorhodopsin, however, showed an opsin shift of 3849 cm-1. It has been found that bacteriorhodopsin is quite unrestrictive in the ionone ring site. The apoprotein seems to prefer chromophores that have the ring portion co-planar with the polyene side chain. The purple membrane has also been modified by treatment with fluorescamine, a surface active reagent specific for amino groups. Reaction under controlled stoichiometric conditions resulted in the formation of a modified pigment. The new pigment showed a band at 390 nm–indicative of fluorescamine reaction with amino group (s) of apoprotein –besides retaining its original absorption band at 560 nm. Analysis of the fluorescamine modified bacteriorhodopsin resulted in the identification of lysine 129 as the modified amino acid residue. Fluorescamine-modified-bacteriorhodopsin suspension did not release protons under photolytic conditions. However, proteoliposomes of fluorescamine-modifiedbacteriorhodopsin were found to show proton uptake, though at a reduced rate.