Artificial Retina Mimbrane Assembly Utilzing Intelligent Materials: Biocompatibility and Electorphysiologic Features

In recent studies there have been various attempts at replacing a damaged retina with an artificial one. This paper outlines the assembly of an artificial retina membrane by incorporating a photorective protein bacteriorhodopsin into an electrochemically syntheszed conducting polymer polypyrrole. An electrophysiologic test was conducted to evaluate the photoresponsiveness of the bacteriorhodopsin and rabbit eyes were used to examine the biocompatibility of the artificial retina. The electrophysiologic test analyzed both wave forms and amplitudes obtained by photostimulating the artificial retina membrane with various light intensites(0.2, 2, 20J). In the biocompatibility test, the artificial membrane was inserted into the anterior chambers(4 eyes) and vitreous cavities(8 eyes) of rabbits. The condition of the eyes was then observed for one month. At the end of the first moonth, the eyes were enucleated and a histological examination was carried out. The electrophysiologic study displayed negative reflection waves, which are characteristic in rhodpsin, and their amplitudes showed a correspondign increase with stronger light intensities. The results of the biocompatibility test demonstrated that inflammatory reactions were not prominent in either the anterior chambers or the vitreous cavities during the first month and the histological examinations revealed no specific findings. In conclusion, a membrane assembled utilizing an electroactive polymer and a phocial retina.

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.

The chromosome-binding site of bacteriorhodopsin. Resonance Raman and surface-enhanced resonance Raman spectroscopy and quantum chemical study.

Surface-enhanced Raman spectra of membrane protein, located in native mem brane, bacteriorhodopsin, adsorbed by silver electrodes and hydrosols have been obtained for the first time. The distance between the retinal Schiff's base and the external side of purple membrane of Halobacteriim halobiim was shown to be 6–9 A. The possible distribition of the point charges aroind protonated retinal Schiff's base has been proposed on the basis of the resonance Raman data and quantim chemical CNDO/S-CI calculations. Such a model contains tyrosine residue located near the retinal Schiff's base and connected with COO– groip via hydrogen bond COO– group acts as a protonated Schiff’s base counterion. The distance between oxygen atoms of COO– group and retinal Schiff's base plane is 2·5-3·0A. The hydrogen bond (O-H. . .O–) length between oxygen atom of OHgroup and oxygen atom of COO_ group has been chosen 2·7±0·1Å Tyrosine hydroxyl group is located at 2·8-3·5 A from retinal Schiff's base plane· It was shown that in contrast to generally accepted Honig and Nakanishi model the spectral properties of Brh570, K610, L550 and Μ4Ϊ2 forms of bacteriorhodopsin photocycle as well as observed tyrosine deprotonation and COO– group protonation during M412 formation can be explained reasonably well by the suggested charge distribution· Furthermore, such a model of bacteriorhodopsin active site microenvironment allows to explain catalyzing of photoinduced protonated retinal Schiff's base deprotonation observed in our preliminary experiments.

Characteristics of fluorescamine modified bacteriorhodopsin.

The light activated proton pump, bacteriorhodopsin was modified with varying amounts of flourescamine, the fluorescamine to protein ratio ranging from 1 to 100. The modified protein was washed free of excess of fluorescamine and reconstituted into phospholipid vesicles to check the proton pumping activity. Although the spectral investigations indicated chemical modification, the circular dichroism measurements pointed to an overall loss of the trimeric structure of the protein. The implications of the present study are that the modifying agent can interact non-specifically with the protein, altering its structural parameters, which in turn affects the function of the protein.