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Anne M Hanneken, Masahiro Kono; Spontaneous in vitro generation of rhodopsin with all-trans retinal. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6048.
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Purpose: Traditional dogma states that rhodopsin is generated following the formation of a protonated Schiff base bond between 11-cis retinal and opsin. We assessed the possibility that rhodopsin could form spontaneously in bleached rod outer segments (ROS) with all-trans retinal.
Methods: Bovine ROS was isolated from frozen retinas using a discontinuous sucrose gradient. ROS was isolated under dim red light conditions to maintain native rhodopsin pigment and bleached, followed by multiple washes with BSA and then buffer alone. Generally, 0.5 µM rod opsin was placed in a cuvette. Differing amounts of all-trans retinal were added to the cuvette and spectra recorded.
Results: Under our conditions, incubation of bleached ROS with all-trans retinal alone generated up to almost 50% rhodopsin or rhodopsin-like pigment (494 nm absorbing species) relative to the amount of free opsin in the cuvette over a 10 min period. This pigment, like rhodopsin, does not dark bleach in the presence of hydroxylamine but readily bleaches with light. Photobleaching of this rhodopsin or rhodopsin-like pigment in the presence of light (>520nm) followed by a second dark incubation over a 40 min period led to regeneration of the rhodopsin or rhodopsin-like pigment, albeit at a significantly slower rate and lower yield which plateaued after 30 min. The amount of the rhodopsin-like pigment generated from all-trans retinal appears to be dose and temperature-dependent, and is reduced in the presence of detergent.
Conclusion: These results were quite unexpected but reproducible. We are working to determine how much of this finding is due to experimental conditions. Is rod opsin able to act as an isomerase by itself? Could this be a result of thermal isomerization with the opsin trapping and accumulating the 11-cis form? Under specific in vitro conditions, rhodopsin appears to be spontaneously generated (and regenerated) in rod outer segments in the presence of all-trans retinal.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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