Abstract
Purpose :
Vision starts with the photoisomerization of the 11-cis-retinylidene chromophore of visual opsins, followed by the hydrolysis of the resultant all-trans-retinylidene product. This hydrolysis is critical for regeneration of apo-opsins with fresh 11-cis chromophore for sustained vision. To determine the rate of this hydrolysis with opsins in either native membranes or detergent micelles, a liquid chromatographic mass spectrometry (LC-MS) was recently developed that was also found to capture the photochemistry of any retinylidene opsin.
Methods :
Rhodopsin in rod outer segment membranes or immunopurified in detergent micelles were treated with sodium borohydride in isopropanol to trap the retinylidene chromophore by reduction. The reduced rhodopsin was isolated by isopropanol precipitation and further analyzed by proteolysis and LC-MS. The alcohol solubilized lipid fraction was analyzed by LC-MS to track the hydrolyzed retinal chromophore. Opsin bound retinal in the protein fraction and hydrolyzed retinal in the lipid fraction were measured to determine hydrolysis rates. Cone opsins and RPE-retinal g protein receptor (RGR) were also immunopurified in detergent micelles and studied for hydrolysis relative to rhodopsin.
Results :
Hydrolysis of the all-trans-retinylidene of photoactivated rhodopsin (Rho*) was significantly slower than that of cone opsins. During 10 sec of illumination at 0 °C, 1.7 ± 0.6% (n = 3) of Rho* spent chromophore was hydrolyzed in native membranes in contrast to 0.6 ± 0.8% (n = 3) in detergent micelles. However, hydrolysis occurred faster in photoactivated cone opsins (Red*, Green*, Blue*) with the extent of hydrolysis being 92.1 ± 0.5% (n = 3) for Red*, 72.2 ± 5.0% (n = 3) for Green*, and 89.2 ± 1.2% (n = 3) for Blue*. The photochemistry of rhodopsin and cone opsins in the isomerization of 11-cis- to all-trans-retinylidene was observed in comparing LC-MS signals before and after the 10 sec illumination. Meanwhile, the converse all-trans to 11-cis photochemistry of RGR was also observed.
Conclusions :
Hydrolysis of the all-trans-retinylidene of photoactivated opsin proteins was captured in either native membranes or detergent micelles. The hydrolysis in photoactivated cone opsins was significantly faster than in Rho*. The 11-cis- to all-trans photochemistry of rhodopsin and cone opsins was observed as well as the converse all-trans to 11-cis photochemistry occurring within RGR.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.