Abstract
Purpose :
Photoexcitation of rhodopsin results in the hydrolysis of all-trans-retinal (ATR) - a potent photosensitizer that, upon absorption of light in the presence of oxygen, generates its reactive form1O2. It is believed that ATR is rapidly reduced to retinol that prevents its photosensitizing effects. The aim of this study was to determine the yields of 1O2 generation by discs isolated from dark-adapted bovine POS as a function of time after photobleaching and compare that kinetics with the kinetics of ATR hydrolysis and enzymatic reduction.
Methods :
Dark-adapted discs from bovine POS were isolated by differential centrifugation. Exposure for 1 min to green light was used to bleach rhodopsin. The hydrolysis of ATR after bleaching of rhodopsin was monitored using intrinsic tryptophan fluorescence at 330 nm excited by 295 nm light. Reduction of ATR was monitored in the presence of NADPH/ATP by retinol fluorescence at 490 nm excited by 324 nm light. Phosphorescence of 1O2 at 1270 nm was monitored after a 5 ns laser pulse from the suspension of dark-adapted discs and at selected times after discs photobleaching.
Results :
The rates of increase in Trp fluorescence after rhodopsin photobleaching were (3.1 ± 0.1)x10-3s-1 and (8.2 ± 0.3)x10-3s-1 at 19°C and 37°C, respectively. In the presence of equimolar NADPH to rhodopsin, retinol fluorescence increased with a rate of (7.00 ± 0.04) x 10-4 s-1. The yield of 1O2 sharply increased immediately after 1 minute photobleaching and a subsequent slower increase followed the kinetics of ATR hydrolysis and reaching a plateau after about 10 min where the yield of 1O2 increaed by an additional 25% in comparison with its yield immediately after bleaching. The reduction of ATR to retinol led to a decrease of 1O2 yields 40 min after bleaching only by 37%.
Conclusions :
Unexpectedly, photobleaching of POS discs results in a rapid increase in the yield of 1O2 , that occurs on a faster timescale than hydrolysis of ATR. A subsequent slow increase in 1O2 yields follows a similar kinetics as the hydrolysis of ATR, suggesting that all-trans-retinylidene form of ATR bound via Schiff-base linkage to Lys296 in opsin can photosensitize singlet oxygen generation and the yields further increase upon its hydrolysis. Moreover, in addition to ATR, retinol can contribute to singlet oxygen generation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.