Abstract
The bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E) is formed as a byproduct of visual cycle in retinal pigment epithelium (RPE). It contributes to golden-yellow fluorescence of the age pigment lipofuscin, which accumulates in RPE. Lipofuscin can generate a variety of reactive oxygen species (ROS) upon blue-light excitation. Although in model systems photoreactivity of A2E has been determined to be low, this bis-retinoid exhibited significant phototoxicity in RPE cells in vitro. Although the mechanism of A2E-mediated phototoxicity remains mostly unknown, we hypothesize that formation of A2E-adducts with different biomolecules may play an important role. In this study, we investigated the photochemical reactivity of A2E and its complex with bovine serum albumin (BSA) using UV–Vis absorption and emission spectroscopy, EPR-spin trapping, EPR-oximetry, time-resolved singlet oxygen phosphorescence, and the fluorogenic CBA probe. Our data show that A2E after complexation with this model protein photogenerated an increased level of ROS, particularly singlet oxygen. We also demonstrated the ability of A2E to oxidize BSA upon excitation with blue light in aqueous model systems. The data suggest that pyridinium bis-retinoid could oxidatively modify cellular proteins under physiological conditions.
Highlights
It has been postulated that the amorphous pigment lipofuscin, which accumulates with age in retinal pigment epithelium (RPE), mediates photochemical reactions, which can contribute to oxidative stress in the outer retina
We investigated the effect of bovine serum albumin (BSA) on photochemical reactivity of A2E using EPR-spin trapping, EPR-oximetry, and singlet oxygen detection
Aerobic photoexcitation of A2E-BSA complexes resulted in the formation of protein hydroperoxides, with the rate being faster in samples without the detergent
Summary
It has been postulated that the amorphous pigment lipofuscin, which accumulates with age in retinal pigment epithelium (RPE), mediates photochemical reactions, which can contribute to oxidative stress in the outer retina. It has been documented that lipofuscin, can generate, upon excitation with blue light, reactive oxygen species (ROS) [1, 2], participate in lipid and protein oxidation, inactivate lysosomal and antioxidant enzymes [3,4,5,6] and inhibit phagocytic activity of RPE cells in vitro [7]. We investigated the effect of bovine serum albumin (BSA) on photochemical reactivity of A2E using EPR-spin trapping, EPR-oximetry, and singlet oxygen detection. We analyzed the capability of A2E to oxidize BSA using the sensitive coumarin boronic acid (CBA) probe and fluorescence measurements
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