Photoreactivity of Bis-retinoid A2E Complexed with a Model Protein in Selected Model Systems

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.

Keywords: A2E; EPR-Spin trapping; Photochemical reactivity; Protein oxidation; Reactive oxygen species; Singlet oxygen.

Scheme 1

The structure of A2E

Fig. 1

UV–Vis spectra of A2E in D2O/1% DDM (black line), with BSA in D₂O-micellar model system (gray line), A2E-BSA complex in D₂O (dashed line) and BSA in D₂O (dotted line)

Fig. 2

Blue light-induced oxygen uptake in the micellar model system containing 50 μM A2E, 100 μM BSA, or a suspension of both in 1% TX-100 (a) and determined rates of oxygen photoconsumption (b)

Fig. 3

Dark and blue light-induced oxygen uptake in the micellar model system containing 50 μM A2E and 100 μM BSA in H₂O or D₂O (a) and determined rates of oxygen photoconsumption (b)

Fig. 4

Blue light-induced oxygen uptake of A2E-BSA complex in D₂O (a) and the comparison of oxygen consumption rates of A2E and BSA in D₂O-micellar system with the A2E and BSA in D₂O-non-micellar system (b)

Fig. 5

Time-resolved luminescence induced by photoexcitation of A2E in D₂O: 1270 nm luminescence decay detected in A2E samples in D₂O/DDM (black line) with addition of 100 µM BSA (light gray line) and with 100 µM BSA but without DDM (lower dark gray line). A2E was excited with 422 nm, 360 µJ laser pulses

Fig. 6

EPR spectra of detected (upper line) and simulated (lower line) DMPO adducts in the samples containing: a A2E in 80% DMSO after 30 min of blue-light irradiation. Hyperfine splitting constants were calculated as: a_N = 12.80 G, a_H^β = 10.37 G, and a_H^γ = 1.25 G; b A2E and BSA in 80% DMSO after 30 min of blue-light irradiation. c Simulations of individual DMPO spin adducts found in the A2E and BSA sample. Hyperfine splitting constants were calculated as: a_N1 = 12.95 G, a_H1^β = 10.44 G, a_H^γ = 1.44 G, a_N2 = 14.01 G, and a_H2^β = 12.28 G

Fig. 7

Kinetics (a) and accumulation rates (b) of DMPO-radical adducts formation in the samples containing A2E or BSA or A2E and BSA in 80% DMSO upon blue-light irradiation. Sample containing A2E and BSA was also blue light-irradiated with 500 units/mL of superoxide dismutase

Fig. 8

Evolution of the COH fluorescence in the samples containing 50 μM A2E and 100 μM BSA in D₂O-micellar model system (a) or D₂O-non-micellar model system (b) and H₂O-micellar model system (c) or H₂O-non-micellar model system (d) after irradiation with 440 nm light for selected time intervals

Fig. 9

Concentration of albumin hydroperoxides generated in samples containing 50 μM A2E and 100 μM BSA in micellar model systems (a) and non micellar model systems (b) after irradiation with 440 nm light for selected time intervals

Fig. 10

The initial formation of the albumin hydroperoxides (V) generated in the samples containing 50 μM A2E and 100 μM BSA in micellar model systems and non-micellar model systems after irradiation with 440 nm light

Fig. 11

Fluorescence spectra of A2E (a), BSA (b), and tryptophan derivatives: N′-formylkynurenine (c) and kynurenine (d) in PB-D₂O, recorded before (black lines) and after photooxidation mediated by A2E at selected time intervals. The excitation wavelengths are given in the top left-hand corners. Emission spectra of tryptophan and its derivatives before the addition of A2E to the BSA sample are marked as black dotted lines