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. 2023 Mar 16;299(5):104614. doi: 10.1016/j.jbc.2023.104614

Figure 4.

Figure 4

Formation and quantification of N-Ret-Phospholipid conjugates in rod outer segment (ROS) membranes.A, representative UV-Visible spectra: (1) Dark-state (unbleached) ROS membranes solubilized in detergent; (2) ROS membranes illuminated with intense light in the presence of hydroxylamine (bleached), followed by the addition of detergent and TFA; (3, 4) Bleached ROS membranes supplemented with external ATR equivalent to 100% of dark-state rhodopsin and incubated at RT for 5 min or 30 min respectively, followed by the addition of TFA and detergent. Dotted blue lines indicate the wavelengths used to determine the concentration of dark-state rhodopsin (500 nm) and the percentage of ATR and N-Ret-PL at the endpoint (385 nm and 435 nm). B, differential spectra between bleached ROS as indicated in (2) in panel A and bleached ROS after the addition of ATR as indicated in (3) or (4) in panel A. C, percentage of N-Ret-PL, primarily N-Ret-PE, formed in bleached ROS membranes supplemented with external ATR. ROS membranes illuminated in the presence of hydroxylamine were supplemented with external ATR equivalent to 100%, 50%, or 25% of the concentration of dark-state rhodopsin and the percentage of N-Ret-PL was determined after 5 min and 30 min reaction time. Data represent mean ± S.D. (n = 3). ATR, all-trans retinal; N-Ret-PE, N-retinylidene-phosphatidylethanolamine; N-Ret-PL, N-retinylidene-phospholipid; TFA, trifluoroacetic acid.