Figure 3.
Spectrophotometric analysis of the slow chromophore exchange in ROS Rho. Purified Rho (5.3 μm) in POPC/CHAPS bicelle buffer B was supplemented with 9CR (49 μm) and incubated at different temperatures (28°C, 36°C, and 44°C). Aliquots were taken at three different time points (106 s or 11.6 days, 2 × 106 s or 23.2 days, and 3 × 106 s or 34.8 days) and repurified to remove excess retinal. The formation of isoRho was quantified by UV-Vis spectroscopy. (A) The normalized difference absorption spectra were obtained by subtracting the drift-corrected spectra of the photobleached Rho-isoRho mixtures from the spectra of the dark-state samples in presence of hydroxylamine and normalizing to –1 at 365 nm. (B) The double-difference spectra (subtracting the difference spectra of Rho-isoRho mixtures from the difference spectrum of pure Rho) reveal the decrease in the 500-nm absorbance of Rho and increase in the 487-nm absorbance of isoRho. The extent of chromophore exchange was calculated from the decrease of 500-nm absorbance in the double-difference spectra.