UV/Vis absorption spectra were recorded with a UV–visible spectrophotometer (UV-2450 and UV-2400, Shimadzu). Samples were kept at 0, 20, or 37°C using a cell holder equipped with a temperature-controlled circulating water bath in order to analyze the thermal reaction of the pigments in detail. The samples were irradiated with either yellow light through a Y-52 cutoff filter (Toshiba) or UV light through a UVD-36 glass filter (AGC Techno Glass) from a 1 kW tungsten halogen lamp (Master HILUX-HR; Rikagaku).
To monitor the process of the photocycle of G188C mutant of bovine rhodopsin, a time-resolved CCD spectrophotometer (C10000 system, Hamamatsu Photonics) was used (Sakai et al., 2012 (link)). Spectra were taken from G188C mutant samples in the dark and at different time points after irradiation (170-μs, yellow light through a Y-52 cutoff filter from a Xenon flash lamp). The temperature of the sample was kept at 37°C by a temperature controller (pqod, QUANTUM Northwest). Absorbance changes at λmax were plotted as a function of time and fitted with a single-exponential function to obtain the time constants for the recovery to the original dark state.
To monitor the process of the photocycle of G188C mutant of bovine rhodopsin, a time-resolved CCD spectrophotometer (C10000 system, Hamamatsu Photonics) was used (Sakai et al., 2012 (link)). Spectra were taken from G188C mutant samples in the dark and at different time points after irradiation (170-μs, yellow light through a Y-52 cutoff filter from a Xenon flash lamp). The temperature of the sample was kept at 37°C by a temperature controller (pqod, QUANTUM Northwest). Absorbance changes at λmax were plotted as a function of time and fitted with a single-exponential function to obtain the time constants for the recovery to the original dark state.
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