Development of high performance photo-diode-array spectrophotometer using weak probe light

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Visual pigment is a prototypical G-protein coupled receptor that receives a light signal from the environment using a light-absorbing chromophore, 11-cis-retinal Recently, we have shown that the low-temperature time-resolved spectroscopy is one of the powerful tools for elucidating the conformational change of visual pigments by monitoring change in absorption spectra. However, low time-resolution (-min) of a scanning type spectrophotometer hampered us from complete detection of change in absorption spectra after the irradiation.In this project, we have developed a unique photo-diode-array (PDA) spectrophotometer to measure the spectral change with higher time-resolution (-sec). For this purpose, intensity of probe light is reduced with a electric shutter system and a glass band-pass filter, to suppress the photoreaction of the sample during the recording of the spectra. A sample cell holder connected to a thermostatic circulator or a optical cryostat was installed into the sample comp … More artment of the spectrophotometer to keep the sample at a constant temperature. Specially designed optical setup including shutters and a movable mirrors are equipped to the compartment to irradiate the sample in the optical cell without removing from cell holder. Using this spectrophotometer, we were able to investigate thermal reactions of meta-intermediates of bovine rhodopsin with time-resolution of 1 sec.Irradiation of rhodopsin sample at - 10 。C caused formation of meta I-intermediate. Transition from meta I-intermediate to meta II was observed with time constant of -10 s which could not be detected with the scanning type spectrophotometer at the temperature. Addition of retinal G-protein, transducin caused accumulation of meta II, suggesting that meta II is a intermediate which interact with transducin. The accumulation process was also observed with time constant of -10 s. Thus, this unique PDA spectrophotometer is a powerful tool for recording change in absorption spectra of biological sample with time-resolution of -s. Less