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

视觉色素是一种典型的G蛋白偶联受体，它通过吸收光的11-顺式视网膜生色团从环境中接收光信号。最近，我们已经证明了低温时间分辨光谱是通过监测吸收光谱的变化来阐明视觉色素构象变化的有力工具之一。然而，扫描式分光光度计的低时间分辨率(-min)阻碍了我们对辐射后吸收光谱变化的完整检测，在本项目中，我们开发了一种独特的光电二极管阵列(PDA)分光光度计来测量更高时间分辨率(-s)的光谱变化。为此，通过电子快门系统和玻璃带通滤光器来降低探测光的强度，以抑制光谱记录过程中样品的光反应。将连接到恒温循环器或光学低温恒温器的样品池保持器安装到样品组合物…中增加分光光度计的配置，使样品保持恒温。专门设计的光学装置，包括百叶窗和可移动的反射镜，配备在隔间，在不从光盒支架上取出样品的情况下将样品照射到光盒中。利用该分光光度计，我们能够研究牛视紫红质中间体的热反应，时间分辨率为1秒。C导致了中间产物的形成。在-10 S的时间常数下，观察到了从Meta I中间体到Meta II的转变，这是扫描式分光光度计在该温度下无法检测到的。视网膜G蛋白、转导蛋白的加入引起Meta II的积聚，提示Meta II是与转导蛋白相互作用的中间产物。该仪器的时间常数为-10 S，是记录生物样品吸收光谱变化的有力工具，时间常数为-10 S。较少

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