Fraaije J G, Kleijn J M, van der Graaf M, Dijt J C
Department of Physical and Colloid Chemistry, Wageningen Agricultural University, The Netherlands.
Biophys J. 1990 May;57(5):965-75. doi: 10.1016/S0006-3495(90)82616-3.
A method for determination of the orientation of adsorbed structure-stable proteins using Total Internal Reflection Fluorescence is outlined. The theory has been elaborated for orientation studies on adsorbed free base cytochrome c, of which the prophyrin can be used as an intrinsic fluorescent label. The ratio of fluorescence intensities at two polarization modes of the incident light (the transverse magnetic and the transverse electric polarization mode, respectively) gives a relation between the orientation angles of the porphyrin relative to the interface. As an illustration of the theory, experimental results on the adsorption of cytochrome c at an optically transparent SnO2 film electrode are presented. It is concluded that the orientation of the molecules can only be affected by the interfacial potential during the process of adsorption, but, once adsorbed, the orientation cannot be changed anymore by variation of the potential.
概述了一种使用全内反射荧光测定吸附的结构稳定蛋白质取向的方法。该理论已针对吸附的游离碱细胞色素c的取向研究进行了阐述，其卟啉可作为固有荧光标记。入射光的两种偏振模式（分别为横向磁偏振模式和横向电偏振模式）下荧光强度的比值给出了卟啉相对于界面的取向角之间的关系。作为该理论的例证，给出了细胞色素c在光学透明的SnO₂薄膜电极上吸附的实验结果。得出的结论是，分子的取向在吸附过程中仅受界面电位影响，但一旦吸附，电位变化就不能再改变其取向。
