DEGRANULATION IN RBL 2H3 CELLS MEASURED THRU CELL CAPACITANCE & VIDEO MICROSCOPY

我们研究了红移突变体的动力学行为， 利用荧光显微镜观察了类似S65 T和EGFP的绿色荧光蛋白（GFP）的荧光， FCS，并发现了一种快速闪烁效应，取决于pH值的 使用缓冲液。 这表明蛋白质花费了相当大的 在黑暗状态下的时间量，来回转换到 在100米公路上的光明州？的时间尺度。 这些发现可能与 在低pH下降低的总体荧光。 关于这种蛋白质的知识表明， 一种质子化形式的发色团，它将吸收转移到 较小的值，其中不会发生FCS激光器的激发。 FCS 从而可以得到精确的动力学和热力学信息， 这个可逆的质子化过程。

We studied the dynamic behavior of red shifted mutants of the fluorescence of green fluorescent protein (GFP) like S65T and EGFP by FCS and discovered a fast blinking effect dependent on the pH of the used buffer. This indicates that the protein spends a considerable amount of time in a dark state, with transitions back and forth to the bright state on the 100 ?s time scale. The findings can be related to the overall fluorescence that decreases at low pH. Structural knowledge about this protein suggests that the dark state is actually a protonated form of the chromophore that shifts the absorption to smaller values where no excitation by the FCS laser can occur. FCS thus allows to derive precise kinetic and thermodynamic information of this reversible protonation process.