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

我们研究了红移突变体的动态行为。 S65T和EGFP等绿色荧光蛋白的荧光检测 FCS发现，快速眨眼的效果依赖于 已用缓冲区。这表明这种蛋白质花费了相当多的时间 处于黑暗状态的时间量，并来回转换到 S时间标尺上的明亮状态。这些发现可以与以下几点相关 在低pH值时减弱的总荧光。结构性 对这种蛋白质的了解表明，暗状态实际上是 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激光激发。功能界别 从而允许推导出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.