KINETICS OF CHROMOPHORE PROTONATION IN EGFP DETERMINED BY FCS

2月22日至26日，堪萨斯州生物物理学会会议上的海报 城市MO荧光相关光谱(Magde等人1972年， 太棒了。莱特牧师。29：705)是唯一适合测定 分子动力学和化学动力学的速率常数 自发的过程。统计物理学规定， 从FCS中使用的小探针体积获得的荧光 由于单个分子的扩散，在平均值附近波动 卷内和卷外和/或由于 分子往返于非荧光形式。自相关 函数因此包含有关化学反应的信息 动力学、扩散系数和平衡化学 浓度。绿色荧光蛋白的荧光高度依赖于 质子化状态和外部条件(Ward等人1982年， 照相。光生物醇。35：803-808)。使用单光子激发 (488 Nm)，共聚焦荧光检测，或者 双光子激发(910 Nm)，我们发现了一个自发的 具有特征时间常数的荧光调制 约200(S。调制的幅度依赖于pH； PH值降低，分子的比例增加 非荧光状态(与整体测量结果一致)。这个 动力学与温度有关，产生的活化能为 约10千卡/摩尔，在D2O中减慢约1.5倍。 从这些结果我们得出结论，观察到的动力学代表了 两种状态的荧光团之间的热相互转化(Brejc 等人的研究。1997年，前列阿卡德。娜塔莉。SCI。美国94：2306-2311)：质子化 (ABS~400 nm，488 nm激发不充分)和非质子化(ABS~490 nm)。 这一过程与最近报道的(Dickson et 等，自然388：355-358)在更慢的时间尺度(~1秒)。

Poster at the Biophysical Society Meeting, Feb 22-26 in Kansas City MO Fluorescence correlation spectroscopy (Magde et al. 1972, Phys. Rev. Lett. 29:705) is uniquely suited for determination of the rate constants of molecular dynamics and chemical kinetics of spontaneous processes. Statistical physics dictates that the fluorescence obtained from the small probe volume employed in FCS fluctuates about a mean value, due to individual molecules diffusing in and out of the volume and/or due to inter-conversion of such molecules to and from a non-fluorescent form. The autocorrelation function therefore contains information about chemical reaction kinetics, coefficients of diffusion and the equilibrium chemical concentrations. GFP fluorescence is highly dependent on the protonation state and external conditions (Ward et al. 1982, Photochem. Photobiol. 35:803-808). Using one-photon excitation (488nm) with confocal fluorescence detection and alternatively two-photon excitation (910nm), we have identified a spontaneous modulation of the fluorescence with a charact eristic time constant around 200 (s. The amplitude of the modulation is pH dependent; with decreasing pH an increasing fraction of molecules is found in a nonfluorescent state (in agreement with bulk measurements). The kinetics are temperature dependent yielding an activation energy of about 10 kcal/mol and are slowed down in D2O by a factor of about 1.5. From these results we conclude that the observed kinetics represent thermal interconversion between two states of the fluorophore (Brejc et al. 1997, Proc. Acad. Natl. Sci. USA 94:2306-2311): protonated (?abs~400nm, not well-excited by 488nm) and unprotonated (?abs~490nm). This process is distinguished from that recently reported (Dickson et al., Nature 388:355-358) at much slower time-scales (~1s).