PHONE CONSULT: SPECT TO DETERMINE BINDING OF ENZYME SYS TO CLATHRIN COATED PITS

海报在生物物理学会会议，2月22日至26日在堪萨斯 城市MO荧光相关光谱法（Magde等人1972， Phys. Rev. Lett. 29：705）是唯一适合于确定 分子动力学和化学动力学的速率常数 自发过程 统计物理学表明， 从FCS中使用的小探针体积获得的荧光 波动的平均值，由于个别分子扩散 进出体积和/或由于这种相互转换 从分子到非荧光形式。 自相关 因此，函数包含有关化学反应的信息 动力学、扩散系数和化学平衡 浓度的 GFP荧光高度依赖于 质子化状态和外部条件（Ward等1982， Photochem. 光生物学 35：803-808）。 使用单光子激发 （488 nm）与共聚焦荧光检测，或者 双光子激发（910 nm），我们已经确定了一个自发的 用特征时间常数调制荧光 约200（s. 调节的幅度是pH依赖性的; 随着pH值的降低，分子的分数增加， 非荧光状态（与批量测量一致）。 的 动力学是温度依赖性的，产生的活化能为 约10千卡/摩尔，在D2 O中减慢约1.5倍。 从这些结果中，我们得出结论，所观察到的动力学代表 荧光团的两种状态之间的热相互转换（Brejc 等人，1997，Proc. Natl. Sci. USA 94：2306-2311）：质子化的 (? abs~ 400 nm，488 nm激发不好）和未质子化（？abs~490nm）。 这一过程不同于最近报道的过程（Dickson et 例如，Nature 388：355-358）在慢得多的时间尺度（~ 1 s）下。

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).