A Novel Improvement in Biochemical Fluorescence Methods: TheUse of a Photodiode Array Fluorometer Together with Global Least Square Analysis of Quenching Data

荧光光谱是最有价值的工具之一 可用于溶液结构和动力学的研究 生物分子与相互作用的热力学和动力学 生化系统 大多数生化系统的排放物是 然而，异质性，这使得解释复杂化 的荧光数据。 作为解决这些问题的手段， 非均匀发射系统，我们建议实施 遵循战略，以提高我们的能力， 解释稳态（强度和各向异性）荧光 淬灭（或增强）研究。 具体而言，我们建议 实施以下战略来增强我们的能力， 获取并解释稳态（强度和各向异性） 荧光猝灭（或增强）研究。 具体地说， 我们建议a）开发计算机软件来执行非线性 以全局方式对荧光数据进行最小二乘分析 (i.e.,多个波长同时拟合），B）至 干扰光电二极管阵列荧光计 计算机，以便方便地获取和存储数据， 一个全面的分析，c）使用计算机控制的分配器， 为了精确和快速地加入猝灭剂或其它试剂， 和d）将这种方法应用于几种荧光猝灭 研究，主要是含有两种荧光蛋白的蛋白质， 色氨酸残基 这种方法不仅对 溶质淬灭研究，也适用于配体结合系统， 并且对于其中探针的荧光被 响应于化学试剂的添加而改变，或响应于 温度、pH值等的变化。

Fluorescence spectroscopy is one of the most valuable tools available for the study of the solution structure and dynamics of biomolecules and the thermodynamics and kinetics of interacting biochemical systems. The emission of most biochemical systems is heterogeneous, however, and this complicates the interpretation of fluorescence data. As a means to resolve these heterogeneously emitting systems we propose to implement the following strategy to enhance our ability to acquire and interpret steady state (intensity and anisotropy) fluorescence quenching (or enhancement) studies. Specifically, we propose to implement the following strategy to enhance our ability to acquire and interpret steady state (intensity and anisotropy) fluorescence quenching (or enhancement) studies. Specifically, we propose a) to develop computer software to perform nonlinear least squares analysis of fluorescence data in a global manner (i.e., multiple wavelengths fitted simultaneously), b) to interfere a photodiode array fluorometer with a personal computer in order to conveniently acquire and store data for such a global analysis, c) to employ a computer-controlled dispenser for the precise and rapid addition of quencher or other reagent, and d) to apply this approach to several fluorescence quenching studies, primarily with proteins that contain two fluorescing tryptophan residues. This approach will be useful not only for solute quenching studies, but also for ligand binding systems, and for any system in which the fluorescence of a probe is changed in response to the addition of a chemical agent, or to a change in temperature, pH, etc..