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

荧光光谱是研究生物分子的溶液结构和动力学以及相互作用的生化体系的热力学和动力学的最有价值的工具之一。然而，大多数生化系统的发射是不均匀的，这使得荧光数据的解释变得复杂。作为解决这些异质发射系统的一种手段，我们建议实施以下策略来增强我们获取和解释稳态(强度和各向异性)荧光猝灭(或增强)研究的能力。具体地说，我们建议实施以下策略来增强我们获取和解释稳态(强度和各向异性)荧光猝灭(或增强)研究的能力。具体地说，我们建议a)开发计算机软件来以全局方式(即，同时拟合多个波长)执行荧光数据的非线性最小二乘分析，B)用个人计算机干扰光电二极管阵列荧光计以便方便地获取和存储用于这种全局分析的数据，C)使用计算机控制的分配器来精确和快速地添加淬火剂或其他试剂，以及d)将该方法应用于几个荧光猝灭研究，主要是含有两个荧光色氨酸残基的蛋白质。这种方法不仅对溶质猝灭研究有用，而且对配体结合体系也是有用的，并且对于其中探针的荧光响应于化学试剂的添加而改变的任何系统，或温度、酸碱度等的变化。

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..