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..
