Resolution of Molecular Dynamics by Time-Resolved Fluorescence Anisotropy: Verification of Two Kinetic Models

Abstract

Two of nine possible kinetic models have been experimentally validated for the general scheme that describes a system with two fluorescent species and two depolarizing processes. These kinetic models define different associations between the emitting species and the possible depolarizing processes. Association model 1 is defined as one species exhibiting the shorter fluorescence lifetime and experiencing only the fastest depolarizing rotation, and the longer-lifetime second species undergoing only the slower rotation. For model 2, one species exhibits the shorter fluorescence lifetime and experiences only the slower rotation and the longer-lifetime second species undergoes only the faster rotation. To confirm model 1, we studied a binary solution of indole and β-cyclodextrin. Model 2 was verified using a binary solution of melatonin and rat F102W parvalbumin. In each case, determination of the correct association model and recovery of appropriate intensity and anisotropy decay parameters required global analysis of related data sets. The indole system required β-cyclodextrin concentration as the independent variable, while emission wavelength was employed for the melatonin/parvalbumin system. To demonstrate further the capabilities of this analysis and kinetic approach, it was shown that the melatonin/parvalbumin system could be converted to a model 1 association by the addition of a quenching agent.