SPECIES-ASSOCIATED SPECTRA AND UPPER AND LOWER BOUNDS ON THE RATE CONSTANTS OF REVERSIBLE INTRAMOLECULAR 2-STATE EXCITED-STATE PROCESSS WITH ADDED QUENCHER - GLOBAL COMPARTMENTAL ANALYSIS OF THE FLUORESCENCE DECAY SURFACE

This paper explains how, in the absence of any a priori information, upper and lower bounds can be specified for the four rate constants describing the kinetics of a reversible intramolecular two-state excited-state process. It is further shown that the steady-state spectrum can be decomposed into unique species-associated spectra. It is demonstrated theoretically that if the fluorescence decay surface includes at least one set of decay traces measured at a minimum of three different quencher concentrations, it is possible to specify limits on the rate constants and to construct the species-associated spectra. The two quenching rate constants must be different. This new analysis method allows one to distinguish reversible from irreversible intramolecular two-state excited-state processes. Practically, the bounds on the rate constants can be obtained by performing a series of global compartmental analyses of a fluorescence decay surface in which one of the rate constants is held fixed at different preset values. Computer-generated fluorescence decay surfaces are used to illustrate how the rate constant bounds are specified and how a steady-state spectrum is decomposed into its two species-associated spectra. This analysis method will be useful for understanding the photophysical behavior of tryptophan, tryptophyl polypeptides, and proteins. Furthermore, using this approach, the kinetics of intramolecular electron transfer and excimer and exciplex formation are now accessible.