The fluorescent indicator PBFI is widely used for the determination of intracellular concentrations of K+. To investigate the binding reaction of K+ to PBFI in the ground and excited states, steady-state and time-resolved measurements were performed. The fluorescence decay surface was analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution at pH 7.2: k01 = 1.1 x 10(9) s-1, k21 = 2.7 x 10(8) M-1s-1, k02 = 1.8 x 10(9) s-1, and k12 = 1.4 x 10(9) s-1. k01 and k02 denote the respective deactivation rate constants of the K+ free and bound forms of PBFI in the excited state. k21 represents the second-order rate constant of binding of K+ to the indicator in the excited state whereas k12 is the first-order rate constant of dissociation of the excited K(+)-PBFI complex. From the estimated values of k12 and k21, the dissociation constant Kd* in the excited state was calculated. It was found that pKd* (-0.7) is smaller than pKd (2.2). The effect of the excited-state reaction can be neglected in the determination of Kd and/or the K+ concentration. Therefore, intracellular K+ concentrations can be accurately determined from fluorimetric measurements by using PBFI as K+ indicator.

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This paper presents a study analyzing the conditions for the recovery of the rate constants and the spectral parameters from the fluorescence decay surface due to three-state excited-state processes. The studied system models a situation when, in addition to a fluorescence indicator which undergoes an excited-state reaction with a co-reactant, a fluorescent impurity is present. It is demonstrated theoretically that, if the fluorescence decay surface contains traces measured in the absence of co-reactant and additionally at two nonzero coreactant concentrations, all five rate constants can always be recovered. In some cases the measurement in the absence of co-reactant is not necessary. If the fluorescence decay surface is collected at three different emission wavelengths and two co-reactant concentrations, the ratios of the absorbances of the free and bound form of the fluorescent indicator can be determined. If two different emission wavelengths are used in combination with three excitation wavelengths (or three co-reactant concentrations), the ratios of the emission weighting factors of the free and bound form of the fluorescent indicator can be obtained. Under both previous conditions no spectral information about the fluorescent impurity can be acquired. The ground-state dissociation constant can be determined from the fluorescence decay traces excited at an isosbestic point.

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