STEADY-STATE FLUORESCENCE METHOD FOR EVALUATING EXCITED-STATE PROTON REACTIONS - APPLICATION TO FLUORESCEIN

Fluorescein is a complex fluorophore in the sense that it displays fou r prototropic forms (cation, neutral, monoanion and dianion) in the pH range 1-9. In experiments with fluorescein-labeled proteins we have s ometimes observed complex nanosecond emission kinetics, which could be due to conversion of the excited monoanion into the excited dianion t hrough an excited state proton exchange with a proton acceptor in the labeled protein. However, the literature is ambiguous on whether this possible excited state proton reaction of fluorescein does occur in pr actice. In this article we describe a general steady-state fluorescenc e method for evaluating excited state proton reactions of simple as we ll as complex pH-sensitive fluorophores and apply it to evaluate excit ed state proton reactions of fluorescein. The method depends on findin g a buffer that can serve as an excited state proton donor-acceptor bu t does not significantly perturb ground state proton equilibrium and e specially does not form ground (or excited state complexes) with the f luorophore. Our results show that the excited monoanion-dianion proton reaction of fluorescein does occur in the presence of phosphate buffe r, which serves as a proton donor-acceptor that does not significantly perturb ground state proton equilibria. The reaction becomes detectab le at phosphate buffer concentrations greater than 20 mM and the react ion efficiency increases with increase in phosphate buffer concentrati ons. The reaction is most clearly demonstrated by adding phosphate buf fer to a solution of fluorescein at constant pH 5.9 with preferential excitation of the monoanion. Under these conditions, the excited monoa nion converts to the dianion during its lifetime. The conversion is de tected experimentally as an increase in dianion and decrease in monoan ion fluorescence intensities with increase in phosphate buffer concent ration. The absorption spectrum is not significantly perturbed by the increase in phosphate buffer concentration. To quantitate the reaction , we have recorded titration graphs of fluorescence intensity versus p H for fluorescein solutions at low (5 mM) and high buffer (1 M) concen trations with preferential excitation of the monoanion and preferentia l detection of the dianion emission. We have also developed theoretica l expressions that relate fluorescence intensity to pH in terms of the concentration of the four prototrophic forms of fluorescein, extincti on coefficients, fluorescence efficiencies and ground and excited stat e pK(a). The theoretical expressions give very good fits to the experi mental data and allow evaluation of fundamental parameters such as pK( a) and fluorescence efficiencies. The analysis of the experimental dat a shows that the excited monoanion-dianion reaction does not significa ntly occur at 5 mM phosphate buffer concentration. However, at 1 M buf fer concentration the reaction is sufficiently fast that it practicall y achieves equilibrium during the lifetimes of the excited fluorescein monoanion and dianion. The pK(a) of the excited monoanion-dianion pr oton reaction is around 6.3. The results and methods presented here sh ould be useful in the development and testing of pH-sensitive labeling fluorophores and fluorescent indicators.