STEADY‐STATE FLUORESCENCE METHOD FOR EVALUATING EXCITED STATE PROTON REACTIONS: APPLICATION TO FLUORESCEIN
Authors: Yguerabide Juan , Talavera Eva , Alvarez Jose Mariaa , Quintero Bartolomea 
Published: January 2008

Abstract Fluorescein is a complex fluorophore in the sense that it displays four prototropic forms (cation, neutral, monoanion and dianion) in the pH range 1–9. In experiments with fluorescein‐labeled proteins we have sometimes observed complex nanosecond emission kinetics, which could be due to conversion of the excited monoanion into the excited dianion through 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 practice. In this article we describe a general steady‐state fluorescence method for evaluating excited state proton reactions of simple as well as complex pH‐sensitive fluorophores and apply it to evaluate excited state proton reactions of fluorescein. The method depends on finding a buffer that can serve as an excited state proton donor‐acceptor but does not significantly perturb ground state proton equilibrium and especially does not form ground (or excited state complexes) with the fluorophore. Our results show that the excited monoanion‐dianion proton reaction of fluorescein does occur in the presence of phosphate buffer, which serves as a proton donor‐acceptor that does not significantly perturb ground state proton equilibria. The reaction becomes detectable at phosphate buffer concentrations greater than 20 mM and the reaction efficiency increases with increase in phosphate buffer concentrations. The reaction is most clearly demonstrated by adding phosphate buffer to a solution of fluorescein at constant pH 5.9 with preferential excitation of the monoanion. Under these conditions, the excited monoanion converts to the dianion during its lifetime. The conversion is detected experimentally as an increase in dianion and decrease in monoanion fluorescence intensities with increase in phosphate buffer concentration. 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 pH for fluorescein solutions at low (5 mM) and high buffer (1 M) concentrations with preferential excitation of the monoanion and preferential detection of the dianion emission. We have also developed theoretical expressions that relate fluorescence intensity to pH in terms of the concentration of the four prototrophic forms of fluorescein, extinction coefficients, fluorescence efficiencies and ground and excited state pKa. The theoretical expressions give very good fits to the experimental data and allow evaluation of fundamental parameters such as pKa and fluorescence efficiencies. The analysis of the experimental data shows that the excited monoanion‐dianion reaction does not significantly occur at 5 mM phosphate buffer concentration. However, at 1 M buffer concentration the reaction is sufficiently fast that it practically achieves equilibrium during the lifetimes of the excited fluorescein monoanion and dianion. The pKa* of the excited monoanion‐dianion proton reaction is around 6.3. The results and methods presented here should be useful in the development and testing of pH‐sensitive labeling fluorophores and fluorescent indicators.