Steady‐state Fluorescence Method for Evaluating Excited State Proton Reactions: Application to Fluorescein

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 p k . The theoretical expressions give very good fits to the experimental data and allow evaluation of fundamental parameters such as pK, 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 p&* of the excited monoanion-dia...

Absorption and Emission Study of 2‘,7‘-Difluorofluorescein and Its Excited-State Buffer-Mediated Proton Exchange Reactions

“…Thus, under these conditions, the excited monoanion is converted to the dianion during its lifetime. Similar behavior was found in fluorescein with phosphate buffer 6,7 and N-acetyl aspartic acid. 8 We also compared, in Figure 8, fluorescence intensity versus pH curves in 1 M acetate buffered media and in the absence of buffer.…”

Fluorescein Excited-State Proton Exchange Reactions: Nanosecond Emission Kinetics and Correlation with Steady-State Fluorescence Intensity

“…Low Phosphate (1 mM) Buffer Concentration. In our previous publication, 2 we found that at low phosphate buffer concentration, the excited monoanion-dianion proton-transfer reaction does not occur to any significant extent. In the present work, we find that the lifetimes and weighting coefficients which we have evaluated at low phosphate buffer concentration are consistent with this mechanism as explained in the next paragraphs.…”

“…At equilibrium, the value of the ratio is determined by the equilibrium relation [D*(t)] eq /[M*(t)] eq ) K A */[H + ] where K A * is the dissociation equilibrium constant for the excited-state proton reaction M* h D* + H + (pK A * ) pK M * ) 6.3). 2 As soon as equilibrium is established, D* and M* must decay monoexponentially and with the same lifetime in order to maintain a constant value for the equilibrium ratio.…”

“…The extinction coefficient vs wavelength graphs were obtained by methods described in our previous publication. 2 To measure the emission spectra of the (s) dianion and (; ‚‚) monoanion, we recorded fluorescence spectra at pH 11 (λex ) 490 nm) and pH 5.0 (λex ) 440 nm). These spectra correspond respectively to the dianion and monoanion.…”

“…In the pH range 6-10, the monoanion-dianion equilibrium prevails, i.e, above pH 6 the concentrations of the cation and neutral forms are essentially zero. 2 When a fluorescein solution is excited by a fast pulse of light, the initial concentrations of the different excited prototropic forms are not at equilibrium values because of (a) preferential excitation of one or more of the forms (depending on exciting wavelength and extinction coefficients) and (b) differences in the values of the equilibrium constants pK a * in the excited state compared to the ground state (pK* M ) 6.3). Therefore, there is a tendency for the different excited forms to interconvert through excited-state proton reactions so as to achieve equilibrium during their lifetimes.…”