Fluorescein changes color in acidic and different basic media due to protonation states
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Peer-reviewed literature investigates the protolytic forms and fluorescence spectra of fluorescein across varying pH ranges, confirming that its spectral properties and color changes depend on protonation states.
Abstract In contrast to the well-studied absorption spectra of different protolytic forms of fluorescein, the complex structure of the fluorescence spectra in a wide pH range is not completely understood because of the interplay between emission and photoinduced proton transfer in the electronic excited states. We provide insight into this interplay through a combined analysis of the experimental data, obtained by absorption and steady-state fluorescence spectroscopy at pH 0.3–10.5, and the time-dependent density functional theory (TD-DFT). The TD-DFT based computational model is validated on dianion and used to model the spectra of other protolytic forms. The protolytic/tautomeric forms of fluorescein are classified according to the partial charges on the triple chromophore ring, and electronic transitions were analyzed in terms of changes in molecular geometries and orbitals. A linear regression analysis between the calculated and experimental results based on both absorption and well-understood dianionic and cationic fluorescence peaks is used to assign the monoanionic (496 nm), neutral quinoid (550 nm) and neutral zwitterionic (483 nm) fluorescence peaks, whose positions were not clear prior to this work. The values of the excited-state dissociation microconstants p k a ∗ for different forms of fluorescein are calculated by means of the Forster cycle in conjunction with the spectroscopic measurements and computational data.
Evidence for conduction of protons along the interface between water and a polar lipid monolayer. Movements of H+ along the polar heads of phospholipids spread in monolayers were compared to movements of H+ in the aqueous subphase. The probe for detecting H+ movement along the monolayer was a pH-sensitive fluorescein chromophore covalently bound to the head group of phosphatidylethanolamine. The behavior of this probe was not affected by the electrical properties of the lipid/water interface. Lateral diffusion of H+ along the phospholipid/water interface was then studied by acid-jump experiments in which advantage was taken of the large size of the monolayer. H+ was injected a few centimeters away from the probe observation area. The time needed for H+ diffusion to the probe was monitored by the change in the fluorescence signal, fluorescein being nonfluorescent in an acid medium. Diffusion of H+ in the bulk phase was monitored by the fluorescence change of water-soluble fluorescein isothiocyanate.
Involvement of a non-proton pump factor (possibly Donnan-type equilibrium) in maintenance of an acidic pH in lysosomes.
Change of the internal pH of isolated lysosomes was measured with fluorescein isothiocyanate-dextran. In buffer of pH 7.0, isolated lysosomes had an acidic pH of about 5.5, which decreased to pH 5.2 on addition of ATP. Addition of bafilomycin inhibited the acidification by H(+)-ATPase and resulted in an increase of the internal pH to 5.5 due to passive diffusion of protons across the lysosomal membrane. However, no further alkalization was observed. The acidic pH (pH 5.5) of isolated lysosomes could be maintained for at least 48 h in the absence of ATP, but increased gradually to pH 5.9-6.4 upon incubation with monovalent cations (K+ or Na+), amines, or ionophores. These results suggest that a non-proton pump factor (possibly Donnan equilibrium) is involved in maintaining the acidic pH of isolated lysosomes.
Published in FEBS letters (1992)
A fluorescent colorimetric pH sensor was developed by a polymerization of a monomeric fluorescein based green emitter (SM1) with a monomeric 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran derived red emitter (SM2) in poly(2-hydroxyethyl methacrylate)-co-polyacrylamide (PHEMA-co-PAM) matrices. Polymerized SM1 (PSM1) in the polymer matrices showed bright emissions at basic conditions and weak emissions at acidic conditions. Polymerized SM2 (PSM2) in the polymer matrices exhibited a vastly different response when compared to PSM1. The emissions of PSM2 are stronger under acidic conditions than those under basic conditions. When SM1 and SM2 were polymerized in the same polymer matrix, a dual emission sensor acting as a ratiometric pH sensor (PSM1,2) was successfully developed. Because the PSM1 and PSM2 exhibited different pH responses and separated emission windows, the changes in the emission colors were clearly observed in their dual color sensor of PSM1,2, which changed emission colors dramatically from green at pH 7 to red at pH 4, which was detected visually and/or by using a color camera under an excitation of 488 nm. In addition to the development of the dual color ratiometric pH sensor, we also studied the effects of different matrix compositions, crosslinkers, and charges on the reporting capabilities of the sensors (sensitivity and pKa).
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