The behavior of 6-propionyl-2-dimethylaminonaphthalene (PRODAN) was studied in homogeneous media and in large unilamellar vesicles (LUVs) of the phospholipid 1,2-di-oleoyl-sn-glycero-3-phosphatidylcholine (DOPC), using absorption, emission, depolarization, and time-resolved spectroscopies. In homogeneous media, the Kamlet and Taft solvatochromic comparison method quantified solute-solvent interactions from the absorption and emission PRODAN bands. These studies demonstrate that the absorption band is sensitive to the polarity-polarizability (pi) and the hydrogen bond donor ability (alpha) parameters of the media. PRODAN in the excited state is even more sensitive to these parameters and to the hydrogen bond acceptor ability (beta) of the media. The transition energy (expressed in kcal/mol) for both absorption and emission bands gives a linear correlation with the well-known polarity parameter E(T30). The results from the absorption and emission bands also reveal that PRODAN aggregates in water. The monomer has two fluorescence lifetimes, 2.27 and 0.65 ns, while the aggregate has a lifetime of 14.6 ns. Using steady-state anisotropy measurements, the calculated volumes of the aggregate and the monomer are 5590 and 222 mL mol(-1), respectively. In DOPC LUVs, PRODAN undergoes a partition process between the water bulk and the DOPC bilayer. We show that the partition constant (K(p)) value is large enough that only at [DOPC] below 0.15 mg/mL PRODAN in water can be detected. PRODAN dissolved in LUVs at [DOPC] > 1 mg/mL exists completely incorporated in its monomer form and senses two different microenvironments within the bilayer: a polar region in the interface near the water and a less polar and also less viscous environment, between the phospholipid tails. These environments were characterized by their fluorescence lifetimes (tau), showing that PRODAN in the polar microenvironment has a tau value of approximately 4 ns while in the less polar region gives a value of 1.2 ns. Moreover, this probe also senses the micropolarity of these two different regions of the bilayer and yields values similar to that of methanol and tetrahydrofuran.
6-propionyl-2-(N,N-dimethyl)aminonaphtahalene, PRODAN, is widely used as a fluorescent molecular probe because of its significant Stokes shift in polar solvents. It is an aromatic compound with intramolecular charge-transfer states (ICT) that can be particularly useful as a sensor. The nature of the emissive states has not yet been established despite the detailed experimental and theoretical investigations done on this fluorophore. In this work, we performed absorption, steady-state, time-resolved fluorescence (TRES) and time-resolved area normalized emission (TRANES) spectroscopies on the molecular probe PRODAN in the anionic water/sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/n-heptane and the cationic water/benzyl-n-hexadecyl dimethylammonium chloride (BHDC)/benzene reverse micelles (RMs). The experiments were done by varying the surfactant concentrations at a fixed molar ratio (W = [H2O]/[Surfactant]) and changing the water content at a constant surfactant concentration. The results obtained varying the surfactant concentration at W = 0 show a bathochromic shift and an increase in the intensity of the PRODAN emission band due to the PRODAN partition process between the external solvent and the RMs interface. The partition constants, Kp, are quantified from the changes in the PRODAN emission spectra and the steady-state anisotropy () with the surfactant concentration in both RMs. The Kp value is larger in the BHDC than the AOT RMs, probably due to the interaction between the cationic polar head of the surfactant and the aromatic ring of PRODAN. The partition process is confirmed with the TRES experiments, where the data fit to a continuous model, and with the time-resolved area normalized emission spectroscopy (TRANES) spectra, where only one isoemissive point is detected. On the other hand, the emission spectra at W = 10 and 20 show a dual fluorescence with a new band that emerges in the low-energy region of the spectra, a band that was previously assigned to the PRODAN emission from the water pool of RMs. Our studies demonstrate that this band is due to the emission from an ICT state of the molecular probe PRODAN located at the interface of the RMs. These results are also confirmed by the lifetime measurements, the TRES experiments where the results fit to a two-state model, and the time-resolved area normalized emission spectroscopy (TRANES) spectra where three or two isoemissive points are detected in the AOT and BHDC RMs, respectively. In the AOT RMs, Kp values obtained at W = 10 and 20 are almost independent of the water content; the values are higher for the BHDC RMs due to the higher micropolarity of this interface.
The kinetics of hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by α-chymotrypsin (α-CT), in reverse
micellar solutions formed by glycerol (GY)−water (38% v/v) mixture/sodium bis(2-ethylhexyl)sulfosuccinate
(AOT)/n-heptane has been determined by spectroscopic measurements. To compare the efficiency of this
reaction with that observed in micelles with water in the core, as well as in the corresponding homogeneous
media, the reaction was also studied in water/AOT/n-heptane reverse micellar solutions and in both
homogeneous media (water and GY−water, 38% v/v mixture). In every media, α-CT was characterized by
the absorption and emission spectra, the fluorescence lifetimes, and the fluorescence anisotropy of its
tryptophan residues. The effect of AOT concentration on the kinetic parameters obtained in the micellar
systems was determined, at a constant molar ratio of the inner polar solvent and surfactant. Moreover,
the data obtained allowed the evaluation of the 2-NA partition constant between the organic and the
micellar pseudophase. It is shown that the addition of GY to the micelle interior results in an increase
in the catalytic properties of α-CT. The fluorescence anisotropy studies in the different media show that
the addition of GY increases the viscosity as compared with the aqueous systems. It seems that the GY
addition to the reverse micellar aggregates results in a decrease of the conformational mobility of α-CT,
which leads to an increase of the enzyme stability and activity.
6-Propionyl-2-(N,N-dimethyl)aminonaphtahalene, PRODAN, is widely used as a fluorescent molecular probe due to its significant Stokes shift in polar solvents. It is an aromatic compound with intramolecular charge-transfer (ICT) states which can be particularly useful as sensors. In this work, we performed absorption, steady-state, time-resolved fluorescence (TRES), and time-resolved area normalized emission (TRANES) spectroscopies on PRODAN dissolved in nonaqueous reverse micelles. The reverse micelles are composed of polar solvents/sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/n-heptane. Sequestered polar solvents included ethylene glycol (EG), propylene glycol (PG), glycerol (GY), formamide (FA), dimethylformamide (DMF), and dimethylacetamide (DMA). The experiments were performed with varying surfactant concentrations at a fixed molar ratio W(S) = [polar solvent]/[AOT]. In every reverse micelle studied, the results show that PRODAN undergoes a partition process between the external solvent and the reverse micelle interface. The partition constants, K(p), are quantified from the changes in the PRODAN emission and/or absorption spectra with the surfactant concentration. The K(p) values depend strongly on the encapsulated polar solvent and correlate quite well with the AOT reverse micelle interface's zones where PRODAN can exist and emits. Thus, the partition toward the reverse micelle interface is strongly favored in DMF and DMA containing micelles where the PRODAN emission comes only from an ICT state. For GY/AOT reverse micelles, the K(p) value is the lowest and only emission from the local excited (LE) state is observed. On the other hand, for EG/AOT, PG/AOT, and water/AOT reverse micelles, the K(p) values are practically the same and emission from both states (LE and ICT) is simultaneously detected. We show here that it is possible to control the PRODAN state emission by simply changing the properties of the AOT reverse micelle interfaces by choosing the appropriate polar solvent to make the reverse micelle media. Indeed, we present experimental evidence with the answer to the long time question about from which state does PRODAN emit, a process that can be controlled using the unique reverse micelle interfaces properties.
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