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Laccase is a versatile enzyme for the oxidation of environmental contaminants and display great potential in many applications; however, it undergoes photo-degradation when irradiated with UVB light. The instability of this biomolecule can be improved by immobilization in different encapsulation media. In the present contribution, reverse micelles of AOT were used with this purpose. Moreover, the use of these kind of media can be relevant in the sense that they be able to mimetic the complexity of contaminated aquatic microenvironments where the enzymes can be used as biological remediation agents.The laccase activity from spectroscopic data, using syringaldazine as substrate has been studied in the absence and in the presence of reverse micelles of 0.15 M AOT/isooctane at W0 ([H2O]/[AOT]) =30, before and after irradiation of the enzyme with UVB light.The kinetic parameters, i.e. Michaelis-Menten constant (KM), catalytic constant (kCAT) and catalytic efficiency (kCAT / KM) were determinate in the micellar system and in aqueous medium and comparatively analyzed. From this comparison, it was found that the enzyme presents a considerably photo-protection when irradiated with UVB light in reverse micelles as compared with the aqueous medium. Moreover, taking into account the distribution of the substrate in the two pseudo-phases (micellar and organic solvent), the activity of the enzyme is significantly preserved in the micellar medium.

The multi-copper Laccase enzyme corresponds to one of the most investigated oxidoreductases for potential uses in xenobiotic bioremediation. In this work, we have investigated the photo-degradation process of Laccase from Trametesversicolor induced by UVB light and the influence on its activity over selected substrates. Laccase undergoes photo-degradation when irradiated with UVB light, and the process depends on the presence of oxygen in the medium. With the kinetic data obtained from stationary and time resolved measurements, a photo-degradation mechanism of auto-sensitization was proposed for the enzyme. Laccase generates singlet oxygen, by UVB light absorption, and this reactive oxygen species can trigger the photo-oxidation of susceptible amino acids residues present in the protein structure. The catalytic activity of Laccase was evaluated before and after UVB photolysis over hydroxy-aromatic compounds and substituted phenols which represent potential pollutants. The dye bromothymol blue, the antibiotic rifampicin and the model compound syringaldazine, were selected as substrates. The values of the kinetic parameters determined in our experiments indicate that the photo-oxidative process of Laccase has a very negative impact on its overall catalytic function. Despite this, we have not found evidence of structural damage by SDS-PAGE and circular dichroism experiments, which indicate that the enzyme retained its secondary structure. We believe that, given the importance of Laccase in environmental bioremediation, the information found about the stability of this kind of biomolecule exposed to UV solar irradiation may be relevant in the technological design and/or optimization of decontamination strategies.

In order to investigate the effect of solar UV light on pharmacological compounds considered as emerging environmental pollutants, we studied the kinetic, mechanistic and toxicological aspects of the direct photolysis of two highly used anti-inflammatory corticosteroids: dexamethasone and prednisone. The photochemical experiments were done in aqueous media under different atmospheric conditions, with the purpose of discerning the participation of oxygen in the photodegradative processes. Moreover, the influence of direct micelles of different surfactants on the behavior of the corticosteroids under UV light was evaluated, and the cytotoxicity of the photoproducts toward the Vero cell line was tested as well. Through static and dynamic spectroscopic techniques, it was possible to detect a dependence between the rate of the photodegradation and the availability of oxygen, since both corticosteroids are capable of photosensitizing the generation of reactive oxygen species, mainly singlet oxygen and the hydroxyl radical, by generating their respective triplet excited state (characterized for the first time in this work). Dexamethasone and prednisone are inert to the action of singlet oxygen but sensitive to the effect of the hydroxyl radical. Also, unimolecular photochemical reactions, namely Norrish type I and II photo-rearrangements, occur due to the presence of carbonyl functional groups in the molecular structures. In the presence of direct micelles of anionic, cationic and non-ionic surfactants, a photoprotective effect was observed for all the studied corticosteroids. The protection phenomenon was attributed to the interaction of the molecules with the micellar interfaces and their overall distribution inside the non-polar micellar core. This behavior seems to be in a close relationship with the dipolar moment of each molecule. On the other hand, the in vitro cytotoxicity bioassays, MTT metabolism and neutral red uptake, reveal that the corticosteroids and their photoproducts obtained under different atmospheric conditions induce cell metabolic alterations at the mitochondrial level, and this effect could be more significant than lysosomal damage. In most cases, photoproducts are found to have greater toxicity than the original drugs. Studies on micellar influence and cytotoxicity also included the corticosteroid prednisolone, whose photodegradation has been previously studied in homogeneous medium.

Photophysical and photochemical properties of the non-steroidal anti-inflammatory drug Etoricoxib was analyzing in aqueous media at different pH values. The mechanistic and kinetic aspects of the photodegradation processes involved, induced by UV irradiation of the drug were also investigated, as well as the generation of reactive oxygen species and transient excited species by the anti-inflammatory. Moreover, due to the fact that the photoproducts can be either less or more toxic than the parent pharmaceutical, the possible cytotoxic effects of etoricoxib and its photoproducts were analyzed in vitro on the Vero cell line. Etoricoxib undergoes pH-dependent photoinduced degradation processes when irradiated with UV light at 233 nm and 285 nm. The greater the degree of protonation, the greater the rate and quantum yield of photodegradation. The use of a variety of stationary and time-resolved techniques allowed us to propose a photodegradation mechanism for the anti-inflammatory. The main pathway involves the generation of an excited singlet state by UV light absorption that allows the formation of two isomeric photoproducts resulting from the photocyclization reaction of the excited etoricoxib. Likewise, the triplet excited state of the drug was detected and the spectrum of this transient species and its quantum yield are reported here for the first time. The triplet state could react with molecular oxygen and generate the reactive specie OH[rad] that lead to the photodegradation of the drug. On the other hand, it was possible to demonstrate that the products of the photocyclization can generated O2(1Δg) and be susceptible to photooxidation mediated by this species. By last, the in vitro cytotoxic test on Vero cell line (Neutral Red and MTT assay) revealed that the photoproducts can alter the viability of the cell by affecting de mitochondrial respiration processes. Fil: Cacciari, Rodolfo Daniel. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina Fil: Menis Candela, Florencia. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentina Fil: Biondi, M. Agostina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina Fil: Reynoso, Eugenia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina Fil: Sabini, Maria Carola. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Montejano, Hernan Alfredo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones en Tecnologías Energéticas y Materiales Avanzados. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Tecnologías Energéticas y Materiales Avanzados; Argentina Fil: Biasutti, Maria Alicia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina

The remodeling of membrane lipids is a mechanism that allows microorganisms to survive in unfavorable environments such as industrial effluents, which often contain inorganic and organic pollutants, like chromium and phenol. In the present work, we evaluated the effect of Cr(VI) and phenol on the membrane of Acinetobacter guillouiae SFC 500-1A, a bacterial strain isolated from tannery sediments where such pollutants can be found. The presence of lipid kinases and phospholipases and the changes in their activities under exposure to these pollutants were determined. Cr(VI) and Cr(VI) + phenol caused the membrane to become more rigid for up to 16 h after exposure. This could be due to an increase in cardiolipin (Ptd2Gro) and a decrease in phosphatidylethanolamine (PtdEtn), which are indicative of more order and rigidity in the membrane. Increased phospholipase A activity (PLA, EC 3.1.1.4) could be responsible for the decrease in PtdEtn levels. Moreover, our results indicate that Cr(VI) and Cr(VI) + phenol trigger the phosphatidic acid (PtdOH) signal. The finding of significantly increased phosphatidylinositol-4-phosphate (PtdIns-4-P) levels means this is likely achieved via PtdIns-PLC/DGK. This report provides the first evidence that A. guillouiae SFC 500-1A is able to sense Cr(VI) and phenol, transduce this signal through changes in the physical state of the membrane, and trigger lipid-signaling events. Fil: Fernandez, Marilina. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentina Fil: Paulucci, Natalia Soledad. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentina Fil: Peppino Margutti, Micaela Yesica. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Biología Molecular. Sección Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Biasutti, Maria Alicia. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina Fil: Racagni, Graciela Esther. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Biología Molecular. Sección Química Biológica; Argentina Fil: Villasuso, Ana Laura. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentina Fil: Agostini, Elizabeth. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentina Fil: González, Paola Solange. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentina

Reactive oxygen species (ROS) have been described in their double physiological function, helping in the maintenance of health as well as contributing to oxidative stress. Diabetes mellitus is a chronical disease nearly related to oxidative stress, whose treatment (in type II variant) consists in the administration of antidiabetic compounds (Andb) such as Gliclazide (Gli) and Glipizide (Glip). In this context, as Andb may be exposed to high ROS concentrations in diabetic patients, we have studied the potential ROS-mediated degradation of Gli and Glip through photosensitized processes, in the presence of Riboflavin (Rf) vitamin. We found that singlet oxygen (O 2 ( 1 ∆ g )) participated in the Rf-sensitized photodegradation of both Andb, and also superoxide radical anion in the case of Gli. Two principal products derived from O 2 ( 1 ∆ g )-mediated degradation of Gli were identified and their chemical structures characterized, through HPLC mass spectrometry. O 2 ( 1 ∆ g )-mediated degradation products and their toxicity was assayed on Vero cell line. These studies demonstrated that neither Gli nor its photoproducts caused cytotoxic effect under the experimental conditions assayed. Our results show strong evidences of ROS-mediated Andb degradation, which may involve the reduction or loss of their therapeutic action, as well as potential cytotoxicity derived from their oxidation products., Facultad de Ciencias Exactas

Photosensitized oxidation of bovine serum albumin (BSA), by using perinaphtenone as a sensitizer, has been studied at pH 7.4 and 11. The selected sensitizer does not present ground-state complexation with BSA and ensures that the mechanism is mediated by O 2 ( 1 △ g ). Strong dependence between BSA?O 2 ( 1 △ g ) photo-oxidation and the pH of the medium has been found. The relative oxygen uptake rate (v − △ O2 ) and the total quenching rate constant (k t ) values are higher at pH 11 than pH 7.4. The enhancement in the alkaline condition is due to conformational changes in the protein and the reactivity of tyrosinate anion with O 2 ( 1 △ g ). Even when the tendency with the pH in the presence of sodium dodecyl sulfate (SDS) micelles is similar to that observed in homogeneous media, an increment on the k t value is detected. This effect may be attributable to the strong interaction of BSA?SDS, which leads to the protein unfolding and could leave more exposed photo-oxidizable amino acids. A protective effect against the O 2 ( 1 △ g )-mediated photo-oxidation was observed in reverse micelles (RMs) of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) by comparing the k t values obtained at W = 10 with respect to the one obtain in homogeneous media. The latter could be mainly explained by the modification in the solvent polarity. Also, another important observation was found, the internal pH inside RMs of AOT sensed through tyrosine absorption was independent of the one used for the formation of the water pool. Hence, the k t values observed at both pH, are quite similar. Fil: Reynoso, Eugenia. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina Fil: Cacciari, Rodolfo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina Fil: Suchetti, Carlos Alberto. Universidad Nacional de Río Cuarto; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Montejano, Hernan Alfredo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones en Tecnologías Energéticas y Materiales Avanzados. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Tecnologías Energéticas y Materiales Avanzados; Argentina Fil: Biasutti, Maria Alicia. Universidad Nacional de Río Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina

The use of biologically active substances with anti-inflammatory properties such as corticosteroids has increased considerably in the last few decades. Particularly, the compound we are interested in, prednisolone (Predn), is a glucocorticoid with high biological activity. This compound absorbs UV radiation and may participate in photochemical processes, which can result in its own decomposition. These processes could result in the formation of free radicals or reactive oxygen species (ROS). On these grounds, the kinetic and mechanistic aspects of the direct photodegradation of Predn have been studied in aqueous medium under different atmospheric conditions by stationary and time-resolved techniques. The mechanism involved in the photodegradation has been elucidated. Predn is capable of generating the excited triplet state 3 Predn∗ as a result of UVB light absorption. In the presence of oxygen, 3 Predn∗ allows the formation of ROS, of which O 2 ( 1 Δ g ) (Φ Δ = 0.014), H 2 O 2 and the radical OH stand out. The latter is generated from the spontaneous dismutation of O 2 - and subsequent homolytic cleavage, photochemically promoted, of H 2 O 2 . Predn undergoes unimolecular photodegradation reactions under an inert argon atmosphere. In this study we found that in the presence of oxygen, the Predn photo-consumption is improved. This implies that the attack by ROS involves a very important additional contribution to the photodegradation of Predn under aerobic conditions. Fil: Cacciari, Rodolfo Daniel. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Reynoso, Eugenia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Montejano, Hernan Alfredo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Biasutti, Maria Alicia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The binding of o-nitroaniline (o-NA) to the micellar interface of n-heptane/1,4-bis(2-ethylhexyl) sulfosuccinate (AOT)/nonaqueous polar solvent reverse micelles was determined by UV-visible spectroscopy. As polar solvents: ethylene glycol (EG), formamide (FA), glycerol (GY), dimethylacetamide (DMA) and dimethylformamide (DMF) were used. The binding constant, Kb, obtained follow the order: DMF ~ DMA > FA ~ WS = 0 > EG > water > GY. It is demonstrated that the o-NA – AOT hydrogen bond interaction is with the AOT sulfonate moiety. Thus, the more accessible that this group is for hydrogen bond interaction the larger is the Kb value.

Kinetic and mechanistic aspects on the stability of the flavones (FL) quercetin (Que), morin (Mor) and rutin (Rut), in methanolic solution and in the presence of reactive oxygen species (ROS) generated by visible light-promoted riboflavin (Rf, vitamin B(2)) photoirradiation were studied. The system was chosen as a model for the evaluation of the in vivo protective effect of biological targets by the flavones. The overall picture includes the vitamin as an endogenous natural photosensitizer. A systematic study on the effect of ROS on FL photostability shows that under work conditions Que is oxidized by singlet molecular oxygen (O(2)((1)Delta(g))), superoxide radical anion (O(2)(-)) and hydrogen peroxide; Mor is degraded by O(2)((1)Delta(g)) and O(2)(-) whereas Rut only reacts with O(2)((1)Delta(g)). Que and Rut, with an extremely poor overall rate constant, are mainly physical quenchers of O(2)((1)Delta(g)). Mor, with O(2)((1)Delta(g))-interception ability slightly lower than the recognized synthetic antioxidant trolox (Tx), behaves as a typical sacrificial scavenger provided that ca 80% of the collisions with O(2)((1)Delta(g)) cause its own degradation, whereas this parameter reaches around 50% in the case of Tx.

The photodynamic effect on tryptophan methyl ester (trpME) and tryptophan octyl ester (trpOE), using the O(2)((1)Delta(g))-photosensitizers Rose Bengal (RB) and Perinaphthenone (PN) has been studied in large unilamellar vesicles (LUVs) of the phospholipid 1,2-di-oleoyl-sn-glycero-3-phosphatidylcholine (DOPC) by stationary photolysis and time-resolved methods. This work reports on the influence of both the site (O(2)((1)Delta(g))) generation and the location of the tryptophan derivatives (trpD), on the photo-oxidation process in a compartmentalized system. The apparent rate constant values for chemical quenching of O(2)((1)Delta(g)) by trpOE (k(r,app)), was higher in vesicles than in water. Also, the ratio between apparent reactive and overall rate constant values for the deactivation of O(2)((1)Delta(g)) (k(r,app)/k(t,app)), increases in vesicles as compared with water, when the oxidative species is generated in the lipidic region or at the interface. Nevertheless, this quotient is lower than the corresponding value in water when O(2)((1)Delta(g)) is generated in the aqueous pseudophase. For trpME, the k(r,app)/k(t,app)values in vesicles and in water are quite similar, confirming the fact that trpME is located in the water pseudophase. Results are discussed in terms of relative protection against O(2)((1)Delta(g)) attack in a microheterogeneous medium as compared with water.

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.

The Rose Bengal-sensitized photooxidations of the dipeptides l-tryptophyl-l-phenylalanine (Trp-Phe), l-tryptophyl-l-tyrosine (Trp-Tyr) and l-tryptophyl-l-tryptophan (Trp-Trp) have been studied in pH 7 water solution using static photolysis and time-resolved methods. Kinetic results indicate that the tryptophan (Trp) moiety interacts with singlet molecular oxygen (O(2)((1)Delta(g))) both through chemical reaction and through physical quenching, and that the photooxidations can be compared with those of equimolecular mixtures of the corresponding free amino acids, with minimum, if any, influence of the peptide bond on the chemical reaction. This is not a common behavior in other di- and polypeptides of photooxidizable amino acids. The ratio between chemical (k(r)) and overall (k(t)) rate constants for the interaction O(2)((1)Delta(g))-dipeptide indicates that Trp-Phe and Trp-Trp are good candidates to suffer photodynamic action, with k(r)/k(t) values of 0.72 and 0.60, respectively (0.65 for free Trp). In the case of Trp-Tyr, a lower k(r)/k(t) value (0.18) has been found, likely as a result of the high component of physical deactivation of O(2)((1)Delta(g)) by the tyrosine moiety. The analysis of the photooxidation products shows that the main target for O(2)((1)Delta(g)) attack is the Trp group and suggests a much lower accumulation of kynurenine-type products, as compared with free Trp. This is possibly because of the occurrence of another accepted alternative pathway of oxidation that gives rise to 3a-oxidized hydrogenated pyrrolo[2,3-b]indoles.

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 (r) 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 behavior of acridine orange base (AOB) in nonaqueous reverse micelles composed of n-heptane/AOT/polar solvent has been performed. Ethylene glycol (EG), propylene glycol (PG), glycerol (GY), formamide (FA), dimethylformamide (DMF), and dimethylacetamide (DMA) were employed as water substitutes. The studies were performed by static and time-resolved emission spectroscopy. Thus, the distribution of AOB between the two pseudophases of the aggregates was quantified by measuring the partition constants from emission spectra at different surfactant concentration. Similar values to those obtained by means of absorption spectroscopy were obtained. This match is indicating that AOB is not experiencing partition during the lifetime of the excited state. Partitioning to the micelles is strongly favored in micelles containing hydrogen-bond donor (HBD) solvents rather than non-HBD solvents. Variations of fluorescence lifetimes with AOT concentration confirm these results. By the solvatochromic behavior of AOB in the different systems it is shown that the microenvironment at the interface is distinct from that of the bulk polar solvent, indicating that the probe senses no “free” solvent. The steady state anisotropy ( 〈 r 〉 ) was measured for EG/AOT/n-heptane and DMF/AOT/n-heptane systems as representatives for HBD and non-HBD polar solvents, respectively. The value of 〈 r 〉 is higher in the micelles containing EG than that obtained with DMF, and increases with AOT concentration. This is explained as due to highly structured polar solvents in the inner core. EG is interacting with the polar heads of AOT through hydrogen-bond interaction, while DMF can only interact with the Na+ counterions. This is confirmed by the time-resolved emission spectra (TRES) of the probe in the micellar systems, in comparison with the bulk solvents.

An exploratory study on the effect of polar solvents upon the partitioning of solutes in nonaqueous reverse micellar solutions composed by n-heptane/AOT/polar solvent has been performed. Ethylene glycol (EG), propylene glycol (PG), glycerol (GY), formamide (FA), dimethylformamide (DMF), and dimethylacetamide (DMA) were employed as water substitutes. The study has been carried out at a single value of the mole ratio between the polar solvent and AOT, Ws = [polar solvent]/[AOT] equal to 2. 9-Anthracenemethanol (9-AM) and acridine orange base (AOB) were the solutes considered. Partitioning of (9-AM) was determined following a fluorescence quenching method. AOB partition constants were evaluated by a method based on the dependence of the absorption spectra with AOT concentration. The results obtained show that, for both solutes, partitioning to the micelles is strongly favored in (EG), (PG), (GY), or (FA), rather than in (DMF) or (DMA) containing micelles. This trend is opposite to that observed for the parti...

The properties of the base acridine orange (AOB) in n-heptane/AOT/water reverse micelles were investigated by using absorption, fluorescence, and single photon counting techniques. For comparison, studies in homogeneous media (water and n-heptane) were also performed. The absorption spectra of AOB in water at pH 10 in the same [AOB] range, only the basic form, AOB, was detected with a band in the visible at λmax = 435 nm, which obeys Lambert−Beer's law. This species was also the only one detected in n-heptane at 417 nm. These results show that only the protonated base dimerizes as confirmed by fluorescence techniques. The absorption spectra of AOB in the micellar media at various W0 (W0 = [H2O]/[AOT]) and pH = 4, working above the operational critical micellar concentration and below [AOT] = 3 × 10-3 M, show the disappearance of the ban...

The antioxidative effectiveness of three hydroxyaromatic derivatives (OHAD), namely phenol, resorcinol and phloroglucinol, as reflected by their scavenging ability of the photochemically generated species singlet molecular oxygen [O(2)((1)Delta(g))], was studied in aqueous solution and micellar media. Kinetic results, obtained through time-resolved phosphorescence detection of O(2)((1)Delta(g)) emission and polarographic methods, at pH 7 and 12 in homogeneous and CTAB micellar media, were compared. Calculated photooxidation quantum efficiencies (phi(r)), ranging from very low values ( approximately 0.01) to relatively high ones (approximately 0.3), depend on the preferential solubilization sites of the hydroxyaromatic derivatives either in the micellar or homogeneous media. OHAD in water pH 7 and in CTAB 0.02 M pH 12 exhibit their highest antioxidative efficiency as O(2)((1)Delta(g)) scavengers. In these conditions, they present the best degree of self protection against O(2)((1)Delta(g))-mediated photooxidation with relatively high values for the overall quenching rate constant and, simultaneously, the lowest phi(r).

Some organic contaminants dissolved in natural waters tend to adsorb on suspended particles and sediments. In order to mimic the photodegradation routes in natural waters of bromoxynil (BXN) adsorbed on silica, we here prepare and characterize silica nanoparticles modified with BXN (NP-BXN). We measure the direct photolysis quantum yield of aqueous BXN at 307 nm (0.064 ± 0.001) and detect the formation of bromide ions as a reaction product. Under similar conditions the photolysis quantum yield of BXN bonded to NP-BXN is much lower (0.0021 ± 0.0004) and does not lead to formation of bromide ions. The rate constant of the reaction of NP-BXN with the excited triplet states of riboflavin, a molecule employed as a proxy of chromophore dissolved organic matter (DOM) was measured in laser flash-photolysis experiments. The rate constants for the overall (kt) and chemical interaction (kr) of singlet oxygen with NP-BXN were also measured. Kinetic computer simulations show that the relevance of the direct and indirect (through reactions with reactive species generated in photoinduced processes) photodegradation routes of BXN is very much affected by sorption on silica. Immobilization of the herbicide on the particles, on one hand, affects the photolysis mechanism and lowers its photolysis quantum yield. On the other hand, the results obtained in aqueous suspensions indicate that immobilization also lowers the rate of collisional encounter, which affects the quenching rate constants of excited triplet states and singlet oxygen with the herbicide. Fil: Escalada, Juan Pablo. Universidad Nacional de la Patagonia Austral; Argentina Fil: Arce, Valeria Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina Fil: Carlos, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina Fil: Porcal, Gabriela Valeria. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina Fil: Biasutti, Maria Alicia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina Fil: Criado, Susana Noemi. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina Fil: Garcia, Norman Andino. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina Fil: Martire, Daniel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina

The production of dichlorophen (2,2′-methylenebis(4-chlorophenol), DCP) and its use as an anthelmintic and in pesticide products result in its direct release to the environment. To the purpose of modelling the possible photodegradation routes of DCP sorbed on sediments or suspended particles, the synthesis and characterization of silica nanoparticles modified with DCP (NP–DCP) is reported. The reactivity of NP–DCP with the excited states of riboflavin, a sensitizer usually present in natural waters, and with singlet oxygen were investigated. Comparison of the kinetic results obtained here to those previously reported for irradiated aqueous solutions of DCP allowed the discussion of the effect of adsorption of the pesticide on its photodegradation. We show with the aid of computer simulations that in natural waters the relevance of the different photodegradation routes dichlorophen is very much affected by attachment to sediments. Fil: Escalada, Juan Pablo. Universidad Nacional de la Patagonia Austral; Argentina Fil: Arce, Valeria Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina Fil: Porcal, Gabriela Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina Fil: Biasutti, Maria Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina Fil: Criado, Susana Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina Fil: Garcia, Norman Andino. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina Fil: Martire, Daniel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina

Nonaqueous microemulsions of AOT in n-hexane and n-heptane by using six polar solvents as water substitutes such as glycerol (GY), ethylene glycol (EG), propylene glycol (PG), formamide (FA), dimethylformamide (DMF), and dimethylacetamide (DMA) were studied and compared with the corresponding aqueous reverse micelles. The microenvironment generated by these systems was sensed by following the solvatochromic behavior of 1-methyl-8-oxyquinolinium betaine (QB). By varying Ws (Ws = [polar solvent]/[AOT]), the existence of an interaction between the hydrogen bond donor solvents EG, PG, and FA and QB was detected by the changes in the absorption spectra. These changes were interpreted as caused by the partition of the probe between the micelle interface and the polar solvent core by hydrogen bond interactions. The hydrogen bond association constants were calculated, being the largest for FA. In the case of GY, as well as for water, QB seems to be anchored at the interface and no partition was detected. For solv...

Reverse micelles, or water-in-oil microemulsions, incorporate a variety of solutes. The distribution of the solute between the surrounding solvent and the micelles can be described in terms of a partition constant based on a two pseudophases model that can be expanded to three pseudophases in micelles with high water to surfactant mole ratios. In these systems, two partition constants can be defined, one between the external solvent and the micellar interface, and other between the external solvent and the inner water pool. The present review describes the methodologies that can be employed to obtain distribution constants, and discuss literature data obtained in these systems. The localization of solutes associated to the micelles, as well as the effects promoted by solute incorporation on micellar properties are also discussed.

The interaction between photochemically generated singlet molecular oxygen [O 2 ( 1 Δ g )] and the relatively recently reported photostable compound, 1′-hydroxy-2′-acetonaphthone (1OH2AN) has been studied. The behavior of the isomer 2′-hydroxy-l-acetonaphthone (2OH1AN) was also investigated for comparative purposes. The former belongs to the family of extremely photostable intramolecular hydrogen bonded systems, much used in polymer photoprotection. Our study analyses the properties of these compounds as generators, by direct photoirradiation, and quenchers of the known oxidative species O 2 ( 1 Δ g ) upon indirect, dye-sensitized photoirradiation. Time resolved phosphorescence detection of O 2 ( 1 Δ g ) and polarographic and spectrophotometric measurements demonstrate that both 1OH2AN and 2OH1AN are excellent O 2 ( 1 Δ g ) quenchers in alkaline media (ionized-phenolic form). Nevertheless the process of quenching has a mainly chemical component, producing photodegradation of both isomers with photooxidation quantum efficiencies of 0.33 and 0.16 for 1OH and 2OH derivatives, respectively. These compounds are poor O 2 ( 1 Δ g ) quenchers when the molecular (non-ionized) phenolic form predominates through an exclusive O 2 ( 1 Δ g )-physical quenching process. The failure of O 2 ( 1 Δ g ) generation upon direct photoirradiation of 1OH2AN constitutes a desirable condition for a polymer photoprotector against photooxidative damage.

The Rose Bengal sensitized photooxidation of 2,3-dihydroxynaphthalene (2,3-DHN) and 2,7-dihydroxynaphthalene (2,7-DHN) in pH 12 water yields 1,1′-dimeric products, the appearance of which can be explained by the coupling of radicals formed by primary electron transfer from the anionic form of each starting dihydroxynaphthalene to singlet molecular oxygen or to the excited sensitizer. The dimer from 2,7-DHN is further oxidised during the irradiation to 6,7-dihydroxyperylene-1,12-quinone (DHPQ). In the case of 2,3-DHN, phthalic acid has also been detected.

The interface polarity of benzyl- n -hexadecyldimethylammonium chloride (BHDC) cationic reversed micelles in benzene was studied and compared with those of the anionic systems of sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT) in benzene and hexane. The nonionic reversed micelles of the poly(oxyethylene) n dodecyl ethers type surfactants Brij 30 in benzene, cyclohexane, and decane and C 12 E 5 in hexane, heptane, and decane were also investigated. 1-Methyl-8-oxyquinolinium betaine (QB) was used as optical probe. The influence of surfactant concentration and W = [H 2 O]/[surfactant] were analyzed as a function of the absorption bands shifts in the visible, B 1 , and in the UV, B 2 , and the absorbances ratio of these bands. At W = 0 the polarity sensed in the micelle interfaces of AOT–hexane and AOT–benzene is entirely similar while for BHDC–benzene is quite smaller. The detected interaction between the QB and BHDC may favor the entrance of QB into the oil side of the micelle interface thus sensing a less polar environment. At W ≥ 10 once the polar heads of the surfactants are completed hydrated, the anionic as well as the cationic surfactants show the same behavior. The polarity of Brij 30 and C 12 E 5 in the aliphatic hydrocarbons is comparable. However at W = 0 their micropolarity is higher than that of AOT–hexane, showing that the formation of hydrogen bond between QB and the free OH groups of these surfactants is the major factor affecting the microenvironment. In all the studied systems even at the maximum possible W , the polarity sensed by QB never reaches the value of pure water, that is, never reaches the bulk water phase prevailing in the micellar core.

A kinetic study on the aerobic (O2(1Δg)-mediated) photooxidation of a series of dihydroxynaphthalenes was carried out. Members of this family of compounds include important environmental contaminants. The interaction with O2(1Δg), determined by both time-resolved phosphorescence and static methods, yields a quenching rate constant in the range of 105–109 M−1 s−1, depending on the solvent polarity, pH, and substitution pattern of the dihydroxynaphthalene. According to experimental evidence, the mechanism of the interaction seems to be mediated by an encounter complex with a considerable charge transfer component. From a mechanistic point of view this characteristic of DHN indicates a behaviour very similar to that of simple substituted phenols and dihydroxybenzenes. Quantum yields for DHN photooxidation (Φr) indicate that these substrates are excellent candidates for O2(1Δg)-mediated degradation under environment conditions. Furthermore, these compounds proved to be good self-sensitizers upon irradiation at 337 nm. Quantum yields (ΦΔ) for the production of O2(1Δg) were determined to be on the order of 0.25 in benzene. Key words: dihydroxynaphthalenes, photooxidation, quenching, sensitization.not available

The microenvironment of the polar core of reverse micelles of the anionic surfactant AOT in n-hexane was studied by following the solvatochromic behavior of 1-methyl-8-oxyquinolinium betaine (QB) and ET(30). A solvatochromic study in pure solvents shows that QB presents two bands which could indicate two different effects. The band in the visible region, B1, which is the band for charge transfer from oxygen to nitrogen, is sensitive, mainly of polarity. The band in the UV region, B2, is most sensitive to the hydrogen bond donor capability of the solvent and it is assigned to the charge transfer of the phenoxide to the ring. These properties are used to establish changes in the probe microenvironment within the reverse micelles. The studies made by varying AOT concentration and the water content of the micellar solution show that QB resides in the micelle interface, indicating its polarity and its hydrogen bond donor ability. These properties at the interface of the AOT micelles in n-hexane increase up to W = 10 and then are practically constant, as demonstrated by the shifts and intensity variations of the B1 and B2 bands with W at a constant AOT concentration. Similar results are obtained with ET(30). However, because the latter is a much more voluminous probe, it is believed to disrupt the system, indicating different places within the micelle than QB.

The photophysics of Safranine-O (3,6-diamino-2,7-dimethyl-5 phenyl phenazinium chloride) (SfH(+)Cl(-)) was investigated in reverse micelles (RMs) of AOT (sodium bis(2-ethylhexyl)sulfosuccinate) with special emphasis on the triplet state processes. The triplet is formed in its monoprotonated form, independently of the pH of the water used to prepare the RMs. While the intersystem crossing quantum yields in RMs are similar to those in organic solvents, the triplet lifetime is much longer. Since the pH in the water pool of AOT RMs is close to 5 and the triplet state of the dye is subjected to proton quenching, the long lifetime indicates that the dye resides in a region where it cannot be reached by protons during its lifetime. All the measurements indicate that the dye is localized in the interface, sensing a medium of micropolarity similar to EtOH : water (3:1) mixtures. The quenching by aliphatic amines was also investigated. While the quenching by the hydrophobic tributylamine is similar to that in methanol, the hydro-soluble triethanolamine is one order of magnitude more effective in RMs than in homogeneous solution. In the latter case the quenching process is interpreted by a very fast intramicellar quenching, the overall kinetics being controlled by the exchange of amine molecules between RMs. Semireduced dye is formed in the quenching process in RMs in the di-protonated state with a comparable quantum yield to the monoprotonated state formed in homogeneous solvents. The results point to the advantage of the reverse micellar system for the generation of active radicals for the initiation of vinyl polymerization, since a much lower concentration of amine can be employed with similar quantum yields.

Within the context of environmentally friendly methods for the elimination of surface-water pollutants, the photodegradation of the phenolic pesticides bromoxynil (BXN) and dichlorophen (DCP) under simulated natural conditions has been studied. The work was done in the presence of the visible-light absorber photosensitizer riboflavin (Rf), usually present in trace quantities in natural waters. Under aerobic conditions, an efficient photooxidation of both pesticides was observed. The relatively intricate photochemical mechanism involves pesticide and oxygen consumption and, to a lesser extent, Rf degradation. The kinetic and mechanistic study supports that both H(2)O(2) and singlet molecular oxygen, O(2)((1)Δ(g)), are involved in the process. Kinetic data for the O(2)((1)Δ(g))-mediated oxidation indicate that BXN and DCP are photodegraded with this species faster than the parent compound phenol, very frequently employed as a model for aquatic contaminants, likely due to their lower pK(a) values. This observation allows the design of phenolic pesticides with different photodegradation rates under environmental conditions.

A study has been made on the properties of human serum albumin (HSA) binding sites and how they are modified by pre-oxidation of the protein with hypochlorite. The oxidation extent was assessed from changes in the protein intrinsic fluorescence and production of carbonyl groups. HSA retains its solute binding capacity even after exposure to relatively large amounts of hypochlorite (up to 40 oxidant molecules per protein). From an analysis of the binding isotherms of dansyl sarcosine (DS) and dansyl-1-sulfonamide (DNSA) to native and hypochlorite treated albumin it is concluded that pre-oxidation of the protein reduces the number of active sites without affecting the binding capacity of the remaining binding sites. From DS and DNSA fluorescence anisotropy, Laurdan anisotropy and generalized polarization measurements, it is concluded that both Sites I and II in the native protein provide very rigid environments to the bound probes. These characteristics of the sites remain even after extensive treatment with hypochlorite. This stubbornness of HSA could allow the protein to maintain its function along its in vivo lifetime.

The rate of hydrolysis of p-nitrophenyl acetate (PNPA) catalyzed by Mucor javanicus lipase has been measured in AOT reverse micellar solutions formulated in aliphatic hydrocarbons, aromatic hydrocarbons and a chlorinated compound. The study has been performed at a single value of W=([water]/[AOT])=6.0. Fluorescence decay measurements of intrinsic enzyme fluorescence, mainly due to tryptophan residues, in the different reverse micellar systems were also carried out, in an attempt to obtain some insight on the effect of the organic solvent on the protein conformation. Differences observed in the kinetics of the fluorescence decays of tryptophan residues of the lipase incorporated to the micelles with the different external organic solvents were also found in the catalytic behaviour of the enzyme. In particular, it is observed that the contribution of the long lived component of the fluorescence decay is considerably higher (ca. 40%) in aliphatic than in aromatic solvents (ca. 15%), indicating significant differences in the protein conformation. This effect of the organic solvent on the protein conformation is also observed in the enzymatic activity, which is higher in the aromatic than in the aliphatic solvents.

The 3,3', 4',7 tetrahydroxiflavone (fisetin) is a natural therapeutically active and fluorescent polyhydroxyflavone, with important spectroscopic and biological behavior. Fisetin shows dual emission, with a normal band (N) from the S1 --S0 transition and the one generated in the excited state (phototautomer; PT) from the intramolecular proton transfer (ESIPT) process. The influence of different interfaces on the ESIPT process of fisetin was investigated in reverse micelles media (RMs) made of the anionic sodium 1,4-bis (2-ethylhexyl) sulfosuccinate (AOT) and cationic benzyl n-hexadecyl dimethylammonium chloride (BHDC) surfactants, in benzene. The studies were carried out by absorption, emission spectroscopy, steady-state anisotropy and time-resolved fluorescence measurements. Fisetin behavior was also investigated in homogeneous media with special emphasis in water and benzene, which are the polar core and the organic pseudofase in the RMs, respectively. In addition, the effect of concentration in benzene and the variation of the pH in water were studied. Fluorescence lifetime measurements show that in water the ESIPT process is independent on the concentration, while in benzene it was possible to detect fluorescent aggregate species (Nas) formed in the ground state. The effect of the pH in water allowed us to identify the anionic fisetin (A-) emission. The studies in RMs show that fisetin interacts specifically with the head of the surfactants, which always results in diminishing the emission of the PT. Also the formation of A- is detected particularly at W00. Appreciable high anisotropy values are obtained in RMs, as compared with those in fluid homogeneous media, which are independent of the water content confirming that fisetin molecules are anchored in the anionic as well as in the cationic interfaces.

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.

Benzylhexadecyl dimethylammonium chloride (BHDC) and sodium bis(2-ethylhexyl) sulfosuccinate (AOT) water-in-oil micro-emulsions were employed to study the influence of medium heterogeneity on singlet molecular oxygen [O(2)((1)Delta(g))]-mediated degradation of 1-hydroxynaphthalene (1-OHN) and 2-hydroxynaphthalene (2-OHN) in their naphthoate form. The kinetic study in the micellar system, that was considered as a closer model for the environment of contaminants in natural polluted waters, consisted of a comparative work with the process in homogeneous solution, by varying surfactant structure and water content of the micro-emulsion. While it is known that 1-OHN and 2-OHN are rapidly and efficiently photo-oxidised in aqueous medium, time-resolved phosphorescence detection of O(2)((1)Delta(g)) and stationary photolysis experiments demonstrate that both the values for the overall and reactive rate constants for the quenching of O(2)((1)Delta(g)) and the photo-oxidation efficiencies are lowered in BHDC micelles, whereas the photo-oxidative process in AOT micro-emulsions was totally inhibited. Results are interpreted and discussed on the basis of different locations of the probe in the micellar environment.

The kinetics of hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by alpha-chymotrypsin (alpha-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, alpha-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 alpha-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 alpha-CT, which leads to an increase of the enzyme stability and activity.