UVB light influence on the laccase enzyme catalytic activity in reverse micelles and in homogeneous aqueous medium.

Laccase is a versatile enzyme widely used for the oxidation of environmental contaminants and exhibits great potential in many others applications; however, it undergoes photo-degradation when irradiated with UVB light. The photo-stability of this biomolecule can be improved by immobilization in different encapsulation media and reverse micelles have been employed with this purpose. The laccase activity using syringaldazine as substrate has been studied in the absence and in the presence of reverse micelles of 0.15 M of sodium 1,4-bis (2-ethylhexyl) sulfosuccinate (AOT) in 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 determined by spectroscopic measurements in the micellar system and in homogeneous aqueous medium. The distribution of the substrate in two pseudo-phases (micelle and organic solvent) was taking into account in the kinetic parameters' determinations. The results obtained indicate that the nano-aggregate system confers a solubilization media in the water core of the micelle, both for the enzyme and the substrate, in which the catalytic function of the enzyme is preserved. On the other hand, in homogeneous aqueous medium kCAT/KM value, it is reduced by ~50% after UVB irradiation of the enzyme, while in micellar medium, less than 10% of the activity was affected. This mean that the enzyme achieves a considerably photo-protection when it is irradiated with UVB light in reverse micelles as compared with the homogeneous aqueous medium. This phenomenon can be mainly due to the confinement of the biomolecule inside the micelle. Physical properties of the nano-environment could affect photochemical reactions.

Effect of UVB solar irradiation on Laccase enzyme: evaluation of the photooxidation process and its impact over the enzymatic activity for pollutants bioremediation.

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.

Influence of pH and micellar systems on the sensitized photo-oxidation of bovine serum albumin.

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 O2 (1 △g ). Strong dependence between BSA-O2 (1 △g ) photo-oxidation and the pH of the medium has been found. The relative oxygen uptake rate (v- â–³ O2 ) and the total quenching rate constant (kt ) 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 O2 (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 kt 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 O2 (1 △g )-mediated photo-oxidation was observed in reverse micelles (RMs) of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) by comparing the kt 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 kt values observed at both pH, are quite similar.

Photodegradation of prednisolone under UVB solar irradiation. Role of photogenerated ROS in the degradation mechanism.

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 3Predn* as a result of UVB light absorption. In the presence of oxygen, 3Predn* allows the formation of ROS, of which O2(1Δg) (ΦΔ = 0.014), H2O2 and the radical OH˙ stand out. The latter is generated from the spontaneous dismutation of O2˙- and subsequent homolytic cleavage, photochemically promoted, of H2O2. 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.

Photodegradation routes of the herbicide bromoxynil in solution and sorbed on silica nanoparticles.

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.

The effect of dichlorophen binding to silica nanoparticles on its photosensitized degradation in water.

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.

Quenching of the triplet state of Safranine-O by aliphatic amines in AOT reverse micelles studied by transient absorption spectroscopy.

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.

A kinetic study of the photodynamic effect on tryptophan methyl ester and tryptophan octyl ester in DOPC vesicles.

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.

An example of how to use AOT reverse micelle interfaces to control a photoinduced intramolecular charge-transfer process.

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.

Kinetics of p-nitrophenyl acetate hydrolysis catalyzed by Mucor javanicus lipase in AOT reverse micellar solutions formulated in different organic solvents.

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.

New insights on the photophysical behavior of PRODAN in anionic and cationic reverse micelles: from which state or states does it emit?

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.

New insights on the behavior of PRODAN in homogeneous media and in large unilamellar vesicles.

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.

The use of acridine orange base (AOB) as molecular probe to characterize nonaqueous AOT reverse micelles.

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.

Effect of reverse micelles on the Rose Bengal-sensitized photo-oxidation of 1- and 2-hydroxynaphthalenes.

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.

Effect of the addition of a nonaqueous polar solvent (glycerol) on enzymatic catalysis in reverse micelles. Hydrolysis of 2-naphthyl acetate by alpha-chymotrypsin.

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.