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.

An inner filter effect based sensing system for the determination of caffeine in beverage samples.

In this work, the inner filter effect (IFE) of caffeine (CF) over the fluorescence signal of glibenclamide (GLB) was used for the determination of CF in beverage samples. The system worked in a turn-off mode since the absorption spectrum of CF overlaps the excitation band of GLB resulting in a decline in its fluorescence signal (λexc = 234 nm, λem = 350 nm). No changes in the fluorescence lifetime of GLB (0.29 ns) were observed in the presence of CF up to 127.6 mg L-1 concentration. The parameters that affect the fluorescence intensity were investigated, such as fluorophore concentration (16 mg L-1), pH (3.2) and temperature (25 °C). Under optimized conditions, the IFE-based approach can determine CF in a range between 1.00 and 100.0 mg L-1, with a detection limit (LOD) of 0.10 mg L-1. The relative standard deviation (% RSD) values for the intra-day and inter-day precision were 0.75 and 1.24, respectively. The new method was successfully tested in the determination of the target analyte in beverage samples without previous treatment. The results were compared with those obtained by a reference method, leading to the conclusion that there were no significant differences at the studied confidence level (α = 0.05).

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.

Measurements of octanol-air partition coefficients, vapor pressures and vaporization enthalpies of the (E) and (Z) isomers of the 2-ethylhexyl 4-methoxycinnamate as parameters of environmental impact assessment.

2-Ethylhexyl 4-methoxycinnamate is one of the UVB blocking agents more widely used in a variety of industrial fields. There are more than one hundred industrial suppliers worldwide. Given the enormous annual consumption of octinoxate, problems that arise due to the accumulation of this compound in nature should be taken into consideration. The GC-RT was used in this work with the aim of determining the vapor pressure, enthalpies of vaporization and octanol-air partition coefficient, for the BBP, DOP, E- and Z-EHMC esters. The results showed that Z-EHMC is almost five times more volatile than E-EHMC. Moreover, BBP, Z-EHMC and E-EHMC can be classified as substances with a relatively low mobility since they lie within the range of 810 and log(PL/Pa)<-4, therefore, a low mobility can be expected. From these parameters, their particle-bound fraction and gas-particle partition coefficient were also derived.

Energy and electron transfer processes in polymeric nanoparticles.

We report herein a study on photoinduced electron transfer (eT) and energy transfer (ET) processes occurring between 9-methylanthracene-acrylate (A) and N,N-dimethylaniline-acrylate (D) derivatives incorporated into polymeric nanoparticles (NP). Five types of NPs were synthesized: PAD0, PAD25, PAD75, PD25, and PD75. All NPs are composed of a crosslinked polymer matrix of methyl methacrylate and ethylene glycol dimethacrylate. In addition, PAD0, PAD25 and PAD75 contain low doping levels of A. For PAD25 and PAD75, 25% and 75% of the mole fraction of methyl methacrylate is replaced by D, respectively. PD25 and PD75 were prepared as above but without A. NPs (diameter 6-9 nm) dispersed in organic solvents were characterized based on their UV-visible absorption, emission, excitation, and excitation anisotropy spectra and time dependent absorption and emission spectroscopy techniques. The emission decay profiles of A and D were always complex. Results indicate that A senses two distinct environments in all NPs. The emission quenching of PAD0 by DMA in DCM solutions is dynamic, and it is apparent that a significant fraction of A is inaccessible to the quencher. The emission of A is efficiently quenched by the presence of D in PAD25 and PAD75. The intra-NP photoinduced eT quenching mechanism has static and dynamic components. Selective excitation of D in PAD25 and PAD75 leads to the formation of the excited state of A via a singlet-singlet ET Föster type mechanism. Results indicate that both intra-NP eT and ET processes are more efficient in PAD75 due to the reduced average D*-A separation in these NPs.

Comparison of the kinetics of electron transfer in the diffusion limit for the singlet and triplet quenching of eosin Y by quinones.

Electron transfer (ET) rate constants were determined by means of lifetime measurements for the fluorescence quenching and by laser flash photolysis for the triplet quenching of the dye eosin Y by benzoquinones in acetonitrile. The results represent a new aspect of the dependence of the rate constants with the driving force in the diffusion limit region. That is, the rate constants for singlet quenching in the highly negative region of ΔGet do not decrease as predicted by Marcus theory, but rather show a small positive dependence on the driving force. However, it is found that, in the same free energy range, the triplet rate constants are lower than those for the singlet process. They also increase with the exergonicity of the reaction, but the dependence with ΔGet is less marked than that found for the singlet reaction. Even at a Gibbs energy change of -1.0 eV the triplet quenching rate constants do not reach the theoretical diffusion limit. The results are analyzed using the current theories for diffusion-mediated ET reactions.

Experimental and theoretical studies on the enantioselectivity of molecularly imprinted polymers prepared with a chiral functional monomer.

A comprehensive study on the enantioseparation of racemic bis[1-phenylethyl]amine (PEA) on a series of molecularly imprinted polymers (MIPs) prepared using the chiral functional monomer (S)-2-(2-methyl-acryloylamino)-3-phenyl propionic acid (MAPP) is reported. MIP-R, MIP-S and MIP-RS, were synthesized separately by imprinting the pure enantiomers (R-, S-PEA) and racemic PEA, respectively, MAPP, EDGMA as crosslinker and chloroform as the porogen. It was found that all MIPs prepared were able to resolve the PEA racemate. Residence times (t(r)) and enantioselectivity factors (α) were estimated from typical elution chromatography experiments. Frontal chromatography experiments were conducted to acquire the adsorption isotherms for both enantiomers on the different MIPs (and on the non-imprinted polymer, NIP). The adsorption isotherms were analyzed using the affinity spectrum (AS) and the expectation-maximization (EM) methods. The study also involved the theoretical evaluation of the MAPP/enantiomers interactions in the pre-polymer mixture. The EM method predicts mono- and bimodal distribution of affinity binding sites depending upon the polymer analyzed. Apparently, the enantioseparation process depends on relatively small differences in the stabilization of the diasteroisomeric ion-pairs PEA/MAPP complexes on the surface of the polymers.

Estimation of the solvent reorganization energy and the absolute energy of solvation of charge-transfer states from their emission spectra.

We report herein the solvent and temperature effects on the emission of the intermolecular exciplexes 1-cyanonaphthalene/triethylamine and 1-methylnaphthalene/triethylamine and the intramolecular exciplexes formed by the bichromophoric compounds diethyl-(3-naphthalen-1-yl-propyl)-amine (I), diethyl-(2-naphthalen-1-yl-ethyl)-amine (II), 3-[ethyl-(2-naphthalen-1-yl-ethyl)-amino]-propionitrile (III) and 3-[(2-cyano-ethyl)-(2-naphthalen-1-yl-ethyl)-amino]-propionitrile (IV). The results are interpreted within the formalism of the semiclassical Marcus theory for radiative back electron transfer (BET) reactions. We show that, following a few simple assumptions, reliable values of the Gibbs free energy changes (DeltaG(epsilon)(-et)) and the solvent reorganization energies (lambda(epsilon)(s)) associated to the BET reactions (and their corresponding enthalpic and entropic contributions) can be estimated directly from the emission of the CT states. We also show that for the 1-cyanonaphthalene/triethylamine exciplex, which exhibits emission in the vapour phase, the experimental calculation of the absolute energy of solvation of the CT state (DeltaG(epsilon)(s)) is also possible. The calculated DeltaG(epsilon)(-et) correlate quite satisfactorily with the corresponding values obtained from independent electrochemical and kinetics experiments. The temperature effects on DeltaG(epsilon)(-et), lambda(epsilon)(s) and DeltaG(epsilon)(s) are discussed qualitatively using different solvation models. The limitations of the present approach for the estimation of DeltaG(epsilon)(-et) and lambda(epsilon)(s) and its possible application to more complex A/D systems are also examined.

Photophysical properties of safranine O in protic solvents.

Spectroscopic and photophysical properties of safranine O (Sf) were investigated in binary water/solvent mixtures. It was found that these properties are strongly solvent-dependent. A blue shift is observed for both the ground-state absorption and the triplet-triplet main absorption band when the solvent polarity augments. At the same time a red shift of the fluorescence emission band takes place. These facts are interpreted in terms of higher dipole moment of the dye molecule in the S(1) state as compared with the S(0) state, while a decrease in the dipole moment of the triplet state T(n) with respect to the triplet state T(1) occurs. The Stokes' shift and the fluorescence lifetime shows a linear correlation with the E(T)(30) parameter, while a non-linear behavior is observed when a correlation with models of a continuous dielectric solvent is attempted. These results suggest the operation of strong specific interactions of Sf with solvent molecules, most likely hydrogen bonding. From fluorescence lifetime and quantum yield determinations, as well as intersystem-crossing quantum yields, the solvent dependence of the photophysical kinetic parameters were obtained. The radiative fluorescence rate constant can be adequately reproduced by calculations based on the UV-Vis absorption and emission spectra, as given by the Strickler-Berg equation.