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On January 4, 2014, during the summer period in South America, an intense forest and dry pasture wildfire occurred nearby the city of Santiago de Chile. On that day the biomass-burning plume was transported by low-intensity winds towards the metropolitan area of Santiago and impacted the concentration of pollutants in this region. In this study, the Weather Research and Forecasting model coupled with Chemistry (WRF/Chem) is implemented to investigate the biomass-burning plume associated with these wildfires nearby Santiago, which impacted the ground-level ozone concentration and exacerbated Santiago's air quality. Meteorological variables simulated by WRF/Chem are compared against surface and radiosonde observations, and the results show that the model reproduces fairly well the observed wind speed, wind direction air temperature and relative humidity for the case studied. Based on an analysis of the transport of an inert tracer released over the locations, and at the time the wildfires were captured by the satellite-borne Moderate Resolution Imaging Spectroradiometer (MODIS), the model reproduced reasonably well the transport of biomass burning plume towards the city of Santiago de Chile within a time delay of two hours as observed in ceilometer data. A six day air quality simulation was performed: the first three days were used to validate the anthropogenic and biogenic emissions, and the last three days (during and after the wildfire event) to analyze the performance of WRF/Chem plume-rise model within FINNv1 fire emission estimations. The model presented a satisfactory performance on the first days of the simulation when contrasted against data from the well-established air quality network over the city of Santiago de Chile. These days represent the urban air quality base case for Santiago de Chile unimpacted by fire emissions. However, for the last three simulation days, which were impacted by the fire emissions, the statistical indices showed a decrease in the model performance. While the model showed a satisfactory evidence that wildfires plumes that originated in the vicinity of Santiago de Chile were transported towards the urban area and impacted the air quality, the model still underpredicted some pollutants substantially, likely due to misrepresentation of fire emission sources during those days. Potential uncertainties may include to the land use/land cover classifications and its characteristics, such as type and density of vegetation assigned to the region, where the fire spots are detected. The variability of the ecosystem type during the fire event might also play a role.

Tris(2,2′-bipyridine) Ru(II) (Ru(bpy) 3 2+ ) phosphorescence is readily quenched by p-substituted phenols in a bimolecular process whose rate, in aqueous solution, is determined by the pH of the solution and the electron-affinity of the substituent. A Hammett’s type plot based on data obtained at pH 10.4 in terms of σ parameters conforms to a parabola with a minimum rate for phenol and two branches, one comprising compounds with negative σ values (electron donors) and other with positive σ values (electron acceptors groups). This anomalous behaviour takes place at a constant pH and it can be explained in terms of two quenching mechanisms: a simple electron transfer from the phenolate to the Ru(II) complex when pKa pH).

The potential activity of mango by-products to inhibit the peroxyl radical-mediated oxidation of tryptophan residues (Trp) of myofibrillar proteins (MP) was studied. Extracts of seeds, peels and pulps were studied in terms of their antioxidant activity, assessed by the oxygen radical absorbance capacity (ORAC) assay employing fluorescein and pyrogallol red as probes (ORAC-FL and ORAC-PGR, respectively). The effect of the extracts of mango by-products on the oxidation of Trp was evaluated by fluorescence spectroscopy. Data obtained employing the ORAC-FL assay showed a low discrimination of the antioxidant activity of the samples (Peel ≈ Seed > Pulp). However, employing the ORAC-PGR assay it was possible to differentiate the antioxidant activity of the samples (Seed > Peel >> Pulp). Extracts obtained from mango seed, peel, and pulp protected Trp of MP when the latter was exposed to AAPH (2,2′-azo-bis(2-amidinopropane) dihydrochloride)-derived free radicals. Such effect was in good agreement with the ORAC-PGR data, showing that this assay could be employed to predict the protection of Trp residues of MP.

In the present work we studied the reaction under gastric conditions of pyrogallol red (PGR), a polyphenolic dye, with nitrous acid (HONO). PGR has been used as a model polyphenol due to its strong UV-visible absorption and its high reactivity towards reactive species (radicals and non-radicals, RS). The reaction was followed by UV-visible spectroscopy and high performance liquid chromatography (HPLC). A clear decrease of the PGR absorbance at 465 nm was observed, evidencing an efficient bleaching of PGR by HONO. In the initial stages of the reaction, each HONO molecule nearly consumed 2.6 PGR molecules while, at long reaction times, ca. 7.0 dye molecules were consumed per each reacted HONO. This result is interpreted in terms of HONO recycling. During the PGR-HONO reaction, nitric oxide was generated in the micromolar range. In addition, the rate of PGR consumption induced by HONO was almost totally abated by argon bubbling, emphasising the role that critical volatile intermediates, such as ŸNO and/or nitrogen dioxide (ŸNO2), play in the bleaching of this phenolic compound.

DPPC and DPPC:SM large unilamellar vesicles (LUVs), prepared by extrusion, readily respond to osmotic shocks (hypo- and hyper-osmotic) by water influx/efflux (evaluated by changes in turbidity) and by entrapped calcein liberation (measured by an increase in dye fluorescence intensity). On the other hand, small unilamellar vesicles (SUVs) prepared by sonication are almost osmotically insensitive. LUVs water transport, both in hypo- and hyper-osmotic conditions, takes place faster than calcein ejection towards the external solvent. Similarly, response to a hypotonic imbalance is faster than that associated to a hypertonic stress. This difference is particularly noticeable for the increase in calcein fluorescence intensity and can be related to the large reorganization of the bilayer needed to form pores and/or to adsorb the dye to the inner leaflet of the vesicle after water efflux. Conversely, addition of SM to the vesicles barely modify the rate of calcein permeation across the bilayer.

The spectroscopic and photophysical properties of rose bengal (RB) encased in bovine serum albumin (BSA) have been examined to evaluate the photosensitized generation of singlet molecular oxygen (1O2). The results show that RB photophysical and photosensitizing properties are highly modulated by the average number of dye molecules per protein (n). At n 蠐1, the dye molecule is tightly located into the hydrophobic nanocavity site I of the BSA molecule with a binding constant Kb = 0.15 ± 0.01 μM-1. The interaction with surrounding amino acids induces heterogeneous decay of both singlet and triplet excited states of RB and partially reduce its triplet quantum yield as compared with that in buffer solution. However, despite of the diffusive barrier imposed by the protein nanocavity to 3O2, the quenching of 3RBBSA generates 1O2 with quantum yield ΦΔ = 0.35 ± 0.05. In turns, the intraprotein generated 1O2 is able to diffuse through the bulk solution, where is dynamically quenched by BSA itself with an overall quenching rate constant of 7.3 × 108 M-1 s-1. However, at n > 1, nonspecific binding of up to ≈6 RB molecules per BSA is produced, allowing efficient static quenching of excited states of RB preventing photosensitization of 1O2. These results provide useful information for development of dye-protein adducts suitable for using as potential intracellular photosensitizers. Fil: Espeche Turbay, Maria Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina Fil: Rey, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina Fil: Argañaraz, Natalia Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina Fil: Moran Vieyra, Faustino Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina Fil: Aspée, Alexis. Universidad de Santiago de Chile; Chile Fil: Lissi, Eduardo A.. Universidad de Santiago de Chile; Chile Fil: Borsarelli, Claudio Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina

The interaction between nanoparticles and molecules plays a key role in determining the activity and performance of a given nanostructure. These interactions are pivotal for a variety of applications including drug delivery, surface manipulation for targeted therapies, and catalysis. However, to this day, gathering precise association parameters for the interaction of the molecules with nanostructures remains elusive and mostly imprecise. In this review, we present a critical discussion of the most commonly used techniques and models intended for determining the association of molecules with nanoparticles. Particular emphasis has been put on discussing the limitations and pitfalls related to determining association constants in this tutorial review. Fil: Ahumada, Manuel. University Of Ottawa; Canadá Fil: Lissi, Eduardo. Universidad de Santiago de Chile; Chile Fil: Montagut, Ana Maria. University Of Ottawa; Canadá Fil: Valenzuela Henríquez, Francisco. Pontificia Universidad Católica de Valparaíso; Chile Fil: Pacioni, Natalia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina Fil: Alarcon, Emilio I.. University of Ottawa; Canadá

Sea anemones synthesize a variety of toxic peptides and proteins of biological interest. The Caribbean Sea anemone Stichodactyla helianthus, produces two pore-forming toxins, Sticholysin I (St I) and Stichloysin II (St II), with the ability to form oligomeric pores in cell and lipid bilayers characteristically lacking cysteine in their amino acid sequences. Recently, two mutants of a recombinant variant of Sticholysin I (rSt I) have been obtained with a Cys residue in functionally relevant regions for the pore-forming activity of the toxin: r St I F15C (in the amino terminal sequence) and r St I R52C (in the binding site). Aiming at characterizing the effects of oxidants in toxins devoid (r St I) or containing -SH moieties (r St I F15C and r St I R52C), we measured their hemolytic activity and pore forming capacity prior and after their incubation with peroxynitrite (ONOO(-)). At low ONOO(-)/Toxin ratios, nearly 0.8 Trp groups are modified by each added peroxynitrite molecule, and the toxin activity is reduced in ca. 20 %. On the other hand, in -SH bearing mutants only 0.5 Trp groups are modified by each peroxynitrite molecule and the toxin activity is only reduced in 10 %. The results indicated that Cys is the initial target of the oxidative damage and that Trp residues in Cys-containing toxins were less damaged than those in r St I. This relative protection of Trp groups correlates with a smaller loss of hemolytic activity and permeabilization ability in liposomes and emphasizes the relevance of Trp groups in the pore forming capacity of the toxins.

There are numerous studies on systems comprising an enzyme encapsulated in unilamellar liposomes and its substrate initially present in the external aqueous media. Most of these studies are focused on enzyme stability and activity in a restricted media. However, the rate of the process is also determined by the capacity of the substrate to permeate towards the liposome inner pool. In spite of this, there are few studies aimed at a quantitative evaluation of the substrate permeation rate and its lifetime inside the liposome pool. In the present work, we describe, in terms of a very simple mechanism, the permeation of glucose and hydrogen peroxide in DPPC unilamellar liposomes. To this aim, we evaluated the rate of the process employing encapsulated glucose oxidase and catalase in the kinetic diffusion controlled limit. Under this condition, the rate of the process becomes zero order in the enzyme and allows a direct evaluation of the rate constant for the permeation process and the lifetime of a substrate molecule incorporated into the liposome inner pool.

The reaction of phenols with nitrite (nitrous acid HONO, or its conjugated base, NO2−) is of importance in stomach fluids (low pH) and in atmospheric hydrometeors (mild acid and basic pH). The initial reaction associated with the oxidation/nitration of 4-substitued phenols promoted by HONO/NO2 depends on the pH of the solution. At low pH, the initial step involves the reaction between HONO and phenol, whereas at basic conditions this involves an electron transfer from the phenoxy anion to nitrogen dioxide (NO2) producing the nitrite anion. The rate of both processes is determined by the donor capacity of the substituent at the 4-position of the phenol, and the data obtained at pH 2.3 follow a linear Hammett-type correlation with a slope equal to –1.23. The partition of the gaseous intermediates (NO and NO2) makes the rate of HONO-mediated oxidation dependent on their gas–liquid distribution. At low pH, the main process is phenol oxidation, even in oxygen-free conditions, and the presence of any 4-substituted phenol decreases the rate of HONO auto-oxidation.

Valdecoxib addition quenches the intrinsic human serum albumin (HSA) fluorescence. This allows an evaluation of the drug-protein association. However, both the number of binding sites and their affinity for the drug depend upon the methodology employed for their evaluation and the employed protein concentration. In this work, we measured the effect of valdecoxib on HSA fluorescence yield over a wide range of experimental conditions and discuss the validity of the binding parameters derived from the different data treatments: Stern-Volmer, Scatchard, double logarithmic, quadratic equation, Benesi-Hilderand, and Encinas-Lissi. It is proposed that a combination of Encinas-Lissi and Scatchard treatments of the data renders the most reliable results. From these data, it is concluded that HSA presents three high-affinity binding sites for valdecoxib (K(as) = 4.5 × 10(4) m(-1)) and several secondary sites of smaller activity.

The anti-peroxyl radical quality of two aqueous rooibos infusions and solutions of their most abundant glycosylated polyphenols was evaluated using pyrogallol red and fluorescein-based oxygen radical absorbance ratios. It was observed that the artificial infusions, prepared using only the most abundant polyphenols present in rooibos and at concentrations similar to those found in the natural infusions, showed greater antioxidant quality than the latter infusions, reaching values close to those reported for tea infusions. Additionally, the antimicrobial activity of the natural and artificial infusions was assessed against three species of bacteria: Gram (+) Staphylococcus epidermidis and Staphylococcus aureus and Gram (−) Escherichia coli. When compared to the natural infusions the artificial beverages did not demonstrate any bacterostatic/cidal activity, suggesting that the antibacterial activity of rooibos is related to compounds other than the glycosylated polyphenols employed in our study.

Changes in the intrinsic protein fluorescence with the additive concentration provide one of the most employed methodologies for the evaluation of the binding constant and the number of binding sites. In the last years, more than 175 studies have been published where the double logarithmic plot shown below is used toward determining the number of equivalent binding sites (n). Log [(F° - F)/F] = log K + n log [Q0 ]. However, the value of n evaluated by this procedure is unrelated to the number of equivalent binding sites; rather it represents the stoichiometry of the binding step. The confusion on the meaning of n arises upon assuming that the binding process is represented by the forward and backward elementary steps shown below, implying that binding of the n solutes takes place simultaneously, i.e. there are no intermediate species. nQ + P ⇆ Qn P. The conclusion that n is unrelated to the number of equivalent binding sites is supported by the fact that in all the systems considered (99% of them) n values are close to one and much smaller than those obtained by ultrafiltration. It is then remarkable, the profusion of publications in peer-reviewed, specialized journals including a conceptual error that confuses Hill's coefficient and/or the stoichiometry of the binding step with the number of independent binding sites. Here, we discuss the origin of this common misconception and provide alternative methods to determine the number of binding sites.

The behavior of 4-aminophthalimide (4-AP), a common molecular probe utilized in solvation dynamics experiments, was revisited in polar aprotic and protic solvents using absorption, steady-state, and timeresolved fluorescence (TRES) techniques. Also, the deuterium isotope effect was investigated using D2O as solvent. The absorption spectra of 4- AP consist of two absorption bands with maxima around 300 nm (B2 band) and 370 nm (B1 band) depending on the environment, while the emission feature consists of a single band. In all the solvents investigated (excluding water), the 4-AP photophysics is similar and the emission spectra are independent of the excitation wavelength used. In water the behavior is unique and the emission spectra maximum is different depending on the excitation wavelength used. The emission maximum is 561.7 nm using the excitation wavelength that correspond to the B2 absorption band maximum (λexcB2 = 303.4 nm) but is 545.7 nm when the excitation wavelength that correspond to the B1 absorption maximum (λexcB1 = 370.0 nm) is used. Moreover, while the fluorescence decays of 4-AP in water exhibit no emission wavelength dependence at λexcB2, the situation is quite different when λexcB1 is used. In this case, we found a time-dependent emission spectrum that shifts to the blue with time. Our results show that the solvent-mediated proton transfer process displays a fundamental role in the 4-AP emission profile and for the first time a mechanism was proposed that fully explains the 4-AP behavior in every solvent including water. The deuterium isotope effect confirms the assumption because the proton-transfer process is dramatically retarded in this solvent. Consequently, we were able to elucidate not only why in water the emission spectra depend on the excitation wavelength but also why the time-dependent emission spectra shift to the blue with time. Thus, our work reveals the importance that the medium has on the behavior of a widespread dye used as chromophore. This is significant since the use of chromophores without understanding its chemistry can induce artifacts into the interpretation of solvation dynamics in heterogeneous environments, in particular, those provided by aqueous biological systems. Fil: Durantini, Andres Matías. 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: Falcone, Ruben Dario. 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: Anunziata, Jorge 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: Abuin, Elsa B.. Universidad de Santiago de Chile; Chile Fil: Lissi, Eduardo A.. Universidad de Santiago de Chile; Chile Fil: Chessa, Juana Josefa. 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: Correa, Nestor Mariano. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Chemical and structural alterations to lysozyme (LYSO), glucose 6-phosphate dehydrogenase (G6PD), and bovine eye lens proteins (BLP) promoted by peroxyl radicals generated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions were investigated. SDS-PAGE analysis of the AAPH-treated proteins revealed the occurrence of protein aggregation, cross-linking, and fragmentation; BLP, which are naturally organized in globular assemblies, were the most affected proteins. Transmission electron microscopy (TEM) analysis of BLP shows the formation of complex protein aggregates after treatment with AAPH. These structural modifications were accompanied by the formation of protein carbonyl groups and protein hydroperoxides. The yield of carbonyls was lower than that for protein hydroperoxide generation and was unrelated to protein fragmentation. The oxidized proteins were also characterized by significant oxidation of Met, Trp, and Tyr (but not other) residues, and low levels of dityrosine. As the dityrosine yield is too low to account for the observed cross-linking, we propose that aggregation is associated with tryptophan oxidation and Trp-derived cross-links. It is also proposed that Trp oxidation products play a fundamental role in nonrandom fragmentation and carbonyl group formation particularly for LYSO and G6PD. These data point to a complex mechanism of peroxyl-radical mediated modification of proteins with monomeric (LYSO), dimeric (G6PD), and multimeric (BLP) structural organization, which not only results in oxidation of protein side chains but also gives rise to radical-mediated protein cross-links and fragmentation, with Trp species being critical intermediates.

Hypochlorite is a strong oxidant able to induce deleterious effects in biological systems. The goal of this work was to investigate the use of PGR and PYR as probes in assays aimed at evaluating antioxidant activities towards hypochorite and apply it to plant extracts employed in Chilean folk medicine. The consumption of PGR and PYR was evaluated from the decrease in the visible absorbance and fluorescence intensity, respectively. Total phenolic content was determined by the Folin Ciocalteau assay. PGR and PYR react with hypochlorite with different kinetics, being considerably faster the consumption of PGR. Different stoichiometric values were also determined: 0.7 molecules of PGR and 0.33 molecules of PYR were bleached per each molecule of added hypochlorite. Both probes were protected by antioxidants, but the rate of PGR bleaching was too fast to perform a kinetic analysis. For PYR, the protection took place without changes in its initial consumption rate, suggesting a competition between the dye and the antioxidant for hypochlorite. Plant extracts protected PYR giving a PYR-HOCl index that follows the order: Fuchsia magellanica » Marrubium vulgare » Tagetes minuta > Chenopodium ambrosoides » Satureja montana > Thymus praecox. Based on both the kinetic data and the protection afforded by pure antioxidants, we selected PYR as the best probe. The proposed methodology allows evaluating an antioxidant capacity index of plant extracts related to the reactivity of the samples towards hypochlorite.

The quality of wine is mainly determined during the alcoholic fermentation that gradually transforms the grape juice into wine. Along this process the yeast goes through several stressful stages which can affect its fermentative ability and industrial performance, affecting wine quality. Based on their actual application on industrial winemaking, commercial Saccharomyces cerevisiae strains (EC1118, QA23, VIN7 and VL3) were used. They were inoculated in batch laboratory fermentations in a model wine solution for evaluating the production of reactive oxygen species (ROS) during the yeast’s alcoholic fermentation. For first time total hydroperoxides were determined by FOX-1 assay to follow ROS generation. The total hydroperoxides accumulated along the 10 days of fermentation peaked up to 10.0 μM in yeast EC1118, of which 1.3 μM was hydrogen peroxide (H 2 O 2 ). The FOX-1 based analytical approach herein presented is a valuable tool for the quantification of ROS oxidative damage during winemaking.

The osmotic condition modulates the properties of liposomes, particularly those related to their stability and response to external agents such as membrane-active proteins or peptides. In a previous work, we have demonstrated that an osmotic shock can increase, per se, water influx/efflux and the exit of the fluorophore calcein entrapped in the aqueous pool of dipalmitoylphosphatidylcholine (DPPC) and DPPC:sphingomyelin (SM) large unilamellar vesicles (LUVs), suggesting a loss of integrity of the liposome bilayer. In the present work, we have extended our study in order to assess how an osmotic imbalance prior to or synchronous with the addition of a recombinant variant of the pore-forming toxin sticholysin I (rSt I) modifies its pore forming capacity in DPPC and DPPC:SM (1:1) LUVs. Our results conclusively show the capacity of hypotonic gradients to improve the pore forming capacity of rSt I molecules, even in pure DPPC liposomes, rendering pore-formation less dependent on the presence of sphyngomyelin. In fact, non-active toxins in DPPC liposomes become active by a hypotonic imbalance in a similar way to those containing SM as a second component.

Addition of cysteine in the mM range to purified oxyhemoglobin, red blood cell lysate or red blood cell suspensions leads to oxidation of the hemoprotein. The rate and extent of the process depend on the initial hemoglobin and cysteine concentrations, and the reaction is limited by the total destruction of the sulfhydryl groups. Similar results are obtained employing glutathione, but the rate of the process is considerably slower. Oxidation of the purified hemoprotein is faster than in the red blood cell lysate. This difference is mainly due to the inhibitory effect of catalase present in the lysate. Addition of sodium azide increases the rate of oxyhemoglobin oxidation in the lysate, while addition of catalase reduces the rate of oxidation of the purified hemoprotein. The results are interpreted in terms of a mechanism comprising the oxidation of the oxyhemoglobin by the -SH group, with concomitant formation of superoxide anion and hydrogen peroxide. These species further contribute to the oxyhemoglobin oxidation. A chain oxidation of the thiol, catalyzed by the hemoprotein, explains the extensive cysteine destruction.

Comparable intracellular concentrations (≈30pmol/10(6) cells) of bovine serum albumin-ZnPc (BSA) adduct outperformed dipalmitoyl-phosphatidyl-choline (DPPC) liposomes containing ZnPc at photodynamic-killing of human cervical cancer cells (HeLa) after only 15min of irradiation using red light (λ>620nm, 30W/cm(2)). This result could not be simply explained in terms of dye aggregation within the carrier, since in the liposomes the dye was considerably less aggregated than in bovine serum albumin, formulation that was capable to induce cell apoptosis upon red light exposure. Thus, using specific organelle staining, our cumulative data points towards intrinsic differences in intra-cellular localization depending on the cargo vehicle used, being ZnPc:BSA preferentially located in the near vicinity of the nucleus and in the Golgi structures, while the liposomal formulation ZnPc:DPPC was preferentially located in cellular membrane and cytoplasm. In addition to those differences, using real-time advanced fluorescence lifetime imaging of HeLa cells loaded with the photosensitizer contained in the different vehicles, we have found that only for the ZnPc:BSA formulation, there was no significant changes in the fluorescence lifetime of the photosensitizer inside the cells. This contrasts with the in situ≈two-fold reduction of the fluorescence lifetime measured for the liposomal ZnPc formulation. Those observations point towards the superiority of the protein to preserve dye aggregation, and its photochemical activity, post-cell uptake, demonstrating the pivotal role of the delivery vehicle at determining the ultimate fate of a photosensitizer.

On 4 and 8 January 2014, at the height of the austral summer, intense wildfires in forests and dry pastures occurred in the Melipilla sector, located about 70 km to the southwest of Santiago, the Chilean capital, affecting more than 6 million inhabitants. Low level winds transported the forest fire plume towards Santiago causing a striking decrease in visibility and a marked increase in the concentration of both primary (PM10 and CO) and secondary (Ozone) pollutants in the urban atmosphere. In particular, ozone maximum concentrations in the Santiago basin reached hourly averages well above 80 ppb, the national air quality standard. This ozone increase took place at the three sampling sites considered in the present study. These large values can be explained in terms of high NOx concentrations and NO2/NO ratios in biomass burning emissions.

The effect of human serum albumin (HSA) addition on the rate of hydrolysis of the synthetic substrate 4-methylumbelliferyl-β-D-N-N'-N″ triacetylchitotrioside ((NAG)(3)-MUF) catalyzed by hen egg white lysozyme has been measured in aqueous solution (citrate buffer 50 mM pH = 5.2 at 37 °C). The presence of HSA leads to a decrease in the rate of the process. The reaction follows a Michaelis-Menten mechanism under all the conditions employed. The catalytic rate constant decreases tenfold when the albumin concentration increases, while the Michaelis constant remains almost constant in the albumin concentration range employed. Ultracentrifugation experiments indicate that the main origin of the observed variation in the kinetic behavior is related to the existence of an HSA-lysozyme interaction. Interestingly, the dependence of the catalytic rate constant with albumin concentration parallels the decrease of the free enzyme concentration. We interpret these results in terms of the presence in the system of two enzyme populations; namely, the HSA associated enzyme which does not react and the free enzyme reacting as in the absence of albumin. Other factors such as association of the substrate to albumin or macromolecular crowding effects due to the presence of albumin are discarded. Theoretical modeling of the structure of the HSA-lysozyme complex shows that the Glu35 and Asp52 residues located in the active site of lysozyme are oriented toward the HSA surface. This conformation will inactivate lysozyme molecules bound to HSA.

Gradients of HONO, NOx (NO + NO2) and O3 were measured during an early summer campaign on a high-rise building in downtown Santiago de Chile. Distinct gradients of decreasing concentrations with altitude were observed for HONO and NOx, while O3 concentrations were found to increase with altitude. From the observed daytime maximum of the HONO/NOx ratio the existence of a strong daytime source of HONO is proposed, thus confirming recent results from another measurement site in downtown Santiago, where a high contribution of HONO to the OH radical initiation sources of >50% was observed. Since the HONO/NOx ratio and its daytime maximum were found to be independent of the altitude, it is concluded that HONO is an important OH radical source in Santiago not only close to the ground surface, but also at higher altitudes of the boundary layer.

Stichodactyla heliantus II (St II) is a haemolytic toxin whose activity depends of the characteristics of red blood cells (RBC). Among the factors that may tune the response of the RBC to the toxin activity stand the oxidative status of the cell. This study investigates how pre-oxidation of RBC modifies St II activity employing two oxidants, peroxynitrite and hypochlorous acid. Results show that peroxynitrite-treated RBC are more resistant to St II activity. On the other hand, hypochlorous acid-treated RBC become more susceptible to St II. This contrasting behaviour of both oxidants is related to the modifications elicited in RBC by both oxidant agents. Peroxynitrite does not modify RBC osmotic fragility but reduces anion transport through band 3 protein. This effect, together with an increase in K+ efflux, can explain the increased resistance to the toxin activity. On the other hand, results obtained with hypochlorous acid can be explained in terms of a disruption of the membrane organization without the compensating effect of a reduction in band 3-mediated anion transport. The present results, obtained employing the effect of a model haemolytic toxin on RBC, emphasize the specificity of the RBC response to different endogenous oxidative agents.

The protective effect of different antioxidants and complex mixtures on the consumption of pyrogallol red (PGR) induced by peroxyl radicals was studied in the absence and presence of Triton X-100 micelles. The presence of micelles decreased significantly the protection of PGR afforded by lipophilic antioxidants (β-carotene, octyl gallate), while no effect of micelles was observed for hydrophilic antioxidants such as Trolox, caffeic acid, gallic acid, and ascorbic acid. In the presence of complex mixtures a clear effect of Triton X-100 micelles was also observed in the protection afforded by wines, tea infusions, and seed extracts of Eugenia jambolana and Myrciaria cauliflora. On the other hand, no effect of micelles was observed for orange juice and pulp fruit extracts. The ORAC (Oxygen Radical Absorbance Capacity) index was evaluated in the absence (ORAC-PGR) and presence of Triton X-100 micelles (ORAC-PGR(MIC)). Triton X-100 micelles affect ORAC-PGR values of antioxidants in a lipophilicity-dependent way. From the obtained results, we conclude that ORAC-PGR and ORAC-PGR(MIC) assays could be considered as an alternative to estimate the antioxidant ability (ORAC-PGR) and to infer the association to Triton X-100 micelles (ORAC-PGR/ORAC-PGR(MIC)) of pure antioxidants and their complex mixtures.

The photobehavior of merocyanine 540 (MC) was studied in homogeneous media (ethanol, buffer and glycerol), and in microheterogenous systems (Triton X-100 micelles and in the presence of human serum albumin) using stationary and time-resolved techniques. Merocyanine 540 in aqueous solution mostly forms aggregates, which in the presence of Triton X-100 or HSA are disaggregated. The extent of binding to HSA and its characteristics were estimated from dye absorption and fluorescence changes following protein addition; the Trp-214 fluorescence quenching was also employed to assess the extent of dye association, and physical separation was employed to evaluate the dye's apparent association constant. These results showed that dye adsorption on HSA takes place at both main protein-binding sites (I and II). This adsorption leads to dye monomerization, changing its photobehavior remarkably, with a noticeable increase in fluorescence and triplet lifetimes. These slower decays can be ascribed to a reduction of the dye photoisomerization rate. In addition, the adsorption of the dye partially protects it from the oxygen present in solution, thus reducing the apparent dye triplet-quenching rate constant. However, singlet oxygen and MC triplet quantum yields remain very low in all the systems tested. Finally, we found that the photoconsumption of merocyanine bound to HSA takes place predominantly by a type I mechanism, being more than seven times more efficient than that taking place in ethanol.

The city of Santiago, Chile experiences frequent high pollution episodes and as a consequence very high ozone concentrations, which are associated with health problems including increasing daily mortality and hospital admissions for respiratory illnesses. The development of ozone abatement strategies requires the determination of the potential of each pollutant to produce ozone, taking into account known mechanisms and chemical kinetics in addition to ambient atmospheric conditions. In this study, the photochemical formation of ozone during a summer campaign carried out from March 8–20, 2005 has been investigated using an urban photochemical box model based on the Master Chemical Mechanism (MCMv3.1). The MCM box model has been constrained with 10 min averages of simultaneous measurements of HONO, HCHO, CO, NO, j(O1D), j(NO2), 31 volatile organic compounds (VOCs) and meteorological parameters. The O3–NOx–VOC sensitivities have been determined by simulating ozone formation at different VOC and NOx concentrations. Ozone sensitivity analyses showed that photochemical ozone formation is VOC-limited under average summertime conditions in Santiago. The results of the model simulations have been compared with a set of potential empirical indicator relationships including H2O2/HNO3, HCHO/NOy and O3/NOz. The ozone forming potential of each measured VOC has been determined using the MCM box model. The impacts of the above study on possible summertime ozone control strategies in Santiago are discussed.

The reactivity of ethyl-, propyl- and octylgallate towards diphenylpicryl hydracil radicals has been measured in homogeneous solution (ethanol) and in Triton X-100 micellar solutions. Similar reactivities were measured in homogeneous solutions, indicating that the length of the alkyl chain does not influence the reactivity of the phenolic groups. On the other hand, different reactivities were measured in micellar solutions, particularly at low (5 mM) surfactant concentrations. Data obtained at different surfactant concentrations allow estimation of partitioning of the gallates and their intramicellar bimolecular rate constant. The values obtained indicate that all the observed differences in reactivity are attributable to differences in substrate partitioning between the micellar and aqueous pseudophases. In fact, similar values of the intramicellar rate constants were obtained, suggesting that the average location of the reactive groups inside the micelles is independent of the solute alkyl chain length. Copyright © 2009 John Wiley & Sons, Ltd.

Sticholysin II (St II) is a toxin from the sea anemona Stichodactyla helianthus that produces erythrocytes lysis at low concentration and its activity depends on the presence of calcium. Calcium may act modifying toxin interaction with erythrocyte membranes or activating cellular processes which may result in a modified St II lytic action. In this study we are reporting that, in the presence of external K + , extracellular calcium decreased St II activity on erythrocytes. On the other hand an increase of intracellular calcium promotes Sty II lytic activity. The effect of intracellular calcium was specifically studied in relation to membrane lipid translocation elicited by scramblases and how this action influence St II lytic activity on erythrocytes. We used 0.5 mmol/L calcium and 10 mmol/L A23187, as calcium ionophore, for scramblases activation and found increased St II activity associated to increase of intracellular calcium. N -ethyl maleimide (activator) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (inhibitor) were used as scramblases modulators in the assays which produced an increase and a decrease of the calcium effect, respectively. Results reported suggest an improved St II membrane pore-forming capacity promoted by intracellular calcium associated to membrane phospholipids translocation.

The oxidation capacity of the highly polluted urban area of Santiago, Chile has been evaluated during a summer measurement campaign carried out from 8–20 March 2005. The hydroxyl (OH) radical budget was evaluated employing a simple quasi-photostationary-state model (PSS) constrained with simultaneous measurements of HONO, HCHO, O3, NO, NO2, j(O1D), j(NO2), 13 alkenes and meteorological parameters. In addition, a zero dimensional photochemical box model based on the Master Chemical Mechanism (MCMv3.1) has been used to estimate production rates and total free radical budgets, including OH, HO2 and RO2. Besides the above parameters, the MCM model has been constrained by the measured CO and volatile organic compounds (VOCs) including alkanes and aromatics. Both models simulate the same OH concentration during daytime indicating that the primary OH sources and sinks included in the simple PSS model predominate. Mixing ratios of the main OH radical precursors were found to be in the range 0.8–7 ppbv (HONO), 0.9–11 ppbv (HCHO) and 0–125 ppbv (O3). The alkenes average mixing ratio was ~58 ppbC accounting for ~12% of the total identified non-methane hydrocarbons (NMHCs). During the daytime (08:00 h–19:00 h), HONO photolysis was shown to be the most important primary OH radical source comprising alone ~55% of the total initial production rate, followed by alkene ozonolysis (~24%) and photolysis of HCHO (~16%) and O3 (~5%). The calculated average and maximum daytime OH production rates from HONO photolysis was 1.7 ppbv h−1 and 3.1 ppbv h−1, respectively. Based on the experimental results a strong photochemical daytime source of HONO is proposed. A detailed analysis of the sources of OH radical precursors has also been carried out.

Zinc phthalocyanine (ZnPc) is a well known Type II (singlet oxygen mediated) hydrophobic photosensitizer with potential use in PDT. We have found that the presence of bovine serum albumin diminishes the aggregation degree of ZnPc in aqueous solution, indicating that albumins could be potentially useful carriers for this type of photosensitizer in PDT. In order to explore the photochemical and photophysical behavior of ZnPc associated to the protein, we have evaluated triplet excited state lifetime and yield, dye bleaching, oxygen consumption, formation of carbonyls and peroxides, and the spontaneous chemiluminescence emitted after photolysis. The results show that dye association to BSA modifies the photophysics and photochemistry of ZnPC. In particular the decreased yield of long lived triplets suggests singlet state and/or static triplet quenching of the bound dye by the host protein.

Oxygen radical absorbance capacity (ORAC) values have been obtained for a series of teas and herbal infusions employing 2,2′-azo-bis(2-amidinopropane) as free radical source, and fluorescein and pyrogallol red as target molecules. The amounts of phenols in the extracts were evaluated by Folin’s methodology. ORAC values are extremely dependent upon the employed target molecule. Even more, relative ORAC values measured for different infusions depend upon the employed methodology. For example, ORAC-fluorescein value of Aloysia citriodora is larger than that of green tea, while if pyrogallol red is employed as target molecule green tea appears as nearly nine times more efficient. Similarly, for extracts with comparable amounts of phenols, herbal infusions are more efficient than teas by ORAC-fluorescein, while opposite conclusions are obtained if ORAC-pyrogallol red values are considered. Extreme care must then be taken for conclusions obtained from ORAC values estimated by employing a single target molecule.

Advanced glycation endproducts (AGEs) are generated in lens proteins following ascorbic acid glycation. Autosensitized photo-processes induced by these AGEs exposed to UVA-visible light at low oxygen concentrations, such as those found in the eye lens, were studied in order to establish their photo-damaging potential. Ascorbic acid glycated bovine lens proteins, irradiated with UVA and/or visible light at 5% oxygen concentration showed an increase in the amount of carbonyl groups and an increase in the emission of chemiluminescence. Both are indicators of the occurrence of amino acid oxidation within the proteins. A good correlation was found between the chemiluminescence intensity and the extent of the glycation process. No modifications in the SDS-PAGE patterns were observed when 6-day glycated proteins were irradiated with UVA-visible light at low oxygen pressure, excluding the occurrence of photochemical crosslinking. When the same samples were derivatized with 2,4-dinitrophenylhydrazine and analyzed by Western blot, using polyclonal antibodies to the DNP moiety, an increase in the carbonyl content restricted to the AGE-crosslinked proteins fractions was observed. These results indicate that photo-oxidations are limited to the nearby environment of the sensitizers. On the contrary, when native lens proteins were irradiated in the presence of small molecular weight models of AGEs, prepared by incubating ascorbate with Nalpha-acetylated lysine and arginine, the occurrence of both photochemically induced crosslinking and oxidative processes were observed, emphasizing the importance of the localization of the sensitizer. Taken together, these results provide information with respect to the actual photosensitizing ability of endogenous AGEs to induce photo-oxidative processes in lens proteins.

The distribution of urocanic acid (UCA) isomers between aqueous solutions and n-octanol, egg yolk phosphatidylcholine (eggPC) liposomes or bovine serum albumin (BSA) has been evaluated. Regarding its partitioning between water and n-octanol, the behaviour of both isomers is very similar, and the amount incorporated to the organic solvent is mostly determined by the fraction of the compound that, in the aqueous phase, is present as uncharged species. This implies that the highest hydrophobicity occurs near the isoelectric point. cis- and trans-UCA are readily incorporated into eggPC unilamellar liposomes. A simple pseudophase treatment of ultrafiltration data renders a binding constant of 0.20 ± 0.04 mL/mg for the trans isomer at pH 7.4. The binding constant decreases, by a factor two, at pH 5.0, suggesting that the negatively charged species is more favourably bound to the liposomes than the neutral species, which is mostly present as zwitterions. The cis-isomer, at both pHs, is less incorporated to the bilayers. trans-UCA and cis-UCA readily bind to BSA at pH 7.4, with binding constants of 3400 M−1 and 6900 M−1, respectively. This result suggests that, as in the octanol/water partitioning, hydrophobic interactions predominate and the degree of binding is determined by the fraction present as uncharged species. A smaller binding constant at pH 5.0 indicates that the charge of the protein is also plying a relevant role.

The effect of three cationic surfactants bearing the same polar head group and different chain length (cetyltrimethyl ammonium bromide (CTAB); tetradecyltrimethylammonium bromide (TTAB); dodecyltrimethylammonium bromide (DTAB)) on the conformation and function of the sea anemone pore-forming toxins sticholysins I and II (St I and St II) was studied by fluorescence and circular dichroism spectroscopy and evaluation of hemolytic activity (HA). Preincubation of the toxins with the longer chain surfactants CTAB and TTAB at concentrations slightly above their critical micelle concentration (CMC) leads to an enhancement of their HA. Significant increases in the fluorescence intensity with a slightly red shift in lambda(max) were observed at concentrations close to the surfactants' CMC, suggesting changes in the environment of the tryptophan residues. The changes in the fluorescence intensity are more noticeable and take place at lower surfactant concentrations for St I, irrespective of the surfactant alkyl chain length, although the differences between St I and St II increase as the surfactant alkyl chain length increases. This is evinced not only by the higher fluorescence intensity values and the lower surfactant concentrations required to reach them, but also by the higher acrylamide-quenching constant values (Ksv) for St I. However, the surfactant's effects on the toxins' HA were not found to be directly related to the observed changes in fluorescence intensity, as well as near- and far-UV-CD spectra. In particular, the latter spectra indicate that changes in HA and in fluorescence behavior take place without noticeable modifications in St I and St II secondary and tertiary structures. The results suggest that the interaction with the surfactants induces only subtle conformational changes in the toxins that favor the formation of lytic competent structures.

The pyraninoxyl radical is readily formed from the MnO2-promoted oxidation of pyranine. The free radical can be formed in high concentrations (mM), and presents a characteristic EPR spectrum that indicates a high spin-density delocalization. It is relatively stable under nitrogen (half-life ca. 50 min) but readily decays in presence of O2. In spite of its high stability, the radical readily reacts with antioxidants (phenols and ascorbic acid) with a partial recovery of the parent pyranine. High concentrations of the pyraninoxyl radical (ca. 9 μM) are present when pyranine is exposed to a free radical source (10 mM 2,2′-azobis[2-amidinopropane], 37°). The fact that these radicals readily react with antioxidants (ascorbic acid and caffeic acid) supports the proposal that protection by antioxidants of peroxyl radical-promoted pyranine bleaching is mainly due to the occurrence of a repair mechanism.

A comparative study of the photosensitizing activity of advanced glycation endproducts (AGEs) prepared by incubating glucose (Glc), threose (Threo) and ascorbate (AH-) in the presence of lysine (Lys) was performed. Photochemical activity was evaluated under low oxygen pressure with the aim to simulate the conditions of the eye lens. AGE-sensitized tryptophan and AH- photodecomposition and glucose 6-phosphate dehydrogenase inactivation were studied. In all systems, glucose-derived AGEs showed the highest photosensitizing efficiency, followed by ascorbate and threose. The presence of different sensitizers in glycation products mixtures was investigated. For this purpose, Trp decomposition quantum yields were determined at 344 and 367 nm. The values obtained at 344 nm are between three and six times higher than those observed at 367 nm, confirming the presence of at least two compounds with different photosensitizing activities in the mixtures. The chemiluminescence associated with the AGE-mediated oxidation of free Trp and Trp residues in human serum albumin was also studied, and a good correlation between the emission of light and the extent of Trp decomposition was found. In conclusion, it is demonstrated that glucose derived AGEs, which can be formed in vivo in the eye lens of diabetic patients and are accumulated in elderly lenses, have a higher photosensitizing efficiency, at low oxygen pressure, than those arising from ascorbate and threose. This high efficiency is especially significant when proteins are employed as photochemical targets, indicating that protein-sensitizer interaction and the local environment around the sensitizers play an important role.

Low level chemiluminescence (CL) is observed after protein oxidation mediated by singlet oxygen produced in Rose Bengal (RB) irradiation. This CL lasts for several minutes after the end of the photolysis. In this work, the mechanism of the process was assessed from the spectral characteristics of the CL and the effect of antioxidants (Trolox or ascorbate), Ebselen (a compound with peroxidase-like activity), azide (a singlet oxygen scavenger) and D2O, added prior to or after RB irradiation. It is concluded that most of the light emission is due to formation of excited states generated in the decomposition of peroxides and/or hydroperoxides accumulated during the photolysis. Experiments carried out in the presence of several amino acids (Cys, Met, His, Tyr and Trp) and di- and tripeptides suggest that peroxides (and/or hydroperoxides) of Trp residues are mainly responsible for the CL observed after singlet oxygen-mediated protein oxidation. The much weaker CL observed after the oxidation of proteins without Trp residues supports this conclusion. A comparison of the results obtained employing free Trp, Ala-Trp and Trp-Ala dipeptides, Ala-Trp-Ala tripeptide and Trp-containing proteins supports the conclusion that blocking the amino group of the Trp moiety strongly increases the efficiency of the chemiluminescent process, producing ≈2.5×10−8 photons per oxidized Trp group in Ala-Trp. A mechanism comprising two chemiluminescent oxidation pathways of Trp residues is proposed to explain the results.