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Dive into the research topics where Ana M. Edwards is active.

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Featured researches published by Ana M. Edwards.


Journal of Photochemistry and Photobiology B-biology | 2001

Effect of visible light on selected enzymes, vitamins and amino acids

Ana M. Edwards; Eduardo Silva

In most cases, the presence of an endogenous photosensitizer is a requirement for visible light modification of biomolecules in animal tissues. Riboflavin (RF) is present in all aerobic cells and is a very efficient photosensitizer, presenting a complex photochemistry with a mixed type I-type II mechanisms. Visible light irradiation in the presence of RF diminished the enzymatic activity of horse radish peroxidase (HRP) only when this glyco-enzyme was deglycosilated. In contrast, the activity of catalase is inactivated via singlet oxygen, and that of lysozyme was efficiently inactivated by a mixed type I-type II mechanisms. The reactive species involved in the RF sensitized photoconversion of lysozyme and the aromatic amino acids tryptophan and tyrosine (both free in solution) is discussed. The role of ascorbate and the effect of RF photosensitized processes in biological complex systems, such as the ocular lens and tumoral cell in culture, is analyzed.


Journal of Photochemistry and Photobiology B-biology | 1994

Riboflavin-sensitized photoprocesses of tryptophan

Eduardo Silva; R.A. Ugarte; A. Andrade; Ana M. Edwards

The photodegradation of tryptophan in oxygen saturated aqueous solution sensitized by riboflavin is accompanied by the generation of the following reactive oxygen species: 1O2, OH., H2O2 and O2.-. When parallel photodegradation experiments are run with 14C-riboflavin in one case and 14C-tryptophan in the other and the irradiation products are separated by Sephadex G-15 and C18-HPLC, the generation of the following species is detected: aggregate forms of riboflavin, indolic products associated to flavins, indolic products of molecular weight higher than tryptophan, formylkynurenine, and other tryptophan photodecomposition products. The significance of the riboflavin anion radical and tryptophan cation radical as intermediates in the photoproduct formation is discussed.


Journal of Photochemistry and Photobiology B-biology | 1994

Visible light effects on tumoral cells in a culture medium enriched with tryptophan and riboflavin

Ana M. Edwards; Eduardo Silva; B. Jofré; Marı́a Inés Becker; A. de Ioannes

When NSO/2 myeloid cell line and teratocarcinoma F9 cells were irradiated in Dulbeccos modified Eagle medium enriched with tryptophan and riboflavin, toxic photoproducts for these tumoral cells were generated. The active participation of 1O2 and .OH was established using specific scavengers and quenchers. A cytotoxic effect was also observed when unirradiated tumoral cells were incubated in a previously irradiated culture medium enriched with tryptophan and riboflavin. When irradiated medium was used alone, enriched only with tryptophan or only with riboflavin, no toxic effect was observed. The relevance of charge transfer processes between triplet riboflavin and tryptophan in the generation of cytotoxic photoproducts is discussed.


Photochemical and Photobiological Sciences | 2009

Photophysics and photochemistry of rose bengal bound to human serum albumin

Emilio Alarcón; Ana M. Edwards; Alexis Aspée; Claudio D. Borsarelli; Eduardo Lissi

Rose bengal (RB) readily binds to human serum albumin (HSA). At low RB concentrations, 90% of the dye is associated to the protein (5 microM), This association takes place in specific binding sites I and/or II. At higher RB concentrations, unspecific binding takes place with up to 10 RB molecules bound per protein molecule. The behavior of excited RB molecules bound to HSA is widely different to that observed in aqueous solution. Furthermore, the data also show that the behavior of bound RB molecules changes with the average number of dye molecules per protein (n). In particular, when n is large, the fluorescence yield is significantly reduced and no measurable long-lived triples and free singlet oxygen formation from bound dyes is detected. These results are related to self-quenching of the singlet and, most likely, excited triplets. All results point to the relevance of intra-protein generated singlet oxygen. However, when the dye is bound to the protein, at low oxygen concentrations such as those prevailing in vivo, trapping by oxygen of the triplet becomes inefficient and type I processes could contribute to the observed photoprocesses.


Journal of Nutritional Biochemistry | 1999

Visible light-induced photooxidation of glucose sensitized by riboflavin

Eduardo Silva; Ana M. Edwards; Danilo Pacheco

We conducted this study to evaluate the oxidation of glucose induced by visible light in the presence of sensitizers such as methylene blue and flavins (i.e., flavin mononucleotide and riboflavin). The concentration of the sensitizers was similar to that of flavin in parenteral nutrients. The photooxidation of glucose sensitized by flavin mononucleotide or riboflavin was greater than that which was observed in the presence of methylene blue, whereas the isotopic effect of deuterium oxide (D(2)O) was enhanced more substantially in the presence of methylene blue than in the presence of flavins. These results show that methylene blue exerts its action through singlet oxygen and that at a high substrate concentration (as was used in this work) flavin mononucleotide and riboflavin act preferentially as type I sensitizers. In the flavin photosensitized processes, the presence of hydrogen peroxide, superoxide anion, and hydroxyl radical was demonstrated. The photooxidation of glucose is favored by an increase in pH, and it also depends on the energy absorbed by the system. By using a specific reagent for glucose (i.e., o-toluidine), it was possible to quantify the photoconversion of glucose. The results obtained in this work should be considered in the management of glucose-containing parenteral nutrients that are exposed to visible light in the presence of a multivitamin complex containing flavin mononucleotide.


Photochemical and Photobiological Sciences | 2009

Photophysics and photochemistry of zinc phthalocyanine/bovine serum albumin adducts

Emilio Alarcón; Ana M. Edwards; Angélica M. Garcia; Marcelo Muñoz; Alexis Aspée; Claudio D. Borsarelli; Eduardo Lissi

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.


Photochemistry and Photobiology | 2003

Study of the Interaction Between Triplet Riboflavin and the α-, βH- and βL-Crystallins of the Eye Lens¶

Gabriela Viteri; Ana M. Edwards; Julio R. De la Fuente; Eduardo Silva

Abstract Time-resolved photolysis studies of riboflavin (RF) were carried out in the presence and absence of α-, βH- and βL-crystallins of bovine eye lens. The transient absorption spectra, recorded 5 μs after the laser pulse, reveal the presence of the absorption band (625–675 nm) of the RF neutral triplet state (τ = 42 μs) accompanied by the appearance of a long-lived absorption (τ = 320 μs) in the 500–600 nm region due to the formation of the semireduced RF radical. The RF excited state is quenched by the crystallin proteins through a mechanism that involves electron transfer from the proteins to the flavin, as shown by the decrease of the triplet RF band with the concomitant increase of the band of its semireduced form. Tryptophan loss on RF-sensitized photooxidation of the crystallins when irradiated with monochromatic visible light (450 nm) in a 5% oxygen atmosphere was studied. A direct correlation was found between the triplet RF quenching rate constants by the different crystallin fractions and the decomposition rate constants for the exposed and partially buried tryptophans in the proteins. The RF-sensitized photooxidation of the crystallins is accompanied by the decrease of the low molecular weight constituents giving rise to its multimeric forms. A direct correlation was observed between the initial rate of decrease of the low molecular weight bands corresponding to the irradiated α-, βH- and βL-crystallins and the quenching constant values of triplet RF by the different crystallins. The correlations found in this study confirm the importance of the Type-I photosensitizing mechanism of the crystallins, when RF acts as a sensitizer at low oxygen concentration, as can occur in the eye lens.


Journal of Photochemistry and Photobiology B-biology | 2000

Lysozyme photo-oxidation by singlet oxygen: properties of the partially inactivated enzyme.

Eduardo Silva; Constanza De Landea; Ana M. Edwards; Eduardo Lissi

This work studies the behaviour of partially inactivated lysozyme formed by the effect of singlet oxygen, which was obtained through the irradiation of the native enzyme solution with polychromatic visible light using Methylene Blue as a sensitizer. The polyacrylamide gel analysis of the partially inactivated lysozyme solution shows the presence of different protein fractions. One of them, which corresponds to 53% of the original enzyme, has the same migration as the native enzyme. The others are a mixture of fractions (47%) that show slower migration to the cathode. When this experiment is carried out in the presence of sodium dodecyl sulfate, only one fraction is obtained, which rules out the presence of covalently aggregated forms of lysozyme. The partially inactivated lysozyme has lost 74% of the fluorescence emission of the tryptophan (Trp) residues. By using the anionic quencher iodide, it is determined that 45 and 36% of the fluorescence emission arising from the native and partially inactivated enzyme, respectively, are due to Trp residues exposed to the solvent. Michaelis-Menten constants (K(in)) of 0.296 and 0.511 (mg/ml) and maximum initial rates (Vmax) of 0.295 and 0.190 (mg/ml min) are determined for the native and the partially inactivated enzyme solutions, respectively. The same inactivation profile is found when the denaturing effect of increasing urea concentration on both the native and partially inactivated lysozyme is studied. It is postulated that the partially inactivated lysozyme solution is composed of one protein fraction with enzymatic activity similar to that of the native enzyme and also of a mixture of fractions (47% of the total enzyme) with very low activity and characterized by a high tryptophan photo-oxidation.


Photochemical and Photobiological Sciences | 2010

Photobehavior of merocyanine 540 bound to human serum albumin

Emilio I. Alarcon; Alexis Aspée; María González-Béjar; Ana M. Edwards; Eduardo Lissi; J. C. Scaiano

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 dyes 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.


Photochemistry and Photobiology | 2004

Enhancement of Riboflavin-mediated Photo-Oxidation of Glucose 6-phosphate Dehydrogenase by Urocanic Acid¶

Eduardo Silva; L Herrera; Ana M. Edwards; Julio R. De la Fuente; Eduardo Lissi

Abstract We have investigated the riboflavin (RF)-sensitized inactivation of glucose 6-phosphate dehydrogenase (G6PD) in the presence and absence of trans-urocanic acid (UCA). The inactivation of the enzyme results from its direct oxidation by the excited triplet RF in a Type-I–photosensitized reaction whose efficiency increases at low oxygen concentration. The addition of histidine to the system produced no change in the inactivation rate, discarding the participation of singlet oxygen in the reaction. On the other hand, the presence of UCA results in its bleaching, with a significant enhancement of RF-mediated inactivation of G6PD. Both the consumption of UCA and G6PD are faster at low oxygen concentrations. UCA also produced a decrease in the sensitizer photodecomposition yield. These results indicate that the enhancement of the RF-mediated G6PD inactivation observed in the presence of UCA is not a singlet oxygen–mediated process. It is proposed that UCA consumption and its effect on G6PD inactivation are due to a complex reaction sequence initiated by a direct oxidation of UCA by the excited sensitizer triplet. The oxidation of the semireduced flavin gives rise to reactive oxygen species (ROS) responsible for the increased rate of the process. This is supported by the protection afforded by several additives with ROS removal capacity: benzoate, superoxide dismutase and catalase.

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Eduardo Silva

Pontifical Catholic University of Chile

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Emilio Alarcón

Pontifical Catholic University of Chile

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Marcelo Muñoz

Pontifical Catholic University of Chile

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Alfredo E. De Ioannes

Pontifical Catholic University of Chile

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Angélica M. Garcia

Pontifical Catholic University of Chile

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Marı́a Inés Becker

Pontifical Catholic University of Chile

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Claudio D. Borsarelli

National Scientific and Technical Research Council

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A. de Ioannes

Pontifical Catholic University of Chile

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