Browsing by Author "Edwards, AM"

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    Lysozyme modification by the Fenton reaction and gamma radiation
    (TAYLOR & FRANCIS LTD, 2002) Edwards, AM; Ruiz, M; Silva, E; Lissi, E
    A comparative study was performed on lysozyme modification after exposure to Fenton reagent (Fe(E)/H2O2) or hydroxyl radicals produced by gamma radiation. The conditions were adjusted to obtain, with both systems, a 50% loss of activity of the modified ensemble. gamma radiation modified almost all types of amino acid residues in the enzyme, with little specificity. The modification order was Tyr >Met = Cys > Lys > Ile + Leu > Gly > Pro = Phe > Thr + Ala > Trp = Ser > Arg > Asp + Glu, with 42 mol of modified residues per initial mole of native enzyme. In contrast, when the enzyme was exposed to the Fenton reaction, only some types of amino acids were modified. Furthermore, a smaller number of residues (13.5) were damaged per initial mole of enzyme. The order of the modified residues was Tyr > Cys > Trp > Met > His > Ile + Leu > Val > Arg. These results demonstrate that the modifications elicited by these two free radical sources follow different mechanisms. An intramolecular free radical chain reaction is proposed to play a dominant role in the oxidative modification of the protein promoted by gamma radiation.
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    Lysozyme photo-oxidation by singlet oxygen: properties of the partially inactivated enzyme
    (ELSEVIER SCIENCE SA, 2000) Silva, E; De Landea, C; Edwards, AM; Lissi, E
    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-m) of 0.296 and 0.511 (mg/ml) and maximum initial rates (V-max) 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. (C) 2000 Elsevier Science S.A. All rights reserved.
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    Visible light induced lipoperoxidation of a parenteral nutrition fat emulsion sensitized by flavins
    (ELSEVIER SCIENCE INC, 1998) Silva, E; Gonzalez, T; Edwards, AM; Zuloaga, F
    The photosensitizing effects of riboflavin, flavin mononucleotide, flavin adenine dinucleotide,and a commercial multivitamin infusate on a lipid emulsion used in parenteral nutrient infusions were studied during exposure to polychromatic visible light. It was found that the efficiency of riboflavin (a polar flavin) as sensitizer of lipid emulsion peroxidation is greater than that of flavin mononucleotide (an ionic flavin). This was determined by measuring the consumption of molecular oxygen and the concentration of thiobarbituric acid-reactive species generated during the irradiation time. These findings are supported by molecular orbital studies of these molecules related to polarity, ionic charges around the different molecular regions, and electrostatic potentials comparisons. Flavin adenine dinucleotide (a more ionic flavin), most likely remains totally excluded from the lipid emulsion due to its polarity and molecular geometry and does not induce lipid peroxidation. The multivitamine complex seems to provide a protective effect on the lipid emulsion exposed to light, attributed to the presence of ascorbic acid, which suffers an intensive photodecomposition. A solution of vitamin C, whose concentration was equivalent to that of the parenteral mixture, consumes an considerable amount of molecular oxygen when it is irradiated with visible light in the presence of flavin mononucleotide. (C) Elsevier Science Inc. 1998.
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    Visible light-induced photooxidation of glucose sensitized by riboflavin
    (ELSEVIER SCIENCE INC, 1999) Silva, E; Edwards, AM; Pacheco, D
    Ne 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 (D2O) 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 las 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 risible light in the presence of a multivitamin complex containing flavin mononucleotide. (C) Elsevier Science Inc. 1999. All rights reserved.