Browsing by Author "Lissi, E"

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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.