title: A kinetic model for the competitive reactions of ozone with amino acid residues in proteins in reverse micelles.
abstract: Lysozyme and 10 other proteins are solubilized in reverse micelles formed by 0.1 M sodium di-2-ethyl-hexylsulfosuccinate and 2.0-2.5 M water (pH 7.4) in isooctane solvent. Exposure of the protein-containing reverse micellar solutions to ozone causes oxidative damage to the proteins, as assessed by the oxidation of tryptophan residues. The oxidation product of the protein-bound tryptophan has a molar absorption coefficient of 3275 +/- 81 M-1 cm-1 (mean +/- S.D., n = 6) at 320 nm. The product is suggested to be a Criegee ozonide or a tautomer of the Criegee ozonide and not N-formylkynurenine. Ozonation of lysozyme in reverse micelles results in the formation of hydrogen peroxide in yields of only approximately 0.07 mol/mol of tryptophan residues oxidized. The recovery of hydrogen peroxide added as an internal standard to the lysozyme-containing reverse micellar solutions ranges from 84 to 88%, whether or not the samples are subjected to ozonation. This suggests that hydrogen peroxide is neither destroyed during the process of ozonation nor consumed by the protein to a significant extent in an adventitious reaction. A kinetic model for the overall reaction of ozone with the proteins is developed, taking into account the concentrations and the reactivities of individual amino acid residues toward ozone. The model predicts the fractional reaction of ozone with tryptophan residues in the proteins, despite differences in amino acid composition, molecular weight, and tertiary structures. The lack of influence of protein structure is confirmed further by the observation that the native lysozyme (with and without external S-carboxymethylcysteine) and S-carboxymethylated lysozyme give identical values of the fractional reaction of ozone with tryptophan residues. The kinetic equations for the competitive reactions of ozone with amino acid residues in proteins, with some minor modification, are applicable to ozonations on complex mixtures of lipids, proteins, and antioxidants.
authors.affiliation: Biodynamics Institute, Louisiana State University, Baton Rouge 70803-1800.
authors.forename: W A
authors.initials: WA
authors.lastname: Pryor
authors.forename: R M
authors.initials: RM
authors.lastname: Uppu
mesh_terms: D000596:Amino Acids; D004143:Dioctyl Sulfosuccinic Acid; D007700:Kinetics; D008433:Mathematics; D008823:Micelles; D008962:Models, Theoretical; D009113:Muramidase / Q000737:chemistry*; D010126:Ozone; D011506:Proteins / Q000737:chemistry*; D013501:Surface-Active Agents; D014364:Tryptophan
chemical_list: D000596:Amino Acids; D008823:Micelles; D011506:Proteins; D013501:Surface-Active Agents; D004143:Dioctyl Sulfosuccinic Acid; D010126:Ozone; D014364:Tryptophan; D009113:Muramidase