97 Journal of Protein Chemistry, Vol. 21, No. 2, February 2002 (© 2002) 0277-8033/02/0200-0097/0 © 2002 Plenum Publishing Corporation Aminoglycosides as Substrates and Inhibitors of Peroxid A Possible Role of These Antibiotics against Myeloperoxidase-Dependent Cytotoxicity Anita Lorrai, 1 Alessandra Padiglia, 1 Rosaria Medda, 1 Andrea Bellelli, 2 Alessandro Arcovito, 2 and Giovanni Floris 1,3 Received October 30, 2001 The kinetics of the catalytic cycle of myeloperoxidase and of horseradish peroxidase reacting wit aminoglycosides have been studied by conventional and stopped-flow spectrophotometry. Amino glycosides acted as one-electron reducing substrates converting compound I, formed when chiometric amounts of hydrogen peroxide were added to the enzyme, to compound II, and com- pound II to the resting, ferric enzyme. The latter gradually decayed into a further spect derivative (l max 5 540 and 403 nm) tentatively identified as a complex of ferric heme with the anti- biotic oxidation product(s), and the resulting enzyme was fully inactivated. Since myeloperoxidas is the only human enzyme known to convert chloride ions into the cytotoxic hypochlorous acid, th data presented in this paper bear relevance to the pharmacological effects of aminoglycoside an biotics, which, while inhibiting bacterial growth, also prevent oxidative cellular damage caused b hypochlorous acid aging as substrates and inhibitors of myeloperoxidase. KEY WORDS: Horseradish peroxidase; myeloperoxidase; chloroamines; hypochlorite; oxidants. tabolism of hormones and alkaloids (Rasmussen et al., 1997). The best known of these enzymes is the isoform C of horseradish peroxidase (HRP}C 4 ) (Welinder, 1985; Shiro et al., 1986; Morishima et al., 1986; Smith et al., 1990; Ryan et al., 1994). In animals, peroxidases occur in saliva, tears, milk, and cervical mucus (Taurog et al., 1970; David and Reisfeld, 1974). A particular per- oxidase is the myeloperoxidase, which seems to be pres- ent in all myeloid leucocytes (Maelhly, 1955; Odajima, 1980; Morozov et al., 1997). This enzyme may be in- volved in the generation of oxidants that play key roles in the defense against invading pathogens and in detoxi- fication of bacterial toxins (Maehly, 1955; Hazen et al., 1996a), although its physiological function is not clearly established. At plasma concentration of chloride ions, human myeloperoxidase converts hydrogen peroxide to hypochlorous acid by catalyzing the reaction 1. INTRODUCTION Peroxidases (E.C. 1.11.1.7, donor: hydrogen peroxide oxidoreductase) are hemoproteins containing ferric pro- toporphyrin IX as the prosthetic group that transfer hy- drogen from hydrogen donors to hydrogen peroxide. Per- oxidases are widely distributed and have been isolated from many higher plants, animal tissues, yeast, and mi- croorganisms. Plant peroxidases are involved in regula- tion of cell growth and differentiation, in cell wall ligni- fication, in oxidation of metabolites essential for certain defense reactions and surface protection, and in the me- 1 Department of Sciences Applied to Biosystems, University of Cagliari, Cagliari, Italy. 2 Department of Biochemical Sciences “A. Rossi Fanelli,” University of Rome “La Sapienza,” and CNR Center of Molecular Biology, Rome, Italy. 3 To whom correspondence should be addressed at Dipartimento di Scienze Applicate ai Biosistemi, Città Universitaria, I–09042 Mon- serrato (CA), Italy; e-mail: florisg@unica.it 4 Abbreviations: HRP, horseradish peroxidase; LDL, low density lipoproteins; MPO, myeloperoxidase.