title: Mechanistic studies of peroxynitrite-mediated tyrosine nitration in membranes using the hydrophobic probe N-t-BOC-L-tyrosine tert-butyl ester.
abstract: Most of the mechanistic studies of tyrosine nitration have been performed in aqueous solution. However, many protein tyrosine residues shown to be nitrated in vitro and in vivo are associated to nonpolar compartments. In this work, we have used the stable hydrophobic tyrosine analogue N-t-BOC-L-tyrosine tert-butyl ester (BTBE) incorporated into phosphatidylcholine (PC) liposomes to study physicochemical and biochemical factors that control peroxynitrite-dependent tyrosine nitration in phospholipid bilayers. Peroxynitrite leads to maximum 3-nitro-BTBE yields (3%) at pH 7.4. In addition, small amounts of 3,3'-di-BTBE were formed at pH 7.4 (0.02%) which increased over alkaline pH; at pH 6, a hydroxylated derivative of BTBE was identified by HPLC-MS analysis. BTBE nitration yields were similar in dilauroyl- and dimyristoyl-PC and were also significant in the polyunsaturated fatty acid-containing egg PC. *OH and *NO2 scavengers inhibited BTBE nitration. In contrast to tyrosine in the aqueous phase, the presence of CO2 decreased BTBE nitration, indicating that CO3*- cannot permeate to the compartment where BTBE is located. On the other hand, micromolar concentrations of hemin and Mn-tccp strongly enhanced BTBE nitration. Electron spin resonance (ESR) detection of the BTBE phenoxyl radical and kinetic modeling of the pH profiles of BTBE nitration and dimerization were in full agreement with a free radical mechanism of oxidation initiated by ONOOH homolysis in the immediacy of or even inside the bilayer and with a diffusion coefficient of BTBE phenoxyl radical 100 times less than for the aqueous phase tyrosyl radical. BTBE was successfully applied as a hydrophobic probe to study nitration mechanisms and will serve to study factors controlling protein and lipid nitration in biomembranes and lipoproteins.
authors.affiliation: Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay.
authors.forename: Silvina
authors.initials: S
authors.lastname: Bartesaghi
authors.forename: Valeria
authors.initials: V
authors.lastname: Valez
authors.forename: Madia
authors.initials: M
authors.lastname: Trujillo
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Peluffo
authors.forename: Natalia
authors.initials: N
authors.lastname: Romero
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: Balaraman
authors.initials: B
authors.lastname: Kalyanaraman
authors.forename: Rafael
authors.initials: R
authors.lastname: Radi
mesh_terms: D002245:Carbon Dioxide; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D005231:Fatty Acids, Unsaturated / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D008566:Membranes / Q000737:chemistry; D015335:Molecular Probes / Q000737:chemistry*; D009584:Nitrogen / Q000737:chemistry; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry; D013057:Spectrum Analysis; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry*
chemical_list: C512163:3,3'-di-(N-t-butoxycarbonyl-tyrosine tert-butyl ester); D005231:Fatty Acids, Unsaturated; D008051:Lipid Bilayers; D008081:Liposomes; D015335:Molecular Probes; C434144:N-t-BOC-tyrosine-t-butyl ester; C512162:N-t-butoxycarbonyl-3-nitrotyrosine tert-butyl ester; D010713:Phosphatidylcholines; D002245:Carbon Dioxide; D030421:Peroxynitrous Acid; D014443:Tyrosine; D009584:Nitrogen