Mechanistic studies of peroxynitrite-mediated tyrosine nitration in membranes using the hydrophobic probe N-t-BOC-L-tyrosine tert-butyl ester

被引:70
作者
Bartesaghi, Silvina
Valez, Valeria
Trujillo, Madia
Peluffo, Gonzalo
Romero, Natalia
Zhang, Hao
Kalyanaraman, Balaraman
Radi, Rafael [1 ]
机构
[1] Univ Republica, Fac Med, Dept Bioquim, Montevideo 11800, Uruguay
[2] Univ Republica, Fac Med, Ctr Free Rad & Biomed Res, Montevideo 11800, Uruguay
[3] Med Coll Wisconsin, Biophys Res Inst, Milwaukee, WI 53226 USA
[4] Med Coll Wisconsin, Free Rad Res Ctr, Milwaukee, WI 53226 USA
关键词
D O I
10.1021/bi060363x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 [生物化学与分子生物学]; 081704 [应用化学];
摘要
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 dilauroy-land dimyristoyl-PC and were also significant in the polyunsaturated fatty acid-containing egg PC. (OH)-O-center dot and (NO2)-N-center dot scavengers inhibited BTBE nitration. In contrast to tyrosine in the aqueous phase, the presence of CO2 decreased BTBE nitration, indicating that CO3 center dot- 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.
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页码:6813 / 6825
页数:13
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