Faculty Collaboration Database

Mechanistic studies of peroxynitrite-mediated tyrosine nitration in membranes using the hydrophobic probe N-t-BOC-L-tyrosine tert-butyl ester. Biochemistry 2006 Jun 06;45(22):6813-25

Date

06/01/2006

Pubmed ID

16734418

DOI

10.1021/bi060363x

Scopus ID

2-s2.0-33744959547 (requires institutional sign-in at Scopus site)   77 Citations

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.

Author List

Bartesaghi S, Valez V, Trujillo M, Peluffo G, Romero N, Zhang H, Kalyanaraman B, Radi R

Author

Balaraman Kalyanaraman PhD Professor in the Biophysics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Carbon Dioxide
Chromatography, High Pressure Liquid
Dimerization
Fatty Acids, Unsaturated
Hydrophobic and Hydrophilic Interactions
Lipid Bilayers
Liposomes
Membranes
Molecular Probes
Nitrogen
Oxidation-Reduction
Peroxynitrous Acid
Phosphatidylcholines
Spectrum Analysis
Tyrosine