Lipid peroxyl radicals mediate tyrosine dimerization and nitration in membranes

Article abstract of DOI:10.1021/tx900446r

Protein tyrosine dimerization and nitration by biologically relevant oxidants usually depend on the intermediate formation of tyrosyl radical ( ^·Tyr). In the case of tyrosine oxidation in proteins associated with hydrophobic biocompartments, the participation of unsaturated fatty acids in the process must be considered since they typically constitute preferential targets for the initial oxidative attack. Thus, we postulate that lipid-derived radicals mediate the one-electron oxidation of tyrosine to ^·Tyr, which can afterward react with another ^·Tyr or with nitrogen dioxide (^·NO ₂) to yield 3,3′-dityrosine or 3-nitrotyrosine within the hydrophobic structure, respectively. To test this hypothesis, we have studied tyrosine oxidation in saturated and unsaturated fatty acid-containing phosphatidylcholine (PC) liposomes with an incorporated hydrophobic tyrosine analogue BTBE (N-t-BOC l-tyrosine tert-butyl ester) and its relationship with lipid peroxidation promoted by three oxidation systems, namely, peroxynitrite, hemin, and 2,2′-azobis (2-amidinopropane) hydrochloride. In all cases, significant tyrosine (BTBE) oxidation was seen in unsaturated PC liposomes, in a way that was largely decreased at low oxygen concentrations. Tyrosine oxidation levels paralleled those of lipid peroxidation (i.e., malondialdehyde and lipid hydroperoxides), lipid-derived radicals and BTBE phenoxyl radicals were simultaneously detected by electron spin resonance spin trapping, supporting an association between the two processes. Indeed, α-tocopherol, a known reactant with lipid peroxyl radicals (LOO^·), inhibited both tyrosine oxidation and lipid peroxidation induced by all three oxidation systems. Moreover, oxidant-stimulated liposomal oxygen consumption was dose dependently inhibited by BTBE but not by its phenylalanine analogue, BPBE (N-t-BOC l-phenylalanine tert-butyl ester), providing direct evidence for the reaction between LOO^· and the phenol moiety in BTBE, with an estimated second-order rate constant of 4.8 - 10³ M¹ s^-1. In summary, the data presented herein demonstrate that LOO ^· mediates tyrosine oxidation processes in hydrophobic biocompartments and provide a new mechanistic insight to understand protein oxidation and nitration in lipoproteins and biomembranes.

Full text of DOI:10.1021/tx900446r

Chem. Res. Toxicol. 2010, 23, 821–835
821
Lipid Peroxyl Radicals Mediate Tyrosine Dimerization and
Nitration in Membranes
Silvina Bartesaghi,†,‡,§ Jorge Wenzel,‡,§ Madia Trujillo,‡,§ Marcos Lo´pez,| Joy Joseph,|
Balaraman Kalyanaraman,| and Rafael Radi*,‡,§
Departamento de Histolog´ıa y Embriolog´ıa, Departamento de Bioqu´ımica, and Center for Free Radical and
Biomedical Research, Facultad de Medicina, UniVersidad de la Repu´blica, AVda. General Flores 2125, 11800
MonteVideo, Uruguay, and Department of Biophysics and Free Radical Research Center, Medical College of
Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226
ReceiVed December 16, 2009
Protein tyrosine dimerization and nitration by biologically relevant oxidants usually depend on the
intermediate formation of tyrosyl radical (•Tyr). In the case of tyrosine oxidation in proteins associated
with hydrophobic biocompartments, the participation of unsaturated fatty acids in the process must be
considered since they typically constitute preferential targets for the initial oxidative attack. Thus, we
•
postulate that lipid-derived radicals mediate the one-electron oxidation of tyrosine to Tyr, which can
afterward react with another •Tyr or with nitrogen dioxide (•NO2) to yield 3,3′-dityrosine or 3-nitrotyrosine
within the hydrophobic structure, respectively. To test this hypothesis, we have studied tyrosine oxidation
in saturated and unsaturated fatty acid-containing phosphatidylcholine (PC) liposomes with an incorporated
hydrophobic tyrosine analogue BTBE (N-t-BOC L-tyrosine tert-butyl ester) and its relationship with lipid
peroxidation promoted by three oxidation systems, namely, peroxynitrite, hemin, and 2,2′-azobis (2-
amidinopropane) hydrochloride. In all cases, significant tyrosine (BTBE) oxidation was seen in unsaturated
PC liposomes, in a way that was largely decreased at low oxygen concentrations. Tyrosine oxidation
levels paralleled those of lipid peroxidation (i.e., malondialdehyde and lipid hydroperoxides), lipid-derived
radicals and BTBE phenoxyl radicals were simultaneously detected by electron spin resonance spin
trapping, supporting an association between the two processes. Indeed, R-tocopherol, a known reactant
with lipid peroxyl radicals (LOO•), inhibited both tyrosine oxidation and lipid peroxidation induced by
all three oxidation systems. Moreover, oxidant-stimulated liposomal oxygen consumption was dose
dependently inhibited by BTBE but not by its phenylalanine analogue, BPBE (N-t-BOC L-phenylalanine
tert-butyl ester), providing direct evidence for the reaction between LOO• and the phenol moiety in BTBE,
with an estimated second-order rate constant of 4.8 × 103 M-1 s-1. In summary, the data presented
herein demonstrate that LOO• mediates tyrosine oxidation processes in hydrophobic biocompartments
and provide a new mechanistic insight to understand protein oxidation and nitration in lipoproteins and
biomembranes.
intermediates both in vitro and in vivo. These tyrosine oxidation
processes depend on the intermediate formation of tyrosyl
radical (•Tyr), a transient species formed by the one-electron
oxidation of tyrosine. For instance, 3,3′-dityrosine formation
results from the termination reaction between two •Tyr radicals
with the formation of a new C-C bond; 3,3′-dityrosine
participates in protein cross-linking (1) and also serves as a
marker for oxidatively damaged proteins. Indeed, elevated levels
of 3,3′-dityrosine can be found as a product of aging, inflam-
mation, exposure to UV and γ-radiation, and other oxidative
stress conditions (2–4). Tyrosine nitration in biological systems
is also a free radical process (5) produced by nitric oxide (•NO)-
derived oxidants such as peroxynitrite2 and nitrogen dioxide
radical (•NO2) (5); typically, the final step in nitration involves
the diffusion-controlled reaction of •Tyr with •NO2. The product
of this reaction, 3-nitro-Tyr, is a footprint of nitro-oxidative
damage in vivo, being revealed as a strong biomarker and
predictor of disease progression in conditions such as inflam-
mation, cardiovascular disease, and neurodegeneration (6–9).
Introduction
Tyrosine dimerization and nitration to 3,3′-dityrosine and
3-nitrotyrosine (3-nitro-Tyr),1 respectively, represent biologically
relevant oxidative post-translational modifications in proteins
generated by the reactions with reactive oxygen and nitrogen
* To whom correspondence should be addressed. Tel: +598-2-9249561.
Fax: +598-2-9249563. E-mail: rradi@fmed.edu.uy.
† Departamento de Histolog´ıa y Embriolog´ıa, Facultad de Medicina,
Universidad de la Repu´blica.
‡ Departamento de Bioqu´ımica, Facultad de Medicina, Universidad de
la Repu´blica.
§ Center for Free Radical and Biomedical Research, Facultad de
Medicina, Universidad de la Repu´blica.
| Medical College of Wisconsin.
1 Abbreviations: BTBE, N-t-BOC L-tyrosine tert butyl ester; BPBE, N-t-
BOC L-phenylalanine tert butyl ester; DLPC, 1,2-dilauroyl-sn-glycero-3-
phosphocholine; PLPC, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocho-
line; EYPC, egg chicken yolk L-R-phosphatidylcholine; SBPC, soybean-
L-R-phosphatidylcholine; dtpa, diethylentriaminepentaacetic acid; •NO, nitric
oxide; O2•-, superoxide; 3-nitro-Tyr, 3-nitrotyrosine; Tyr, tyrosyl radical;
•
PC, phosphatidylcholine; RP-HPLC, reverse-phase high-performance liquid
chromatography; ABAP, 2,2′-azobis (2-amidinopropane) hydrochloride;
MDA, malondialdehyde; FOX, ferrous oxide-xylenol orange assay; LOO•,
lipid peroxyl radical; LO•, lipid alkoxyl radical; TBARS, thiobarbituric acid
reactive species; ESR, electron spin resonance; BHT, butylated hydroxy-
toluene; MNP, 2-methyl nitrosopropane; BOC, butoxypyrocarbonate.
2 IUPAC-recommended names for peroxynitrite anion (ONOO-) and
peroxynitrous acid (ONOOH) (pKa ) 6.8) are oxoperoxonitrate (1-) and
hydrogen oxoperoxonitrate, respectively. The term peroxynitrite is used to
refer to the sum of ONOO- and ONOOH.
10.1021/tx900446r  2010 American Chemical Society
Published on Web 02/19/2010

822 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Bartesaghi et al.
Protein tyrosine nitration could result in dramatic changes in
protein structure and can affect biological activity either by a
loss [e.g., Mn-SOD (10–12)] or by a gain of function [e.g., nerve
growth factor (13) and cytochrome c (14, 15); recently reviewed
in ref 16].
glutathione (22). In addition, there is a differential distribution
of oxidizing species in the lipid vs aqueous phase: While •NO2
can readily diffuse, concentrate, and react in the hydrophobic
compartment (40), CO3•- and hemeproteins have limited action
due to the restricted permeation and steric restrictions, respec-
tively. Another important aspect to consider is the restricted
lateral diffusion of •Tyr in the organized structure of membranes,
which limits the dimerization process and results in smaller 3,3′-
dityrosine/3-nitro-Tyr ratios than those observed in aqueous
phases (∼1/100-1/400) (41).
As 3,3′-dityrosine and 3-nitro-Tyr formation require the
•
intermediacy of Tyr, both tyrosine oxidation products can be
•
formed simultaneously in oxidizing environments where NO
and reactive oxygen intermediate radicals (e.g., superoxide
radical, O2•-, hydrogen peroxide, H2O2) coexist. Specifically,
this chemistry can be performed by peroxynitrite, a powerful
oxidant and cytotoxic species formed in vivo by the diffusion-
The need to further investigate tyrosine oxidation mechanisms
in hydrophobic environments has led to the development of
probes such as hydrophobic tyrosine analogues (41, 42) and
tyrosine-containing transmembrane peptides (42). In this regard,
N-t-BOC L-tyrosine tert-butyl ester (BTBE) is a stable tyrosine
analogue that we have previously used to study peroxynitrite
and MPO-mediated tyrosine oxidation in lipid phases including
liposomes and biomembranes (41–43). BTBE can be efficiently
incorporated (>98%) into phosphatidylcholine (PC) liposomes,
and the formation of BTBE-derived phenoxyl radicals and
oxidation products (i.e., 3-nitro-BTBE, 3,3′-di-BTBE, and
3-hydroxy-BTBE) has been evaluated (41–43). Previous ob-
servations (41, 43) and kinetic considerations prompted us to
investigate in depth how the unsaturated fatty acids present in
high concentrations in membranes and other hydrophobic
compartments may influence peroxynitrite-mediated tyrosine
oxidation mechanisms and yields. Indeed, peroxynitrite is a
known inductor of lipid peroxidation via free radical reactions
(44) initiated after the homolysis of ONOOH to •OH and •NO2
(21); unsaturated fatty acids readily react with •OH
(k ) 1 × 1010 M-1 s-1) (45) and to a lesser extent with •NO2 (k
) 2 × 105 M-1 s-1 for linoleate at pH 9.4) (46, 47), both of
which promote hydrogen abstraction from a bis-allylic hydrogen
to initiate a chain reaction. Therefore, at a first glance, an
increased level of unsaturation in PC liposomes could result in
a decrease of tyrosine oxidation yields. However, peroxynitrite-
dependent BTBE nitration and dimerization yields were still
high in PC liposomes containing a large polyunsaturated fatty
acid content (41). These data indicate that a simple competition
kinetic model does not apply and suggest that lipid-derived
radicals mediate tyrosine oxidation within the membrane.
Indeed, lipid peroxidation is an oxygen-dependent process that
results in the formation of peroxyl (LOO•) (eqs 1-3) and,
secondarily, alkoxyl radicals (LO•) (48–50).
•
•-
controlled reaction between NO and O2 (5, 17–19). Peroxy-
nitrite does not directly react with tyrosine (20) but promotes
tyrosine dimerization and nitration due to the reactions of
peroxynitrite-derived species such as hydroxyl radical (•OH),
carbonate radical (CO3•-), •NO2 and the high oxidation state of
+
redox-active metal centers [Me(n+1) dO, where Me is Fe, Cu,
or Mn] (recently reviewed in ref 21) that can oxidize tyrosine
•
•
•
to yield Tyr, which then combines with another Tyr or NO2
to yield 3,3′-dityrosine or 3-nitro-Tyr, respectively (22). For free
tyrosine or tyrosine analogues in aqueous solution, the 3,3′-
dityrosine/3-nitro-Tyr ratio after peroxynitrite exposure typically
range in values of 1/20-1/25; however, this ratio may change
if peroxynitrite is added as a single bolus or by slow infusion,
depends on the tyrosine and peroxynitrite concentration, and
may become smaller in proteins, reflecting the relative ease of
the nitration reaction with respect to the dimerization reaction,
due to diffusional and steric limitations. In addition, other
tyrosine oxidation products from peroxynitrite can be formed,
including the hydroxylated derivative 3,4-dihydroxyphenyl-
alanine (DOPA) (23). Of note, 3-nitro-Tyr was initially con-
sidered a specific marker of peroxynitrite; however, there is now
agreement that tyrosine nitration can also occur biologically by
peroxynitrite-independent mechanisms, which include hydrogen
peroxide (H2O2)-dependent nitrite oxidation catalyzed by heme
(24) and hemoperoxidases [e.g., myeloperoxidase (25) and
eosinophil peroxidase (26); reviewed in ref 5], pathways that
also lead to the formation of 3,3′-dityrosine.
Protein tyrosine dimerization and nitration sites and yields
depend on the protein structure, oxidation mechanism, and the
environment where the protein tyrosine residues are located
(22, 27). In this last regard, most of the mechanistic studies of
oxidation for free and protein tyrosines have been performed
in aqueous solution (10, 11, 28). However, many protein tyrosine
residues shown to be dimerized and nitrated either in vitro and
in vivo are associated with nonpolar compartments, such as red
cell membrane proteins (29–31), mitochondrial membrane
proteins (32–34), sarcoplasmic reticulum Ca2+ ATPase, mi-
crosomal glutathione S-transferase (35), and apolipoproteins A
and B (3, 25, 36). Within these proteins, in some cases, oxidized
tyrosines have been shown in cytosolic or extracellular domains
[e.g., Tyr 192 and apoA-I (36)] but in other cases in domains
closely related to lipids [e.g., Tyr 39 in R-synuclein (37)]. Also,
reversible interactions of phospholipids with proteins can
modulate tyrosine oxidation yields and sites, as observed for
the cases of apoA-I (36), R-synuclein (38), and matrix metal-
loproteinase MMP-13 during wound repair (39).
LH + X• f L• + XH
L• + O2 f LOO•
(1)
(2)
LOO• + L′H f Lꢀ• + LOOH
(3)
These lipid-derived radicals are reactive species that can in turn
oxidize biological targets (RH) including protein side chains
(51). According to the one-electron redox potential of alkoxyl
•
•
(E°LO /LOH ) 1.76 V) and peroxyl radicals (E°LOO /LOOH ) 1.02
V), it is thermodynamically possible for both species to oxidize
•
tyrosine to the phenoxyl radical (E°Tyr /TyrH ) 0.88 V), while
•
the alkyl radicals (E°L /LH ) 0.6 V) are not able to perform that
Physicochemical factors controlling tyrosine oxidation in
hydrophobic biocompartments such as biomembranes and
lipoproteins differ from those in aqueous solution. For example,
hydrophobic phases contain a high concentration of unsaturated
fatty acids and exclude key antioxidant molecules that are potent
inhibitors of tyrosine oxidation in aqueous phases such as
oxidation. Herein, we postulate that LOO• is capable of oxidizing
tyrosine to •Tyr, therefore, fueling the tyrosine oxidation pathway
in hydrophobic biocompartments. To test this hypothesis, we
have studied BTBE dimerization and nitration in PC liposomes
of different unsaturation degrees promoted by three different
oxidation systems, namely, peroxynitrite, hemin, and the peroxyl

Lipid Peroxidation and Tyrosine Nitration in Membranes
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 823
R-tocopherol-containing liposomes, R-tocopherol (in ethanol) was
added at the desired concentration to the lipid solution in chloroform
with or without BTBE, and liposomes were prepared thereafter as
indicated above. Liposomes (30 mM PC and 0.3 mM BTBE) were
exposed to peroxynitrite, hemin, or ABAP under different conditions
throughout the work. BTBE and BTBE-derived products (e.g.,
3-nitro-BTBE and 3,3′-di-BTBE) were extracted with chloroform,
methanol, and 5 M NaCl as reported previously [1:2:4:0.4, sample:
methanol:chloroform:NaCl v/v with recovery efficiencies for all
compounds >95% (42)]. Samples were then dried and stored at
-20 °C. Immediately before HPLC separation, samples were
resuspended in 100 µL of a mixture containing 85% methanol and
15% KPi (15 mM), pH 3. Experiments with liposomes were
performed at 25 °C, a temperature above the transition phase
temperatures of the different liposomes and at 37 °C when ABAP
was used as an oxidant.
Oxidation Systems. BTBE-containing PC liposomes were
oxidized by the addition of either peroxynitrite, hemin, or the
organic peroxyl radical donor ABAP. Peroxynitrite was added as
a single bolus under vigorous vortexing [t1/2 ) 2.5 s at 25 °C (55)]
or by slow infusion using a motor-driven syringe system (Kd
Scientific) under continuous stirring. Because of the addition of
alkaline peroxynitrite solutions, the final pH was also checked at
the end of the incubation to ensure that there were no significant
variations (<0.1 pH units). Hemin was added directly to the PC
liposomes; EYPC and SBPC liposomes always contain a basal level
of preformed lipid hydroperoxides that serve as the redox substrate
for hemin. Alternatively, tert-butyl hydroperoxide was used as a
hemin reactant in DLPC liposomes. Finally, in the case of ABAP,
samples were incubated at 37 °C for 2-3 h to achieve a final ABAP
concentration of 0-40 mM. ABAP-dependent oxygen consumption
was measured using a high-resolution oxymeter (Oroboros 2K)
yielding a flux of 0.3 µM/min of peroxyl radicals. Experiments
under low oxygen tension (ca. 5 µM) were carried out by
extensively purging samples under argon for 30 min.
Lipid Peroxidation Analysis. Malondialdehyde (MDA), a
byproduct of lipid peroxidation, was measured as a thiobarbituric
acid reactive substance at 532 nm (ε ) 150000 M-1 cm-1), as
previously described (44). Calibration curves and assessment of
MDA content were performed with known amounts of MDA
obtained from the acid hydrolysis of 1,1,3,3-tetramethoxypropane
in 20% acetic acid, pH 3.5. To prevent further peroxidation of lipids
during assay procedures, 0.05% (w/v) of butylated hydroxytoluene
(BHT) was added to the thiobarbituric acid reactive species
(TBARS) reagent. Lipid hydroperoxide formation was evaluated
by the ferrous oxide-xylenol orange assay (FOX) assay (56). Briefly,
50 µL of the liposome sample was added to 950 µL of FOX reagent
consisting of 100 mM xylenol orange, 250 mM Fe2+ (ferrous
ammonium sulfate), 25 mM H2SO4, and 4 mM BHT in 90% (v/v)
methanol. Reaction mixtures were incubated for 1 h at room
temperature, and the absorbance was measured at 560 nm. The
concentration of lipid hydroperoxides was estimated with the
apparent extinction coefficient of 43000 M -1 cm -1 (56). Oxygen
consumption during lipid peroxidation processes was measured by
high-resolution oxymetry using an Oxygraph 2K (Oroboros Instru-
ments, Austria).
radical donor 2,2′-azobis (2-amidinopropane) hydrochloride
(ABAP), and their relationship with the lipid peroxidation
process.
Experimental Procedures
Chemicals. Diethylentriaminepentaacetic acid (dtpa), manganese
dioxide, sodium bicarbonate, mono- and dibasic potassium phos-
phate, L-tyrosine, 3-nitro-Tyr, R-tochopherol, 2-methyl nitrosopro-
pane (MNP), 1,1,3,3 tetramethoxypropane, and hemin were pur-
chased from Sigma. BTBE, 3-nitro-N-t-BOC L-tyrosine tert-butyl
ester (3-nitro-BTBE) and 3,3′-di-N-t-BOC L-tyrosine tert-butyl ester
(3,3′-di-BTBE) were prepared and handled as previously (42). N-t-
BOC L-phenylalanine tert-butyl ester (BPBE) was synthesized for
the first time using an identical procedure as the one previously
described for BTBE but starting from commercially available
(Sigma) L-phenylalanine-t-butylester (42). Stock BTBE solutions
(1 M) were prepared in methanol immediately before use. 3,3′-
Dityrosine was synthesized by incubating 0.5 mM L-tyrosine, 0.2
mg/mL (4.5 µM) horseradish peroxidase, and 500 µM H2O2 in 50
mM phosphate buffer (pH 7.4) for 20 min at 25 °C. The resulting
mixture was centrifuged through a Centricon tube (molecular mass
cutoff 5000 Da) to remove the enzyme, and the concentration of
3,3′-dityrosine was obtained and determined spectrophotometrically
at 315 nm using an extinction coefficient of 5700 M-1 cm-1 (pH
7.4) and 8380 M-1 cm-1 (pH 9.9) (52). 1,2-Dilauroyl-sn-glycero-
3-phosphocholine (DLPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-
phosphocholine (PLPC), and egg yolk and soybean PC [egg chicken
yolk L-R-phosphatidylcholine (EYPC) and soybean-L-R-phosphati-
dylcholine (SBPC)] were from Avanti Polar Lipids. Organic
solvents for the synthesis of standards and chromatography were
from Baker or Mallinckrodt. All other compounds were reagent
grade.
A stock hemin solution was freshly prepared in 0.1 N NaOH
and kept in the dark at 4 °C until use. ABAP was purchased from
Wako Chemicals USA. A fresh stock solution (100 mM) was
prepared in water, and incubations were performed at 37 °C for
the indicated times. Argon and nitrogen gases were purchased from
AGA Chemical Co. (Uruguay). All solutions were prepared with
highly pure deionized nanopure water to minimize trace metal
contamination.
Peroxynitrite Synthesis and Quantitation. Peroxynitrite was
synthesized in a quenched-flow reactor from sodium nitrite (NaNO2)
and hydrogen peroxide (H2O2) under acidic conditions as described
previously (53). The H2O2 remaining from the synthesis was
eliminated by treating the stock solutions of peroxynitrite with
granular manganese dioxide, and the alkaline peroxynitrite stock
solution was kept at -20 °C until use. Peroxynitrite concentrations
were determined spectrophotometrically at 302 nm (ε ) 1670 M-1
cm-1) (44, 54). The nitrite concentration in the preparations was
typically lower than 20% with respect to peroxynitrite. The control
of nitrite levels was critical for obtaining reproducible data as, if
•
present in excess, it can react with OH and other oxidants and
yield •NO2 (41). In control experiments, peroxynitrite was allowed
to decompose to nitrate in 100 mM phosphate buffer, pH 7.4, before
use, that is, “reverse order addition” of peroxynitrite.
HPLC Analysis. BTBE, 3-nitro-BTBE, and 3,3′-di-BTBE were
separated on a Agilent 1200 system equipped with UV-vis and
fluorescence detectors by reverse-phase HPLC using a Agilent
Eclipse XDB-C18 5 µm column (150 mm length, 4.6 mm i.d.).
Mobile phase A consisted of 15 mM phosphate buffer, pH 3, and
mobile phase B consisted of methanol. Chromatographic conditions
were as follows: flow, 1 mL/min; 75% mobile phase B for 25 min,
followed by a linear increase to 100% mobile phase B for 10 min.
UV-vis settings for BTBE were 280 nm and ε ) 1200 M-1 cm-1
and for 3-nitro-BTBE were 360 nm and ε ) 1500 M-1 cm-1. 3,3′-
BTBE Incorporation into Liposomes and Oxidizing Systems.
BTBE incorporation into liposomes was carried out as in refs 41
and 42 with minor modifications. Briefly, a methanolic solution of
BTBE (0.35 mM) was added to 35 mM PC lipids dissolved in
chloroform. Under these conditions, more than 98% BTBE was
incorporated (42). The mixture was then dried under a stream of
nitrogen gas. Previous studies from our group (41, 42) already
indicated that BTBE incorporation yields and formation of oxidation
products from peroxynitrite were comparable in unilamellar and
multilamellar liposomes and therefore not dependent on the
membrane morphology. Therefore, because of the more simple
preparation procedure, reported experiments were carried out mainly
with multilamellar liposomes. Multilamellar liposomes were formed
by thoroughly mixing the dried lipid with 100 mM sodium
phosphate buffer (pH 7.4) plus 0.1 mM dtpa. For experiments with
di-BTBE was detected fluorimetrically at λex ) 294 nm and λ
em
) 401 nm. Authentic 3-nitro-BTBE and 3,3′-di-BTBE were used
as standards.
3-Nitro-Tyr and 3,3′-dityrosine were separated by reverse-phase
high-performance liquid chromatography (RP-HPLC), using a

824 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Bartesaghi et al.
Partisil ODS-3 10 µm (250 mm length, 4.6 mm i.d.) C18 column
as previously reported with minor modifications (41). Briefly,
separation of tyrosine oxidation products was performed by isocratic
RP-HPLC. Chromatographic conditions were as follows: flow, 1
mL/min; 97% mobile phase A (KPi, 15 mM, pH 3) and 3% mobile
phase B (methanol) for 30 min. 3-Nitro-Tyr was measured by UV
detection (280 and 360 nm), and 3,3′-dityrosine was measured
fluorometrically (λex ) 280 nm and λem ) 400 nm). Authentic 3,3′-
dityrosine and 3-nitro-Tyr were used as standards. Artifactual BTBE
or tyrosine nitration during the chromatographic separation proce-
dure due to nitrite-dependent nitration at acidic pH were ruled out
by appropriate controls using predecomposed peroxynitrite.
ESR Spin Trapping Measurements. ESR spectra were recorded
at room temperature on a Bruker EMX spectrometer operating at
9.8 GHz. Typical spectrometer parameters were as follows: sweep
width, 100 G; center field, 3505 G; time constant, 20.48 ms; scan
time, 42 s; modulation amplitude, 1.0 G; modulation frequency,
100 kHz; receiver gain, 1 × 106; and microwave power, 20 mW.
Samples were subsequently transferred to a 50 µL capillary tube
for ESR measurements.
MNP (20 mM) was used as a spin trap; its photolysis may occur
during the experiments and leads to the formation of a three line
signal corresponding to the di-tert-butyl nitroxide radical, but it
has a different aN of 17.1 G. To minimize this, all liposomes
preparations and solutions were covered with aluminum foil, and
spectra were recorded in the dark; therefore, the signal due to
photolysis was not seen under our experimental conditions.
General Experimental Conditions. Experiments were typically
carried out in the presence of BTBE (0.3 mM) in PC liposomes
(30 mM) in 100 mM sodium phosphate plus 0.1 mM dtpa (pH
7.4) and 25 °C unless otherwise stated.
Data Analysis. All experiments reported herein were repeated
a minimum of three times. Results are expressed as mean values
with the corresponding standard deviations. Graphics and data
analysis were performed using Origin 8.0.
Results
Participation of Lipid-Derived Radicals on BTBE Oxida-
tion. When BTBE-containing DLPC liposomes were treated
with peroxynitrite (1 mM), 3-nitro-BTBE and 3,3′-di-BTBE
were formed in yields of up to 2.5 and 0.01% with respect to
initial peroxynitrite concentration, respectively (Figure 1A,B),
in agreement with previous results (41, 57). Significant levels
of both BTBE oxidation products were also formed in EYPC
(Figure 1) and SBPC, which contain a significant proportion of
unsaturated fatty acids, ∼24 and 57%, respectively. Thus, in
spite of the fact that for EYPC and SBPC (30 mM) unsaturated
fatty acids correspond to ∼15 and 34 mM, respectively, a much
larger concentration than that of BTBE (0.3 mM), the BTBE
oxidation process was still operative.
To assess the participation of lipid-derived radicals in BTBE
oxidation, experiments were also performed under low oxygen
tensions (Figure 1), which should result in an inhibition of LOO•
formation (eq 2) in both saturated3 (58) and unsaturated fatty
acids. Indeed, under these conditions, BTBE nitration and
dimerization yields were substantially decreased in either
saturated and unsaturated fatty acid-containing liposomes (Figure
1A,B). In parallel, we evaluated lipid peroxidation in the samples
by quantitating MDA, a well-known breakdown product of
peroxidized lipids. Peroxynitrite caused MDA formation in
BTBE-containing EYPC and SBPC liposomes (Figure 1C), in
Figure 1. Effect of oxygen on peroxynitrite-dependent BTBE oxidation
and lipid peroxidation. BTBE (0.3 mM) was incorporated into the
different liposomes DLPC, EYPC, and SBPC (30 mM) and exposed
to peroxynitrite (1 mM) in the presence of oxygen (200 µM) or under
low oxygen tensions (∼5 µM). Samples were analyzed for (A) 3-nitro-
BTBE, (B) 3,3′-di-BTBE, and (C) MDA contents. The data on MDA
show its increase over basal levels upon addition of peroxynitrite. Basal
levels in EYPC, SBPC and DLPC were 4.5, 5 and 0 µM, respectively.
Structures of 3-nitro-BTBE and 3,3′-di-BTBE are indicated in the
corresponding panels.
3 Saturated fatty acids (e.g., lauric acid in DLPC) will also react with
•OH at fast rates [k ∼ 5 × 108 M-1 s-1 (58)] to yield the alkyl radical. This
can evolve to lauric acid peroxyl radical that, however, will not be capable
of propagating a lipid peroxidation chain reaction in DLPC liposomes due
to the lack of unsaturated fatty acids. Still, the peroxyl radical could be
potentially reactive with other targets such as BTBE (vide infra).
agreement with our previous observations (41); importantly,
MDA levels were reduced under low oxygen concentration,
revealing the inhibition of the lipid peroxidation process. As

Lipid Peroxidation and Tyrosine Nitration in Membranes
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 825
expected, no MDA was detected in DLPC samples. Thus, in
unsaturated fatty acid-containing liposomes (EYPC and SBPC),
BTBE oxidation and lipid peroxidation occurred simultaneously
and were both inhibited at low oxygen levels. Lipid hydroper-
oxide levels were significantly higher (over 1 order of magni-
tude) than those obtained for BTBE oxidation products, in
agreement with the idea that lipid peroxidation is the main
process within the membrane. On the other hand, when tyrosine
(0.3 mM) was exposed to peroxynitrite (1 mM) in 100 mM
phosphate buffer (pH 7.3) and 0.1 mM DTPA, oxygen did not
influence tyrosine nitration and dimerization yields (∼6 and
0.25%) with respect to peroxynitrite in the aqueous phase (not
shown). Overall, the data point to the formation of LOO• as
key intermediates in the BTBE oxidation process.
Electron spin resonance (ESR) spin trapping was used to
detect the lipid-derived and BTBE phenoxyl radicals formed
during peroxynitrite exposures using MNP as described previ-
ously (41, 59). In DPLC liposomes, no ESR signal was obtained
in the absence of BTBE when exposed to peroxynitrite (Figure
2, line a). On the other hand, BTBE-containing DLPC liposomes
treated with peroxynitrite resulted in an anisotropic three line
signal, confirming the formation of a partially immobilized
phenoxyl radical in the interior of the membrane (Figure 2, line
b) and in agreement with our previous work (41, 60). When
peroxynitrite-treated BTBE containing DLPC liposomes was
dissolved in ethanol (60), a sharper and clear three line signal
was obtained, which, despite the low signal-to-noise ratio,
allowed the determination of a hyperfine constant of 13.8 G;
this is consistent with a MNP adduct with the one-electron
oxidation of BTBE (61). In EYPC liposomes, peroxynitrite
caused the formation of a MNP adduct in the absence of BTBE,
compatible with the formation of MNP-lipid alkyl (carbon-
centered adduct) radical adducts (Figure 2, line c) with an
estimated hyperfine splitting constant aN ∼ 15 G (59). When
BTBE was incorporated into liposomes, the signal was even
larger (Figure 2, line d), supporting the coexistence of lipid-
derived and BTBE phenoxyl radicals and confirming the
temporal association between the lipid peroxidation and the
BTBE oxidation processes. No ESR spectrum was obtained in
DLPC or EYPC liposomes when treated with decomposed
peroxynitrite (Figure 2, lines e and f) or if MNP was added
after peroxynitrite (Figure 2, line g) or if peroxynitrite was added
to MNP only (without liposomes, not shown).
Figure 2. ESR spin trapping of BTBE phenoxyl and LOO•. (A) Reaction
mixtures consisting of BTBE (2.5 mM) incorporated into 45 mM DLPC
liposomes in 100 mM phosphate buffer (pH 7.4) containing dtpa (0.1 mM)
were treated with 20 mM MNP spin trap and rapidly mixed with 5 mM
peroxynitrite. Samples were subsequently transferred to a 50 µL capillary
tube for EPR measurements. (a) DLPC liposomes plus peroxynitrite, (b)
BTBE-containing DLPC liposomes plus peroxynitrite, (c) EYPC liposomes
plus peroxynitrite, (d) BTBE-containing EYPC liposomes plus peroxyni-
trite, (e) same as b with reverse order addition of peroxynitrite, (f) same
as d with reverse order addition of peroxynitrite, and (g) peroxynitrite only.
(B) The structures of the MNP-phenoxyl and MNP-lipid alkyl radical
adducts are shown and correspond to the signals obtained in lines b and c
of panel A, respectively. Both spin adducts are present in line d.
Slow Infusion vs Bolus Addition of Peroxynitrite in
Lipid Peroxidation and BTBE Oxidation. Peroxynitrite has
a short half-life in 100 mM phosphate buffer, pH 7.4, and
25 °C (t1/2 ) 2.5 s) due to the proton-catalyzed homolysis to
•
•OH and NO2 in 30% yields (55). Thus, the addition of
peroxynitrite to reaction mixtures as a single bolus may result
in a high initial concentration of radicals, and the extent of
oxidation processes in target molecules do not necessarily reflect
the expected outcome in more biologically relevant conditions
where peroxynitrite is formed as a continuous flow (62, 63).
This consideration becomes particularly important in lipid
peroxidation processes that depend on propagation reactions
where an initial large flux of radicals will prematurely terminate
the process. Thus, experiments were performed to study lipid
peroxidation and BTBE oxidation yields with peroxynitrite
addition as a single bolus or by slow infusion for up to 30 min
(1 µM/min). Peroxynitrite addition to BTBE-containing EYPC
liposomes resulted in dose-dependent formation of MDA (Figure
3A); MDA yields were increased 2-3-fold when peroxynitrite
was added as a slow infusion. Similarly, BTBE nitration and
dimerization (Figure 3B-D) yields in both EYPC and DLPC
by peroxynitrite infusion were up to 3-fold higher that with bolus
addition. Moreover, BTBE oxidation (Figure 3E) and MDA
formation (not shown) yields in EYPC were significantly
reduced under low oxygen tensions when peroxynitrite was
added as a slow infusion. The data further support an association
between the lipid peroxidation and the BTBE oxidation processes.
Effect of Phospholipid Unsaturation Degree in Peroxyni-
trite-Mediated BTBE Oxidation. BTBE oxidation was studied
as a function of fatty acid unsaturation by using liposomes
containing variable mixtures of DLPC and PLPC. Nitration and
dimerization yields were significant through all the phospholipid
unsaturation range (0-100%). A tendency toward increased
BTBE oxidation yields was observed at around 40% PLPC
content, while a slight decrease was observed at 100% PLPC.
Thus, in spite of a largely variable content of unsaturated fatty
acids in the liposomal mixtures (from 0 to 30 mM linolenic

826 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Bartesaghi et al.
Figure 3. Peroxynitrite-mediated oxidations: slow infusion versus bolus addition. BTBE (0.3 mM) was incorporated into EYPC and DLPC liposomes
(30 mM) and exposed to peroxynitrite either as a single bolus (9) or by slow infusion (O) to achieve final concentrations of 0.2-2 mM. Samples
were analyzed for 3-nitro-BTBE, 3,3′-di-BTBE, and MDA contents. EYPC: (A) MDA, (B) 3-nitro-BTBE, and (C) 3,3′-di-BTBE. DLPC: (D)
3-nitro-BTBE and 3,3′-di-BTBE (inset). (E) BTBE- (0.3 mM) containing liposomes were exposed to slow infusion of peroxynitrite (1 mM) in the
presence and absence of oxygen, and 3-nitro-BTBE and 3,3′-di-BTBE were measured as previously.
acid), BTBE oxidation was consistently present. Thus, the data
support that a simple kinetic competition model, where unsatur-
ated fatty acids simply outcompete BTBE for peroxynitrite-
derived radicals, does not apply and that secondary reactions
of lipid-derived radicals with BTBE must contribute to nitration
and dimerization reactions (Figure 4).

Lipid Peroxidation and Tyrosine Nitration in Membranes
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 827
Figure 4. Lipid unsaturation degree and BTBE oxidation. BTBE
nitration (9) and dimerization (O) were studied as a function of fatty
acid unsaturation by using mixtures of DLPC and PLPC (0-100%
PLPC) liposomes containing 0.3 mM BTBE and treated with perox-
ynitrite (1 mM).
Scheme 1. Lipid Hydroperoxide Reactions with Hemin and
Lipid-Derived Radicals Formationa
Figure 5. Hemin-induced lipid peroxidation and BTBE oxidation.
BTBE- (0.3 mM) containing DLPC and EYPC liposomes (30 mM)
were exposed to hemin (5-20 µM) in 100 mM sodium phosphate, pH
7.3, plus 0.1 mM DTPA. Samples were analyzed for (A) 3,3′-di-BTBE
and (B) MDA contents. The arrow indicates the values corresponding
to DLPC liposomes that were zero for both measurements under all
reaction conditions.
a Lipid hydroperoxide (LOOH) can react with hemin either in Fe3+ or
Fe2+ redox states to yield LOO• or LO•, respectively. Secondarily, hemin
was detected. Thus, the data imply the participation of LOO•
radicals formed from the reaction of hemin with lipid hydrop-
eroxides (Scheme 1) in the BTBE dimerization process.
Second, we performed experiments using the organic peroxyl
radical donor, ABAP, which generates a flux of peroxyl radicals
by thermolysis.
•-
in the reduced state (Fe2+) can yield O2 that can further participate in
redox reactions.
Hemin and ABAP-Induced Lipid Peroxidation. To further
demonstrate that lipid peroxidation processes are related with
BTBE oxidation, we exposed BTBE-containing liposomes to
two other oxidation systems, namely, hemin and ABAP, both
of which lead to the formation of LOO• in unsaturated fatty
acid-containing liposomes. Free hemin readily interacts with
membranes (64) and initiates lipid peroxidation in unsaturated
liposomes by reaction with LOOH always present in variable
extents in EYPC and SBPC (typical hydroperoxide contents in
well-stored preparations represent in our 30 mM liposome
samples ∼45-80 µM, 0.075-0.13%, total unsaturated fatty acid
as determined by the FOX assay). Hemin (Fe3+) promotes the
one-electron oxidation of LOOH to yield LOO• and hemin in
the Fe2+ redox state (Scheme 1). In turn, the reduced hemin
can generate LO• by reduction of LOOH.
Secondary reactions in the system generate variable amounts
of O2•-, H2O2, and heme-Fe4+dO that can amplify the process
(64). In EYPC liposomes, hemin induced 3,3′-di-BTBE and
MDA formation in a dose-dependent manner (Figure 5). On
the other hand, in DLPC liposomes (saturated), where hemin
can not cause lipid peroxidation, no formation of 3,3′-di-BTBE
A-NdN-A f 2A•
2A• + O2 f AOO•
(4)
(5)
AOO• + LH f AOOH + L•
(6)
ABAP initiates lipid peroxidation in unsaturated fatty acid-
containing liposomes such as EYPC, by the reaction of the
ABAP-derived peroxyl radicals (AOO•) with an unsaturated
alkyl chain to yield the alkyl radical (L•), which then (eqs 4-6),
after oxygen addition, forms LOO•. The addition of ABAP to
BTBE-containing liposomes resulted in the formation of 3,3′-
di-BTBE in both DLPC and EYPC liposomes (Table 1 and
Figure 6A), but MDA formation was observed in EYPC
liposomes only. Thus, either ABAP-derived (in the DLPC
experiment) or lipid-derived (in the EYPC one) peroxyl radicals
react with BTBE to yield BTBE-derived phenoxyl radical and

828 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Table 1. ABAP-Mediated BTBE Oxidationa
Bartesaghi et al.
EYPC
DLPC
condition
control
NO2-BTBE (µM)
di-BTBE (µM)
NO2-BTBE (µM)
di-BTBE (µM)
0
0
0
0
ABAP
0
0.011 ( 0.001
0
0.79 ( 0.164
0.537 ( 0.029
0.32 ( 0.065
ND
-
ABAP + NO2
2.5 ( 0.76
0
1.46 ( 0.8
0
0.043 ( 0.008
16.15 ( 1.39
0
9.43 ( 2
0
ABAP - O2
0
NDb
0
-
ABAP + NO2 - O2
-
NO2
0
a BTBE (0.3 mM) was incorporated into DLPC and EYPC liposomes (30 mM), and the mixture was incubated for 2 h at 37 °C with ABAP (10 mM,
0.3 µM/min flux of peroxyl radicals) in the presence and absence of nitrite (NO2-) (20 mM), and 3-nitro-BTBE and 3,3′-di-BTBE contents were
measured. The same experiment was performed in the presence of low oxygen tensions (∼5 µM). b ND, not determined.
subsequently 3,3′-di-BTBE. Interestingly, samples treated with
ABAP plus nitrite resulted in the formation of both 3,3′-di-
BTBE and 3-NO2-BTBE, supporting that ABAP-derived peroxyl
liposomes resulted in a significant decrease in oxidant-induced
oxygen consumption rates (Figure 8B). The extent of inhibition
in oxygen consumption by R-tocopherol is compatible with the
previously reported rate constants of LOO• with adjacent alkyl
chains (k ) 36 M-1 s-1) (68) and with R-tocopherol (k ) 5 ×
105 M-1 s-1) (66). The inhibitory action of R-tocopherol was
also observed if added exogenously during the time course of
the oxygen consumption studies but was less potent than when
preincorporated, due to the suboptimal penetration in the
preformed liposome structure within the time frame of the ex-
periment. Remarkably, the incorporation of BTBE to the EYPC
liposomes resulted in a dose-dependent inhibition of both hemin-
and ABAP-induced oxygen consumption rates, which is con-
sistent with the reaction of BTBE with lipid-derived radicals
(Figure 8B). To discard subtle structural effects in the liposomal
membranes due to BTBE incorporation, experiments were
performed using a phenylalanine hydrophobic analogue (BTPE)
preincorporated to the liposomes (Figure 8C). In this case,
oxygen consumption rates were identical to the one observed
in control EYPC (without BTBE), which is in agreement with
a direct participation of the phenolic-OH moiety in the BTBE
in reaction leading to inhibition of lipid peroxidation-dependent
oxygen consumption, which is not present in the phenylalanine
derivative (Figure 8C).
•
radicals can also oxidize nitrite to NO2 (Tables 1 and 2) (65).
Low oxygen levels decreased BTBE oxidation yields induced
by ABAP plus nitrite, in both DLPC and EYPC liposomes,
confirming the role of LOO• in the process. Control experiments
with nitrite only showed no formation of BTBE oxidation
products (Table 1).
To specifically address the reaction of organic peroxyl radicals
with tyrosine, we studied the effect of ABAP on free tyrosine
oxidation in aqueous phase (Figure 6B). Tyrosine exposure to
ABAP-derived peroxyl radicals resulted in the formation of 3,3′-
dityrosine (Figure 6B, line b); when nitrite was added to the
incubation mixture, both tyrosine oxidation products, 3-nitro-
Tyr and 3,3′-dityrosine, were formed (Figure 6B, line c).
Tyrosine nitration and dimerization yields were lower with free
tyrosine in aqueous solution than those observed for BTBE in
liposomes, which suggest that tyrosine oxidation by peroxyl
radicals more easily occurs in nonpolar environments.
Effect of r-Tocopherol on BTBE Oxidation and Lipid
Peroxidation. R-Tocopherol is a well-known lipophilic chain-
breaking antioxidant that reacts with LOO• with a rate constant
of 5 × 105 M-1 s-1 (66) to yield lipid hydroperoxides and the
stable R-tocopheroxyl radical (eq 7):
The dose-dependent inhibition of oxygen consumption by
increasing concentrations of preincorporated BTBE in EYPC
liposomes was further analyzed to obtain an estimated second-
order rate constant for the reaction of LOO• with BTBE. A plot
that relates the inhibition fraction in oxygen consumption as a
function of BTBE concentration is in agreement with a direct
competition of BTBE for the LOO• (Figure 8D). These data
allow the estimation of an apparent second-order rate constant
of 4.8 × 103 M-1 s-1 for the following reaction:
LOO• + R-TOH f LOOH + R-TO•
(7)
R-Tocopherol incorporated into EYPC liposomes inhibited
peroxynitrite-mediated BTBE nitration (Figure 7A), dimerization
(not shown), and lipid peroxidation (Figure 7B) in a dose-
dependent manner. Interestingly, R-tocopherol also inhibited
BTBE oxidation in DLPC liposomes, where no lipid peroxi-
dation occurs. The effect of R-tocopherol is explained by the
reaction between R-tocopherol with LOO•. In the case of DLPC,
LOO• + BTBE-OH f LOOH + BTBE-O• (8)
•
the reaction of R-tocopherol with NO2 [k ) 1 × 105 M-1 s-1
(67)] also becomes relevant to explain the inhibition of BTBE
oxidation.
This rate constant value compares well with an independent
determination considering the R-tocopherol studies. From data
in Figure 8B,C, one concludes that 87% inhibition is obtained
with 5 µM R-tocopherol or 1 mM BTBE. Considering the
stoichiometric factor n ) 2 for R-tocopherol (69), thus, 2 ×
ktoc [R-tocopherol] ) kBTBE [BTBE], so 2 × ktoc [R-tocopherol]/
[BTBE] ) kBTBE ) 5 × 103 M-1 s-1. In addition, both MDA
and hydroperoxide yields were decreased in the presence of
BTBE, in agreement with a reaction between BTBE and LOO•
(Figure 8E,F) and with the oxygen consumption data.
Oxygen Consumption Studies and Kinetic Determina-
tion of LOO• Reaction with BTBE. Experiments were
performed to measure the oxygen consumption associated with
lipid peroxidation initiated by hemin and ABAP and the
potential chain-breaking reaction of BTBE. If LOO• react to a
significant extent with BTBE, then, the hydrophobic tyrosine
analogue should inhibit oxygen consumption. In EYPC (but not
in DPLC) liposomes (in the absence of BTBE), a basal decrease
in oxygen concentration is shown. Oxygen consumption is
observed upon the addition of either ABAP or hemin (Figure
8A), in agreement with the initiation and propagation phases
of lipid peroxidation. In contrast, no further oxygen consumption
was observed in DLPC liposomes after ABAP or hemin addition
(Figure 8A). The incorporation of R-tocopherol to EYPC
Discussion
Previously identified and biologically relevant one-electron
•
oxidants for tyrosine in aqueous environments include OH,
CO3•-, and •NO2, compounds I and II of hemeperoxidases, and

Lipid Peroxidation and Tyrosine Nitration in Membranes
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 829
lipid-derived radicals generated during lipid peroxidation pro-
•
cesses can mediate tyrosine oxidation to Tyr, the first step in
the path to 3,3′-dityrosine or 3-nitro-Tyr.
Lipid peroxidation was initiated by three independent oxida-
tion systems, namely, peroxynitrite, hemin, and ABAP. Peroxy-
nitrite promotes lipid peroxidation and tyrosine nitration in
hydrophobic compartments secondary to homolysis of peroxy-
•
•
nitrous acid (ONOOH) to OH and NO2 inside or at close
proximity of the lipid phase (41, 44, 70). In EYPC, for instance,
the large concentration ratio of unsaturated fatty acid/BTBE (50/
1) and the participating rate constants (see Table 2) determine
that the initial attack of peroxynitrite-derived radicals occurs at
the fatty acid moieties; this initial reaction yields the alkyl
(pentadienyl) radical that upon oxygen addition evolves to LOO•,
which propagates the process (44). Alternatively, lipid-derived
radicals were generated by the reaction of hemin with pre-
existing lipid hydroperoxides to directly yield LOO• or by ABAP
whose peroxyl radical derivative diffuses to the liposomes and
reacts with unsaturated alkyl chains to initiate lipid peroxidation
as well (71). LOO• are known to react at fast rates with some
phenolic compounds, including R-tocopherol and other dietary
compounds that may exert antioxidant actions in vitro and in
vivo. For example, linoleate peroxyl radicals react with phenolic
antioxidant compounds such as R-tocopherol, curcumin, and
quercetin with rate constants in the range ∼106-107 M-1 s-1
(72–75). In the case of tyrosine, another phenolic compound,
the reaction with LOO• is thermodynamically possible; however,
tyrosine is not a particularly good hydrogen donor; therefore,
the reaction may be kinetically hindered. Interestingly, hydro-
bobic phases may increase the reactivity of phenolic compounds
such as tyrosine for LOO• (see below). Thus, extensive studies
were performed herein to unambiguously show the existence
of the LOO• reaction with the hydrophobic tyrosine analogue
BTBE.4
The evidence that we have gathered herein to support the
participation of LOO• in the tyrosine oxidation processes
promoted by the three different tested oxidation systems can
be summarized as follows: (a) Tyrosine (BTBE) oxidation was
seen in significant extents in unsaturated fatty acid-containing
PC liposomes, (b) tyrosine oxidation in liposomes (but not in
aqueous phase) was decreased under low oxygen concentrations,
•
(c) lipid-derived and Tyr were simultaneously detected, (d)
R-tocopherol strongly inhibited tyrosine oxidation, (e) BTBE
(but not BPTE) partially inhibited lipid peroxidation processes,
(f) changes in the levels of tyrosine oxidation and lipid
peroxidation products followed a parallel trend, and (g) the data
are in agreement with an overall process involving the free
radical mechanisms of lipid peroxidation and tyrosine oxidation
plus a “connecting reaction” represented by one-electron oxida-
tion of tyrosine by LOO• (eq 7) (see also Table 2).
Figure 6. ABAP-induced BTBE and tyrosine oxidation. (A) (a) BTBE-
(0.3 mM) containing DLPC liposomes in 100 mM sodium phosphate
(pH 7.3) plus 0.1 mM DTPA were exposed to (b) ABAP (20 mM) in
the absence and (c) presence of nitrite (40 mM), and 3-nitro-BTBE
and 3,3′-di-BTBE were measured. (B) (a) Tyrosine (0.3 mM) in 100
mM sodium phosphate (pH 7.3) plus 0.1 mM DTPA was exposed to
(b) ABAP (20 mM) in the absence and (c) presence of nitrite (40 mM),
and 3-nitro-Tyr and 3,3′-dityrosine were measured.
With regard to the reactivity of peroxyl radicals with tyrosine,
the data showed that ABAP-derived peroxyl radicals were
already capable of oxidizing both BTBE in DLPC (saturated)
liposomes as well as free tyrosine (Table 1 and Figure 6), but
reaction yields were much larger for BTBE, compatible with
the concept that the reaction of phenolic compounds with
oxidizing radicals is influenced by a kinetic solvent effect
(76, 77). Indeed, different phenolic compounds (e.g., quercetin
high oxidation states of transition metal centers (5). However,
in the case of tyrosine oxidation in proteins associated with
hydrophobic biocompartments such as membranes or lipopro-
teins, (a) some of the oxidants may not easily reach or permeate
the lipid phase, and (b) unsaturated fatty acids, due to their
reactivity and large concentration, constitute preferential targets
for the initial oxidative attack. Thus, mechanisms by which
tyrosine residues become dimerized and nitrated in hydrophobic
biocompartments seem to have unique characteristics from those
previously described in hydrophilic phases. In this work, we
have explored, using a validated model system of PC liposomes
containing a hydrophobic tyrosine analogue (BTBE), whether
4 Alkoxyl radicals (LO•) are also formed in lipid peroxidation processes
via one-electron reduction of lipid hydroperoxides (48) or breakdown of
tetroxide intermediates formed by the coupling of lipid two peroxyl radicals;
alkoxyl radicals are far more potent oxidizing species than LOO• but mainly
rearrange to epoxyallylic radicals (50), which in turn, after coupling to O2,
become a secondary peroxyl radical.

830 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Bartesaghi et al.
Table 2. Main Reactions Involved in Tyrosine Oxidation in Hydrophobic Environments and the Connection to Lipid
Peroxidationa
reaction
k (M-1 s-1
)
reference
•
Lb + OH f L• + OH-
1 × 1010
45
2 × 105
46
90
•
-
L + NO2 f L• + NO2
1.24 × 1010
Tyr + OH f Tyr + OH-
•
•
6.5 × 108
3.2 × 105
90
46
•
•
Tyr + OH f TyrOHc + OH-
•
•
-
Tyr + NO2 f Tyr + NO2
3 × 109
46
•
•Tyr + NO2 f 3-nitro-Tyr
2•Tyr f 3,3′-di-Tyr
L• + O2 f LOO•
2.25 × 108
2.25 × 106
3 × 108
91
41
92
d
•
LOO• + L f LOOH + L
37
68
92
107
2LOO• f LOOH + O2
5 × 107
5 × 108
92
92
LOO• + L• f LOOL
2L• f LL
4.5 × 106
65e
-
•
LOO• + NO2 f LOOH + NO2
4.8 × 103
this work
•
LOO• + Tyr f LOOH + Tyr
5 × 105
1 × 105
67
67
R-tocopherolf + LOO• f R-tocopheryl• + LOOH
•
-
R-tocopherol + NO2 f R-tocopheryl•+NO2
3.8 × 109
66
•
R-tocopherol + OH f R-tocopheryl• + OH-
a Reactions involving lipids refer to those containing unsaturated fatty acid unless otherwise indicated. b Lipids composed of saturated fatty acid are
also oxidized by hydroxyl radicals at similar rates (58). c This radical rapidly dehydrates and evolves to •Tyr under the experimental conditions
employed herein. d Value for tyrosine dimerization in membranes, expected to occur at least 100 times slower than in the aqueous phase. e This rate
constant has been reported for acetylperoxyl radical reduction by nitrite. f The radical scavenging mechanisms of R-tocopherol are indicated.

Lipid Peroxidation and Tyrosine Nitration in Membranes
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 831
of tyrosine and the lack of an “induction period” in oxygen
consumption studies when lipid peroxidation was initiated in
BTBE-containing liposomes; this moderate rate constant (1/200
of that of R-tocopherol) is still larger than that of LOO• with
LH [e.g., the reaction of linoleic acid peroxyl radical with
linoleic acid, k ) 36 M-1 s-1 in 70% EtOH (68)]. The reactions
of halogenated organic peroxyl radicals (known to be more
reactive than LOO•) with tyrosine have been reported at
alkaline pH, where most of the tyrosine is in the phenolate
form (pKTyr-OH ∼ 10), with values approaching 108 M-1 s-1
(79). Oxidation of the protonated form of the phenol by
peroxyl radicals (as expected at neutral pH or when inside a
hydrophobic structure) is significantly less favorable, in full
agreement with the kBTBE value in the range of 103 M-1 s-1
obtained.
In a previous work (57), the nitration of a transmembrane
peptide containing tyrosine in position eight from the C terminus
(ca. Y-8 at a depth of ∼20 Å from the surface) induced by the
peroxynitrite donor 3-morpholinosydnonimine hydrochloride
(SIN-1) was progressively inhibited with increasing unsaturation
degree of the DLPC:PLPC mixtures. A closer look to the data
indicates that the extent of inhibition was significantly less than
that predicted by a simple competition of Y-8 with unsatur-
ated fatty acids [e.g., at 40% liposome unsaturation and
considering the reported rate constant values for hydroxyl
radical and nitrogen dioxide-dependent fatty acid and tyrosine
oxidation (Table 2), over 90% inhibition of tyrosine oxidation
would be expected, but only 40% inhibition was actually
observed].
The relative yields of tyrosine dimerization and oxidation are
largely affected by the biomembrane structure (41–43, 80), and
we have estimated a diffusion coefficient (D) for BTBE in PC
liposomes as ca. 5 µm2 s-1 (41). Thus, the rate constant value
is lowered 100-200-fold (k ∼ 1-2 × 106 M-1 s-1) with respect
•
to the corresponding one of Tyr (k ) 2.25 × 108 M-1 s-1).
Intermolecular tyrosine dimerization will be even less likely in
integral peptides and proteins as D values become >103-104
times smaller than in solution depending on protein size (e.g.,
D ∼ 0.1-0.5 µm2 s-1) (81, 82) and in line with the lack of
tyrosine dimerization on transmembrane peptides treated with
peroxynitrite (57). On the other hand, •NO2 concentrates to some
extent in hydrophobic environments (83), and the D value (D
•NO2) is ∼1500 µm2 s-1, very close to that of the aqueous phase
of 4500 µm2 s-1 (84); thus, nitration in membranes is not
kinetically impeded, a concept that is in perfect agreement with
the large 3-nitro- BTBE/3,3′diBTBE ratios found during per-
oxynitrite exposures.
Figure 7. Effect of R-tocopherol on lipid peroxidation and BTBE
oxidation. The indicated concentrations of R-tocopherol (0.1-1 mM)
were preincorporated to different EYPC and DLPC preparations (30
mM) and treated with peroxynitrite (1 mM); samples were analyzed
for (A) 3-nitro-BTBE and (B) MDA contents.
and epicatechin) react with peroxyl radicals at similar rates than
R-tocopherol in nonpolar solvents but not in hydrogen-bonding
solvents. In solvents that are strong hydrogen bond acceptors,
the rate constants of peroxyl radicals with phenolic compounds
dramatically decline and may even become undetectable5 (78).
Polar solvents (a) interfere with the intramolecular stabilization
(H-bonding) of the phenoxyl radical and (b) generate steric
hindrance for the approach of the peroxyl radical to the solvent-
complexed phenol, which reduces the rate constant of H-atom
abstraction. Such considerations become important in hetero-
geneous phases of lipid bilayers, where the localization of the
phenolic groups will influence their effectiveness to trap peroxyl
radicals. In the case of LOO• generated in EYPC, an estimated
second-order rate constant for the reaction with BTBE was
obtained by oxygen consumption studies, using two different
approaches considering independent “primary targets”, namely,
unsaturated fatty acids (kLH ∼ 10-50 M-1 s-1) and R-tocopherol
[kR-toc ) 5 × 105 M-1 s-1 (66)] (Figure 8). The estimated kBTBE
of 4.8 × 103 M-1 s-1 is compatible with the redox properties
Significant evidence supports that lipid peroxidation and
tyrosine oxidation processes are associated with oxidative stress-
related processes and pathophysiology (31, 43, 85, 86); for
instance, membrane protein tyrosine (and BTBE) oxidation and
nitration were evidenced in red blood cells exposed to perox-
ynitrite, in a process that was inhibited under low oxygen
tensions (43). In a related way, accumulation of protein 3-nitro-
Tyr and 4-hydroxynonenal protein adducts has been simulta-
neously detected in liver and kidney in a model of type I diabetes
where peroxynitrite has been identified as a key mediator of
lipid peroxidation (86). Future work should address whether
both processes in vivo are mechanistically linked as suggested
by our in vitro model studies. Our data also indicate that
R-tocopherol can potently modulate tyrosine oxidation yields
in membranes by inhibiting lipid peroxidation. In this regard,
it is important to consider that the extent of inhibition is a
function of R-tocopherol levels that under biologically relevant
5 For instance, a decrease by a factor of 36 in the k value has been
observed for the reaction of R-tocopherol with cumylperoxyl radicals when
changing from hexane to tert-butyl alcohol (78).

832 Chem. Res. Toxicol., Vol. 23, No. 4, 2010
Bartesaghi et al.
Figure 8. Oxygen consumption studies. Oxygen consumption was measured in a 2K Oxygraph to assess ABAP- (10 mM) and hemin- (1 µM)
induced lipid peroxidation in 100 mM sodium phosphate (pH 7.3) plus 0.1 mM DTPA. (A) EYPC and DLPC liposomes (6.25 mM). (B) BTBE (0.3
mM) or R-tocopherol (0.3 mM) containing EYPC liposomes. The arrow indicates the exogenous addition of 0.25 mM R-tocopherol; the transient
increase in oxygen levels observed in this record is due to oxygen dissolved in the volume of reagent added (C) BPBE (0.3 mM) and control EYPC
liposomes. (D) EYPC liposomes in the absence and presence of different concentrations of BTBE (0.1-3 mM) were exposed to ABAP (10 mM)
and hemin (1 µM), and oxygen consumption inhibition was evaluated as a function of BTBE concentration. (E) EYPC liposomes with or without
BTBE (0.3 mM) were exposed to hemin (30 min) and ABAP (2.5 h), and the lipid hydroperoxide content was measured. Inset: UV-vis spectra
of the same samples shown in E: (a) -BTBE, (b) +BTBE, (c) +BTBE + hemin, (d) +BTBE + ABAP, (e) -BTBE + ABAP, and (f) -BTBE
+ hemin. (F) The same samples as panel E in which the MDA content was measured. The structure of BPBE is indicated in the corresponding
panel.
conditions [ca. 1 molecule of R-tocopherol per 100-1000
molecules of membrane phospholipid (87)] may not be sufficient
to fully prevent tyrosine oxidation events in either biomembranes
(43, 86) or model membranes (this work, see Figure 7A at 0-0.3

Lipid Peroxidation and Tyrosine Nitration in Membranes
Chem. Res. Toxicol., Vol. 23, No. 4, 2010 833
Figure 9. Proposed reaction mechanism by which LOO• participate in tyrosine oxidation.
mM R-tocopherol for 30 mM DLPC). Thus, we predict that an
increase of cell/tissue levels of R-tocopherol should result in
attenuation of protein tyrosine oxidation in both biomembranes
and lipoproteins.
supported by a fellowship of ANII. R.R. is a Howard Hughes
International Research Scholar.
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