Marla SS, Lee J, Groves JT. Peroxynitrite rapidly permeates phospholipid membranes. Proc Natl Acad Sci U S A.

Abstract

Peroxynitrite (ONOO-) is a potent oxidant implicated in a number of pathophysiological processes. The activity of ONOO- is related to its accessibility to biological targets before its spontaneous decomposition (t1/2 approximately 1 s at pH 7.4, 37 degrees C). Using model phospholipid vesicular systems and manganese porphyrins as reporter molecules, we demonstrated that ONOO- freely crosses phospholipid membranes. The calculated permeability coefficient for ONOO- is approximately 8.0 x 10(-4) cm.s-1, which compares well with that of water and is approximately 400 times greater than that of superoxide. We suggest that ONOO- is a significant biological effector molecule not only because of its reactivity but also because of its high diffusibility.

Full text

Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 14243–14248, December 1997
Chemistry
Peroxynitrite rapidly permeates phospholipid membranes
SUDHAKAR S. MARLA*, JINBO LEE*, AND JOHN T. GROVES
†
Department of Chemistry, Princeton University, Princeton, NJ 08544
Edited by Ronald Breslow, Columbia University, New York, NY, and approved October 14, 1997 (received for review August 22, 1997)
ABSTRACT Peroxynitrite (ONOO
2
) is a potent oxidant
implicated in a number of pathophysiological processes. The
activity of ONOO
2
is related to its accessibility to biological
targets before its spontaneous decomposition (t
1y2
' 1satpH
7.4, 37°C). Using model phospholipid vesicular systems and
manganese porphyrins as reporter molecules, we demon-
strated that ONOO
2
freely crosses phospholipid membranes.
The calculated permeability coefficient for ONOO
2
is '8.0 3
10
24
cmzs
21
, which compares well with that of water and is
'400 times greater than that of superoxide. We suggest that
ONOO
2
is a significant biological effector molecule not only
because of its reactivity but also because of its high diffus-
ibility.
Peroxynitrite (ONOO
2
) has emerged as an important member
of the family of reactive oxygen and nitrogen species (1–5)
since the recognition of its rapid formation from nitric oxide
(NO
2z
) and superoxide anion (O
2
2z
) (6) The production of
ONOO
2
in vivo has been demonstrated in the macrophage
immune response (7, 8) and under conditions of oxidative
stress such as ischemiayreperfusion (9–11). The reactions of
ONOO
2
with biological substrates are known to include the
nitration of tyrosine residues in proteins (12) and the oxidation
of redox metal centers (13, 14), DNA (15, 16), lipids (17),
sulfhydryls (18), and methionine (19). In light of this reactivity,
ONOO
2
has been implicated in a number of pathological
conditions including neurological disorders (20–23) such as
Alzheimer disease and amyotrophic lateral sclerosis, in ath-
erosclerosis (24, 25), and a variety of conditions precipitated
by endothelial injury (26). Furthermore, since nitration of
tyrosine has been shown to block tyrosine phosphorylation, a
key event in signal transduction cascades, the role of ONOO
2
as a signal molecule has been under investigation (27, 28). It
also has been demonstrated that ONOO
2
nitrates and inac-
tivates manganese superoxide dismutase in chronic rejection
of human renal allografts, which was proposed to be a general
mechanism for the amplification of ONOO
2
oxidative damage
(29). Given the short lifetime of ONOO
2
(t
1y2
' 1 s at pH 7.4,
37°C) (30), a diffusion distance of '100
m
m has been estimated
in physiological buffers (31). However, cells are compartmen-
talized into membrane-protected organelles (32). Thus, an
important determinant of toxicity or signaling effectiveness of
ONOO
2
will lie in its invasiveness and ability to access
biological targets. Here, we demonstrate that ONOO
2
can
diffuse freely across phospholipid membrane bilayers to react
with target substrates. Thus, the significance of ONOO
2
as a
biological effector molecule will derive not only from its
reactivity but also its diffusibility.
MATERIALS AND METHODS
Materials. 5,10,15,20-Tetrakis(N-methyl-49-pyridyl)por-
phinatomanganese(III) chloride [Mn(III)TMPyP(Cl)] and
Fe(III)TMPyP(Cl) were purchased from Midcentury (Posen,
TX). Peroxymonosulfate (HSO
5
2
or OXONE), hypochlorite
(OCl
2
), and 1-O-octyl-
b
-D-glucopyranoside were obtained
from Aldrich. Sephadex G25 was obtained from Pharmacia.
Anhydrous monosodium phosphate, anhydrous disodium
phosphate, Sepharose 4B, dimyristol
L-
a
-phosphatidylcholine
(DMPC), and Liposome Kit Positive (lyophilized powder
containing 63
m
mol of L-
a
-phosphatidylcholine, 18
m
mol of
stearyl amine, and 9
m
mol of cholesterol) were purchased from
Sigma. (
a
,
b
,
a
,
b
-Meso-tetrakis[O-((3
b
-hydroxy-5-cholenyl)-
amido)phenyl]-porphyrinato)manganese(III) chloride
[Mn(III)ChP(Cl)] was synthesized according to literature
methods (33). The sodium salt of ONOO
2
was prepared from
the reaction of acidic H
2
O
2
with sodium nitrite following a
published procedure (34). All solvents were analytical grade.
Water used in all the experiments was distilled and deionized
(Milli-Q, Millipore).
Vesicles Preparation. Small unilamellar vesicles (SUV) were
prepared by using the method of ultrasonication of DMPC thin
films containing Mn(III)ChP (35). The porphyrin was replaced
by retinoic acid (40
m
M) or tocopherol (80
m
M) in the lipid thin
films for SUV used in the Fe(III)TMPyP protection experi-
ments. Buffer solutions were added to the dry lipid thin films
and sonicated using a probe tip sonicator (Branson model 250)
for '5 min at 20–22°C. SUV generated were allowed to
equilibrate for 1y2 h before centrifugation to remove partic-
ulate matter. The final concentration of DMPC in the vesicular
solutions was 1.67 mM. The Mn(III)ChP concentration was
varied between 2 and 8
m
M.
Large unilamellar vesicles (LUV) were prepared using the
detergent removal method (36) with modifications. In brief, a
mixture of
L-
a
-phosphatidylcholine (63
m
mol), stearyl amine
(18
m
mol), and cholesterol (9
m
mol) was dissolved in a
phosphate buffer (50 mM, pH 7.4) solution containing Mn(II-
I)TMPyP (200
m
M) and 1-O-octyl-
b
-D-glucopyranoside (900
m
mol). This solution was applied to a Sephadex G-25 column
(1 3 20 cm) presaturated with an eluant consisting of Mn(II-
I)TMPyP (200
m
M) in phosphate buffer (50 mM, pH 7.4). The
exclusion of the detergent on the column resulted in the
formation of LUV containing Mn(III)TMPyP (200
m
M) in the
entrapped internal volume. The vesicles were eluted from the
column by using the same solution used to presaturate the
column. The vesicles were purified further on a Sepharose 4B
column (2 3 10 cm) using phosphate buffer (50 mM, pH 7.4).
Neutral LUV without the positively charged stearyl amine
were prepared similarly. Transmission electron micrographs of
the LUV with entrapped Mn(III)TMPyP and SUV were
obtained after fixing with 0.5% OsO
4
and staining with 2%
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1997 by The National Academy of Sciences 0027-8424y97y9414243-6$2.00y0
PNAS is available online at http:yywww.pnas.org.
This paper was submitted directly (Track II) to the Proceedings office.
Abbreviations: ONOO
2
, peroxynitrite; Mn(III)ChP(Cl), (
a
,
b
,
a
,
b
-
meso-tetrakis[O-((3
b
-hydroxy-5-cholenyl)amido) phenyl]-porphyri-
nato)manganese(III) chloride; Mn(III)TMPyP, 5,10,15,20-tetrakis(N-
methyl-49-pyridyl)porphinatomanganese(III) chloride; HSO
5
2
, per-
oxymonosulfate; SUV, small unilamellar vesicles; LUV, large
unilamellar vesicles.
*S.S.M. and J.L. contributed equally to this work.
†
To whom reprint requests should be addressed. e-mail: jtgroves@
princeton.edu.
14243

uranyl acetate. Typical SUV diameters were 35 nm, and the
LUV diameters were 150 nm.
Stopped-Flow Spectrophotometry. Time-resolved UV-vis
spectra were recorded on a HI-TECH SF-61 DX2 stopped-
flow spectrophotometer (HI-TECH, Salisbury, U.K.) by using
the photo-diode array fast scan mode. The spectral resolution
was '1 nm. The stoichiometric oxidation of Mn(III)TMPyP by
ONOO
2
was achieved by stopped-flow single mixing to give
final concentrations of 5
m
M Mn(III)TMPyP and 6.7
m
M
ONOO
2
in phosphate buffer (25 mM, pH 7.4). The reaction
was followed by UV-vis (0–0.5 s, 100 scans, 0.005-s integration
time). The kinetic profiles of the Mn(III)TMPyP oxidation by
ONOO
2
in solution or inside the LUV were collected by using
the stopped-flow photomultiplier mode. All measurements
were performed at ambient temperatures (22–24°C). The
reactions were conducted in either a phosphate buffer (25 mM,
pH 7.4) or a Tris buffer (25 mM, pH 8.2). Typically, the
concentration of Mn(III)TMPyP was held constant (2
m
M)
while the ONOO
2
concentration was varied (50–250
m
M) to
provide pseudo first-order conditions. For experiments involv-
ing Mn(III)TMPyP entrapped in LUV, the concentration of
Mn(III)TMPyP in the bulk solution was used (typically 2–2.5
m
M). The decay of Mn(III)TMPyP and the appearance of
oxoMn(IV) complexes were monitored at 462 nm and 428 nm,
respectively. The kinetic profiles could be fitted nicely into a
single exponential to give the pseudo first-order rate constants
[k (s
21
)] under different ONOO
2
concentrations. The oxi-
dants H
2
O
2
, perchlorate (OCl
2
), and peroxymonosulfate
(HSO
5
2
) were used for the control experiments.
The reactions of the membrane-bound Mn(III)ChP with
ONOO
2
also were performed in the stopped-flow photomul-
tiplier mode by following changes at 474 nm [Mn(III)ChP] and
FIG. 1. A schematic representation of the reporter manganese porphyrins in the model vesicular systems. Mn(III)TMPyP, when entrapped inside
the vesicles, is prevented from sampling the bulk volume during the experiments. Mn(III)ChP spans the membrane with the position of the
macrocycle at the center of the membrane and porphyrin plane parallel to the membrane surface (33). The reaction of the manganese(III) porphyrin
with peroxynitrite to afford oxomanganese(IV) and NO
2
is depicted below.
14244 Chemistry: Marla et al. Proc. Natl. Acad. Sci. USA 94 (1997)

424 nm [oxoMn(IV) species]. The initial rate of oxoMn(IV)
formation was linearly dependent on the ONOO
2
concentra-
tion. This allowed an estimation of the second-order rate
constant (k) for the oxidation reaction after taking into
account the spontaneous decay rates of ONOO
2
and the
oxoMn(IV) species.
Protection of Membrane Components by Fe(III)TMPyP.
Retinoic acid (40
m
M) and tocopherol (80
m
M) were incor-
porated into SUV preparations containing DMPC (1.67 mM).
The reactions of ONOO
2
with retinoic acid were followed at
340 nm, and those with tocopherol were followed at 300 nm.
The membrane-bound substrates were mixed with 250
m
M
ONOO
2
, which resulted in a complete loss of chromophore in
both cases indicating oxidation. For the protection experi-
ments, the concentration of Fe(III)TMPyP was varied between
2 and 10
m
M. Under these conditions, the ONOO
2
concen-
tration was exhausted quickly (t
1
y
2
5 0.16 2 0.035).
RESULTS AND DISCUSSION
The membrane permeability of ONOO
2
has been examined
in a phospholipid vesicular model system by exploiting the
diagnostic reactions of manganese porphyrins we have de-
scribed recently (37, 38). Manganese porphyrins known to
occupy different regions of the membrane system have been
used to assess the accessibility of ONOO
2
to these sites. Thus,
a membrane-spanning steroidal porphyrin [Mn(III)ChP] (33,
39) and a water-soluble [Mn(III)TMPyP] porphyrin were
deployed within the hydrophobic bilayer and inside the aque-
ous interior compartment, respectively, of unilamellar vesicles
(Fig. 1). Oxidation of Mn(III) to the corresponding oxo
Mn(IV) species was followed by observing the signature
changes in the UV-vis spectra on addition of ONOO
2
to the
medium.
The solution reaction of Mn(III)TMPyP with ONOO
2
was
found to be fast and stoichiometric by rapid mixing stopped-
flow spectrophotometry (Fig. 2). The depletion of Mn(III)-
TMPyP to generate the oxoMn(IV) species followed clear
stoichiometric behavior with isosbestic points at 389, 448, 536,
and 603 nm. Kinetic measurements determined that the in-
trinsic rate of Mn(III)TMPyP oxidation by ONOO
2
was 1.8 3
10
6
M
21
zs
21
at pH 7.4. Notably, the rates for the ONOO
2
oxidation of metalloporphyrins and heme peroxidases like
myeloperoxidase are the fastest reactions reported for
ONOO
2
(14). Control experiments showed that H
2
O
2
was
incapable of oxidizing Mn(III)TMPyP to oxoMn(IV) species
under these conditions.
The rate of oxidation of Mn(III)ChP by ONOO
2
,inthe
bilayer region, was much slower (k 5 3 3 10
4
M
21
zs
21
) but
demonstrated the ability of ONOO
2
to enter and react within
the hydrophobic region of the membrane (Fig. 1). Likewise,
retinoic acid and tocopherol were oxidized readily by ONOO
2
under these conditions. Mn(III)TMPyP redox coupled with
antioxidants, which catalytically reduces ONOO
2
, has been
shown to afford a dose-dependent protection of these lipid
components (40). Fe(III)TMPyP, which has been reported to
isomerize ONOO
2
to nitrate (41), also effectively prevented
the oxidation of these lipid components by ONOO
2
. Liposo-
mal assemblies of both manganese and iron porphyrin amphi-
philes have been found to be effective as well but only when the
molecular design of the membrane anchor allowed the charged
porphyrin moiety to occupy the aqueous region of the mem-
brane interface (42).
Significantly, the reaction of ONOO
2
with Mn(III)TMPyP
entrapped within LUV resulted in the efficient production of
the oxoMn(IV) species (Fig. 3A). Similar results were obtained
with the oxidant OCl
2
. By contrast, HSO
5
2
(43) failed to react
under these conditions (Fig. 3 B and C), demonstrating that the
LUV were impermeable to this less chaotropic and less basic
anion. The high membrane permeability of ONOO
2
and OCl
2
may be attributed to the relatively high pKa of their conjugate
acids, 6.8 (18, 19) and 7.4 (44), respectively. The order
described by the Eisenman sequence I for anions crossing
artificial membranes is: SCN
2
. I
2
. NO
3
2
. Br
2
. Cl
2
.
F
2
. CH
3
OSO
3
2
' CH
3
SO
3
2
' SO
4
22
(45). Whereas HSO
5
2
behaves like the membrane-impermeable CH
3
SO
3
2
, ONOO
2
resembles NO
3
2
in its permeability. Curiously, however, the
oxidation rate of the entrapped Mn(III)TMPyP by ONOO
2
showed little dependence on pH. The second-order rate
constants at pH 7.4 (25 mM, phosphate) and at pH 8.2 (25 mM
Tris) were k
obsd
5 0.8 3 10
6
M
21
zs
21
(R 5 0.996) and k
obsd
5
1.2 3 10
6
M
21
zs
21
(R 5 0.996), respectively. We also deter-
mined that the presence of the positively charged stearyl amine
did not assist in ONOO
2
transport across membranes because
its removal to generate neutral vesicles did not change the
observed rates of the oxidation reactions. We suggest that the
hydrophobic vesicular barrier may modulate the pKa of
ONOO
2
(46), thus facilitating the transport of the neutral
HOONO species across membranes.
Comparison of rates of reaction between Mn(III)TMPyP
and ONOO
2
in solution and in the Mn(III)TMPyPyLUV
system (Fig. 3D) showed that membrane entrapment retarded
the rate of manganese oxidation by only 2-fold. Accordingly,
the barrier offered to ONOO
2
transport by the membrane is
small. This rate difference allows an estimate of the perme-
ability coefficient (P) for ONOO
2
crossing a membrane
bilayer using Fick’s Law. The permeability coefficient is de-
fined as P 5 JyDc, where J (molzcm
22
zs
21
) is the flux of
ONOO
2
across unit surface area of the membrane and Dc
(molyL) is the difference in the concentration ([ONOO
2
]
out
2
FIG. 2. Time resolved UV-vis profile of the stoichiometric oxida-
tion of Mn(III)TMPyP by ONOO
2
to generate the oxoMn(IV)
species. Stopped-flow mixing was used to give final concentrations of
Mn(III)TMPyP (5
m
M) and ONOO
2
(6.7
m
M) in 25 mM phosphate
(pH 7.4) buffer. The reaction was followed by UV-vis (0–0.5 s, 100
scans, 0.005-s integration time) by using a HI-TECH SF-61 DX2
rapid-mixing stopped-flow spectrophotometer (1 in 10 scans shown for
clarity). The rate constant for the oxidation of Mn(III)TMPyP by
ONOO
2
obtained under pseudo first-order conditions at pH 7.4 was
k
intrinsic
5 1.8 3 10
6
M
21
zs
21
(see Fig. 3D).
Chemistry: Marla et al. Proc. Natl. Acad. Sci. USA 94 (1997) 14245

FIG. 3. ONOO
2
is shown to cross membranes freely. Difference spectra of the UV-vis absorbances before and after addition of oxidants to
LUV—encapsulated Mn(III)TMPyP. (A) ONOO
2
(1 mM) addition resulted in an instantaneous oxidation of Mn(III) (462 nm) to the oxoMn
species (428 nm), indicating that ONOO
2
diffused rapidly across the lipid bilayer. Rapid oxidation was seen between pH 7.4 and 8.8, suggesting
that the ONOO
2
anion as well as HOONO can cross membranes (pKa ONOO
2
6.8). Similar results were obtained with OCl
2
.(B) Addition of
HSO
5
2
(1 mM) to the Mn(III)TMPyPyLUV resulted in no oxidation, verifying that the LUV prevented intermixing of the entrapped porphyrin
and HSO
5
2
in this construct. (C) Oxidation by HSO
5
2
was observed only after the LUV had been sonicated to relocate Mn(III)TMPyP into the
bulk solution. (D) Comparison of the pseudo first-order rates for the solution reaction compared with the reaction in LUV. All of the experiments
were conducted using rapid mixing stopped-flow methods as described in Materials and Methods. Reaction rates obtained from the slope of the
pseudo first-order plots were: k
intrinsic
5 1.8 3 10
6
M
21
zs
21
(R 5 0.999); and k
obsd
5 0.8 3 10
6
M
21
zs
21
(R 5 0.996).
14246 Chemistry: Marla et al. Proc. Natl. Acad. Sci. USA 94 (1997)

[ONOO
2
]
in
). The ratio of these concentrations is directly
related to k
obsd
and k
intrinsic
, the observed and intrinsic rate
constants for the reaction of ONOO
2
with Mn(III)TMPyP we
have measured (Eq. 1).
[ONOO
2
]
in
[ONOO
2
]
out
5
k
obsd
k
intrinsic
5 0.48 [1]
In the steady state, the membrane crossing rate of ONOO
2
was
taken to be equal to the rate of the oxidation of the entrapped
porphyrin inside the LUV, reflecting the barrier offered by the
lipid bilayer to the diffusion of ONOO
2
(Eq. 2). Thus, P is
expressed as in Eq. 3, where A is the surface area of vesicles,
and V
in
is the volume entrapped in LUV.
d
dt
@ONOO
2
]
flux
5 k
intrinsic
z[MNTMPyP]
in
z[ONOO
2
]
in
[2]
P 5
d
dt
[ONOO
2
]
flux
z
V
in
AzDc
5
k
intrinsic
z[MnTMPyP]
in
z[ONOO
2
]
in
zV
in
AzDc
[3]
Calculated values for A 5 4.0 3 10
6
cm
2
yL and V
in
5 10 mlyL
derive from the observed radius of the vesicles and afforded a
value for P 5 8.0 3 10
24
cmzs
21
at pH 7.4. Simulations using
this permeability coefficient agreed well with the experimental
data. A simple, sequential reaction model was assumed, taking
into consideration the ONOO
2
diffusion across phospholipid
membrane and subsequent reaction with the entrapped
Mn(III)-TMPyP.
We also have considered the recently reported reaction
between ONOO
2
and CO
2
to generate an unstable interme-
diate, presumably ONOOCO
2
2
(k ' 1 3 10
4
M
21
zs
21
) (47, 48).
Because the physiological concentration of HCO
3
2
is relatively
high ('25 mM) and is in equilibrium with CO
2
, this reaction
has been suggested to be an important pathway for ONOO
2
and a determinant of toxicity. However, the rate of diffusion
for ONOO
2
crossing lipid bilayers determined here is at least
30 times faster than the ONOO
2
reaction with the physiolog-
ical concentration of CO
2
(see Table 1). Preliminary experi-
ments showed that the oxidation of manganese by ONOO
2
in
the Mn(III)TMPyPyLUV system remained unaffected by 25
mM HCO
3
2
, indicating further that the transmembrane diffu-
sion of ONOO
2
is faster than its reaction with CO
2
. Therefore,
we conclude that the transmembrane diffusion of ONOO
2
is
considerably faster than all documented reaction rates of
ONOO
2
with its biological targets.
Significantly, the permeability coefficient P obtained for
ONOO
2
in this way is close to that reported for water (49) and
is '400 times faster than that of superoxide (50, 51) (Table 1).
Accordingly, the availability of superoxide across biological
membranes is limited both by its relatively slow diffusion and
the high concentrations of superoxide dismutase within the
cell (25, 52, 53). The high membrane permeability observed
here for ONOO
2
may have important consequences regarding
its biochemistry and pathophysiology. Thus, in marked con-
trast to superoxide ion (54), ONOO
2
can be expected to have
free access to cell interiors across membranes and over dis-
tances of cellular dimensions, necessary properties for a bio-
logical effector molecule.
We thank Dr. M. K. Stern, Monsanto Chemical Co., for stimulating
discussions. Support of this research by the National Institutes of
Health (GM36928) is acknowledged gratefully.
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Table 1. Permeability across lipid bilayers
P (cmzs
21
) k
diff
5
AzP
V
in
(s
21
)
H
2
O 2.3 3 10
23
920*
ONOO
2
8.0 3 10
24
320
O
2
2z
2.1 3 10
26
0.84*
A, membrane surface area (cm
2
yL); P, permeability coefficient
(cmzs
21
); k
diff
, rate of ONOO
2
exchange across the membrane; V
in
,
entrapped volume (mLyL).
*These values were calculated by using A and V
in
parameters of our
LUV system and published values of permeability coefficients (49–
51).
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