Kinetics of Peroxynitrite Reaction with Amino Acids and Human
Serum Albumin*
Beatriz
Alvarez
§¶,
Gerardo
Ferrer-Sueta
,
Bruce
A.
Freeman**, and
Rafael
Radi§
From the
Laboratorio de Enzimología, Unidad
Asociada Enzimología, and
Departamento de
Fisicoquímica Biológica, Facultad de Ciencias,
§ Departamento de Bioquímica, Facultad de Medicina,
Universidad de la República, 11800 Montevideo, Uruguay and the
** Departments of Anesthesiology and Biochemistry and the UAB Center for
Free Radical Biology, the University of Alabama at Birmingham,
Birmingham, Alabama 35233
 |
ABSTRACT |
An initial rate approach was used to study the
reaction of peroxynitrite with human serum albumin (HSA) through
stopped-flow spectrophotometry. At pH 7.4 and 37 °C, the second
order rate constant for peroxynitrite reaction with HSA was 9.7 ± 1.1 × 103 M
1
s
1. The rate constants for sulfhydryl-blocked HSA and for
the single sulfhydryl were 5.9 ± 0.3 and 3.8 ± 0.8 × 103 M
1 s
1,
respectively. The corresponding values for bovine serum albumin were
also determined. The reactivity of sulfhydryl-blocked HSA increased at
acidic pH, whereas plots of the rate constant with the sulfhydryl
versus pH were bell-shaped. The kinetics of peroxynitrite reaction with all free L-amino acids were determined under
pseudo-first order conditions. The most reactive amino acids were
cysteine, methionine, and tryptophan. Histidine, leucine, and
phenylalanine (and by extension tyrosine) did not affect peroxynitrite
decay rate, whereas for the remaining amino acids plots of
kobs versus concentration were
hyperbolic. The sum of the contributions of the constituent amino acids
of the protein to HSA reactivity was comparable to the experimentally
determined rate constant, where cysteine and methionine (seven residues
in 585) accounted for an estimated 65% of the reactivity. Nitration of
aromatic amino acids occurred in HSA following peroxynitrite reaction,
with nitration of sulfhydryl-blocked HSA 2-fold higher than native HSA.
Carbon dioxide accelerated peroxynitrite decomposition, enhanced
aromatic amino acid nitration, and partially inhibited sulfhydryl
oxidation of HSA. Nitration in the presence of carbon dioxide increased when the sulfhydryl was blocked. Thus, cysteine 34 was a preferential target of peroxynitrite both in the presence and in the absence of
carbon dioxide.
 |
INTRODUCTION |
Peroxynitrite anion (ONOO
) and its conjugated
acid, peroxynitrous acid (ONOOH, pKa = 6.8) (1), are
the products of nitric oxide (·NO) and superoxide (O
2)
radical reaction (2).
Peroxynitrite1 is a potent
and versatile oxidizing and nitrating agent, and several lines of
evidence point at peroxynitrite as a key biomolecule in mediating the
reactivity and toxicity of superoxide and nitric oxide (3-6).
Peroxynitrite anion is a relatively stable species; its hydronated form
readily isomerizes to nitrate at a rate of 4.5 s
1 at
37 °C (7). Target molecules are proposed to be oxidized by
peroxynitrite via two main pathways. First, ground-state peroxynitrite can oxidize the substrates in a process that is first order in peroxynitrite and first order in substrate. In the second pathway, peroxynitrous acid rearranges in a rate-limiting step into a highly reactive species, which is the ultimate oxidant. This process is first
order in peroxynitrite and zero order in substrate (1, 8-10).
To better understand the mechanisms of peroxynitrite cytotoxicity and
its role in pathological processes, it is important to characterize its
reactivity toward different biomolecules. In this sense, proteins are
key targets of oxidative stress. Exposure to oxidants such as hydrogen
peroxide leads to amino acid modifications and subsequent changes in
protein structure and function. These alterations have been related to
protein turnover, aging, and diverse pathological processes (11-13).
Peroxynitrite reaction with proteins can nitrate as well as oxidize
residues. Thus, nitrotyrosine detection has been used as a probe for
peroxynitrite formation in vivo (14). The reactions of
peroxynitrite with free cysteine, methionine, tyrosine, and tryptophan
are characterized (1, 15-19), with no data available for other amino
acids. Peroxynitrite-mediated oxidation and nitration of amino acids
proceeds through both one- and two-electron mechanisms, the former
leading to the formation of protein radicals (20-22). In addition to
modifying amino acids, peroxynitrite can affect prosthetic groups.
Metalloproteins react with peroxynitrite at rates of 105 to
107 M
1 s
1 (23).
These reactions are among the fastest reported for peroxynitrite and
can lead to reversible redox modification of metal centers, as for
cytochrome c (24) or disruption of the center followed by
liberation of the metal, as for aconitase and alcohol dehydrogenase (4,
25).
Human serum albumin (HSA)2
was used as model for probing peroxynitrite reactivity toward proteins.
HSA is the most abundant protein in plasma. Its physiological roles
include the maintenance of colloid osmotic pressure and the transport
of different ligands. In addition, HSA may have important roles as an
extracellular antioxidant, by ligating free metals and scavenging
reactive species and serving as a transport molecule for nitric oxide.
This well characterized 66-kDa protein has 18 tyrosines, 6 methionines, 1 tryptophan, 17 sulfur bridges, and only 1 free cysteine in a total of
585 amino acids, and no prosthetic groups.
In this paper, the kinetics of peroxynitrite reaction were studied for
all free L-amino acids, HSA, BSA, and their single sulfhydryls. In addition, the influence of carbon dioxide/bicarbonate on the interactions between peroxynitrite and albumin was assessed. This study enables us to rationalize peroxynitrite reactivity toward
proteins on kinetic terms and also contributes to understanding the
fate of peroxynitrite in the intravascular space.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
Human and bovine serum albumin (fraction V) were
obtained from Sigma. Mercuric chloride was obtained from Merck. To
prevent metal interference, 0.1 mM
diethylenetriaminepentaacetic acid (dtpa) was added to the solutions.
Peroxynitrite was synthesized from hydrogen peroxide and nitrous acid
as described (1, 8). Contaminating hydrogen peroxide was eliminated
with manganese dioxide, and peroxynitrite concentration was determined
at 302 nm (
= 1.67 mM
1 cm
1)
(26).
Albumin Preparation--
HSA was defatted with charcoal in acid
solution (27). To reduce sulfhydryls oxidized during purification and
storage, HSA was treated overnight with 10 mM
2-mercaptoethanol at 4 °C, and excess reductant was removed by gel
filtration on Sephadex G-25 using 0.01 M potassium
phosphate, 0.1 mM dtpa, pH 7.4. HSA typically was 1 mM and had a sulfhydryl to albumin molar ratio
(R) of 0.6-0.8, as described previously (1). The sulfhydryl
of HSA was blocked by the addition of an equimolar amount of mercuric
chloride (HgCl2), leaving no residual sulfhydryls.
Alternatively, HSA was incubated for 30 min at 20 °C with a 7-fold
excess of N-ethylmaleimide (NEM), followed by gel
filtration. In this case, the sulfhydryl to albumin ratio was
diminished to 0.015.
Biochemical Analyses--
Albumin concentration was measured by
absorbance at 279 nm (
= 0.531 (28) and 0.667 (29),
(g/liter)
1 cm
1 for HSA and BSA,
respectively). The molecular weights were 66,486 for HSA (30) and
66,429 for BSA (31). The amino acid compositions were obtained from
GenBank (accession numbers A06977 for HSA and M73993 for BSA).
Sulfhydryls were quantified using 5,5'-dithiobis-(2-nitrobenzoic acid)
(
412 = 13.6 mM
1
cm
1) (32). Carbon dioxide-containing solutions were
prepared immediately before the experiment and used within 10 min to
minimize diffusion of CO2 out of the solution (33).
Nitration of tyrosine residues in HSA was assessed by measuring the
increase in absorbance at 430 nm after alkalinization to pH
10 (
430 = 4.4 mM
1
cm
1) (17).
Kinetic Studies--
The kinetics of peroxynitrite decomposition
were studied in a stopped-flow spectrophotometer (Applied Photophysics,
SF17MV) at 302 nm. Fits to kinetic traces were performed with the
software provided with the instrument. Time-dependent
spectra were obtained with a photodiode array (Applied Photophysics).
All experiments were performed at 37 °C, and the pH was measured
before and after reactions.
Simulations--
Computer simulations for a given reaction
scheme and a set of rate constants were produced with Gepasi version
3.1 (34, 35).
Data Analyses--
All experiments were repeated at least three
times, and representative data are reported. Results are expressed as
mean ± S.D. Graphics and mathematical fits to experimental data
were performed with SlideWrite (Advanced Graphics Software) or Origin (Microcal Software).
 |
RESULTS |
Kinetics of Peroxynitrite Reaction with HSA and Its Single
Sulfhydryl at pH 7.4 and 37 °C--
The usual approach to study the
rate of peroxynitrite reaction with target molecules is through an
integral rate method, under pseudo-first order conditions (36).
Peroxynitrite decomposition then follows an exponential function
versus time, and the rate constant is determined through the
fit of the kinetic trace to this function. For peroxynitrite reaction
with HSA, it was not possible to achieve pseudo-first order conditions,
since the limiting maximal concentration of HSA solutions (about 1 mM) did not permit a 10-fold excess over peroxynitrite in
stopped-flow experiments. The kinetic traces of peroxynitrite (0.25 mM) decomposition in the presence of 0.139 mM
HSA (r = 0.608) (Fig. 1)
did not follow a single exponential function, since the logarithmic
plots were not linear (Fig. 1, inset). Rather, the rate of
peroxynitrite decay was initially faster as peroxynitrite reacted with
both the single sulfhydryl residue present in substoichiometric amounts and with the other amino acids of the protein. When sulfhydryl-blocked HSA was used (pretreated with mercuric chloride), the decay of peroxynitrite was slower than for native HSA.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 1.
Kinetic traces of peroxynitrite decomposition
in the presence of HSA. Peroxynitrite (0.25 mM) was
mixed with phosphate buffer, 0.05 M, pH 7.45 ± 0.01, 0.1 mM dtpa, at 37 °C, alone (---) and in the presence
of 0.139 mM native HSA (R = 0.608)
(···) or sulfhydryl-blocked HSA (- - -). Inset,
logarithmic plot of the same data up to 0.75 s. In the presence of
HSA, deviations from linearity are observed after 0.2 s.
|
|
Hence, the kinetics of peroxynitrite-HSA reaction could not be studied
through the integral rate method. Instead, an initial rate approach was
used, using relatively similar concentrations of peroxynitrite and HSA.
Since the absorbance at 302 nm (A) is proportional to
peroxynitrite concentration and the change in albumin absorbance is
negligible at this wavelength in the experimental conditions, the
initial rate of peroxynitrite decomposition would be as shown in
Equation 1.
|
(Eq. 1)
|
where dA/dt is the initial change in
absorbance at 302 nm, and Ao and
A
are the initial and final values, respectively. Following the initial rate assumption that the
concentration of reagents remains effectively constant during this
period, the rate constant could be determined by Equation 2.
|
(Eq. 2)
|
In order to ensure accuracy of the rate constant determinations,
200 absorbance measurements were acquired during the initial part of
the reaction (
20% decrease in peroxynitrite absorbance), and 200 further points were acquired until more than 99.9% of peroxynitrite
had decomposed. The initial slopes were determined for the period of
linear decrease in absorbance, which corresponded to 10-15% of
peroxynitrite decomposition. In the absence of HSA, the rate constants
of peroxynitrite decomposition determined from the initial rate method
compared well with the values determined from the integral rate method.
For instance, the initial rate method
yielded values of kobs = 1.11 ± 0.09 s
1 (n = 7), with the integral method
giving a kobs of 0.99 ± 0.03 s
1. Differences in the values calculated from both
methods were a maximum of 10%, with the initial rate method
systematically having a greater degree of dispersion than the integral
one, as revealed by the increase in the standard deviation. In the
presence of relatively high concentrations of sulfhydryl-blocked HSA,
peroxynitrite decay better approximated an exponential function, due to
the high overall concentration of target residues. In this case, the values determined through both kinetic approaches were in good agreement. To validate further the initial rate method, we confirmed that values of kobs determined at 320 nm were
the same as those determined at 302 nm.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2.
Rate constants for peroxynitrite reaction
with HSA and its sulfhydryl at pH 7.4. Peroxynitrite (0.25 mM) was mixed with phosphate buffer, 0.05 M, pH
7.47 ± 0.02, 0.1 mM dtpa, at 37 °C, alone ( )
and in the presence of varying concentrations of native HSA
(R = 0.636) ( ) or sulfhydryl-blocked HSA ( ). Data
are the mean ± S.D. (n 7) of representative
results from three different experiments.
|
|
For the following reaction schemes (Equations 3-5)
|
(Eq. 3)
|
|
(Eq. 4)
|
|
(Eq. 5)
|
k1, k2, and
k3 are the rate constants of peroxynitrite
isomerization and reaction with sulfhydryl-blocked HSA and with the sulfhydryl, respectively. Defining k'1,
k'2, and k'3 as the
corresponding apparent rate constants at a fixed given pH of 7.4 and
37 °C, the observed rate constant of peroxynitrite disappearance
would be as shown in Equation 6.
|
(Eq. 6)
|
Since R was defined as the sulfhydryl to albumin molar
ratio, then (see Equation 7)
|
(Eq. 7)
|
As observed in Fig. 2, kobs determined
through the initial rate approach increased linearly with HSA
concentration, in agreement with Equation 7. For native HSA, the second
order rate constant of peroxynitrite reaction with HSA
(R = 0.636) at pH 7.47 ± 0.02 and 37 °C was
determined from the slope of the plot to be 8.3 ± 0.3 × 103 M
1 s
1. The plot
was consistently observed to intercept the y axis at a value
lower than that for peroxynitrite spontaneous decomposition, as noted
previously for other molecules (15, 37, 38). For sulfhydryl-blocked HSA
(pretreated with mercuric chloride), the slope of the plot decreased.
In this case, Equation 7 simplifies to Equation 8.
|
(Eq. 8)
|
From Equation 8, k'2, the rate constant of
peroxynitrite reaction with sulfhydryl-blocked HSA was 5.9 ± 0.3 × 103 M
1
s
1. According to Equations 7 and 8,
k'3, the rate constant of peroxynitrite reaction
with the single sulfhydryl of HSA, can be calculated by dividing the
difference in the slopes with and without mercury treatment by
R. In this way, k'3 was determined to
be 3.8 ± 0.8 × 103 M
1
s
1. By adding the values of the rate constants with
sulfhydryl-blocked HSA and with the sulfhydryl, the total rate constant
of peroxynitrite reaction with HSA, assuming R = 1, can
be calculated to be 9.7 ± 1.1 × 103
M
1 s
1.
Kinetics of Peroxynitrite Reaction with BSA--
The rate
constants of peroxynitrite reaction with BSA at pH = 7.43 ± 0.03 and 37 °C were determined in the same way as with HSA (data not
shown), and the total rate constant with BSA (assuming R = 1) was determined to be 7.5 ± 0.8 × 103 M
1 s
1. The rate
constant with the single sulfhydryl of BSA was
k'3 = 3.9 ± 0.5 × 103
M
1 s
1. This value is in good
agreement with the one determined for HSA and higher than the one
reported previously for BSA of 2.6-2.8 × 103
M
1 s
1 (1). The rate constant
for peroxynitrite reaction with sulfhydryl-blocked BSA was
k'2 = 3.6 ± 0.3 × 103
M
1 s
1, significantly lower than
that for HSA and again higher than the previously reported value of
2.5 × 103 M
1
s
1 (1). The differences with the previous values are
likely to be due to the use of an integral rate approach.
Kinetics of Peroxynitrite Reaction with the Free Amino
Acids--
The kinetics of peroxynitrite decomposition in the presence
of all the L-amino acids was studied under pseudo-first
order conditions at pH = 7.40 ± 0.05 and 37 °C. The
initial peroxynitrite concentration was 0.2-0.6 mM, and
the amino acid concentration ranged from 0 to 100 mM where
solubility permitted. Leu, Phe, Met, and Trp were studied at lower
concentrations (up to 67, 60, 25, and 3.5 mM,
respectively). Tyr and cystine could not be studied because of their
low solubility. The decrease in absorbance at 302 nm was monitored for
more than 8 t1/2, and absorbance versus time traces
were fitted to a single exponential function in order to obtain
kobs.
For the amino acid Cys, kobs increased linearly
with concentration as before (1). For Met, kobs
showed a hyperbolic plus linear dependence on concentration as
previously at 25 °C (16). This is the case for Trp as well (19),
although in this work the low concentration range used in order to
prevent interference from nitrotryptophan absorbance did not allow us
to observe the linear dependence. Ala, Arg, Asn, Asp, Gln, Glu, Gly,
Ile, Lys, Pro, Ser, Thr, and Val displayed hyperbolic behavior, as
exemplified for Pro in Fig. 3. His, Leu,
and Phe did not affect peroxynitrite decay rate to any detectable
extent in the conditions studied. This is expected for Tyr as well
according to the results obtained for Phe (this work), phenol, and
salicylate (18).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Pseudo-first order rate constant for
peroxynitrite decay in the presence of proline as a representative
amino acid. Peroxynitrite (0.6 mM) was mixed with
increasing concentrations of proline in phosphate buffer, pH = 7.42 ± 0.01, 0.1 mM dtpa. Experimental data ( ,
mean ± S.D., n = 9) were fitted to a rectangular
hyperbola.
|
|
From the plots of kobs versus amino
acid concentration, three parameters, a1,
a2, and a3, were adjusted
by nonlinear regression to the function shown in Equation 9.
|
(Eq. 9)
|
where [aa] is the amino acid concentration,
a1 represents the difference between
kobs at infinite (k
)
and zero (k'1) amino acid concentrations,
a2 represents the concentration of amino acid,
where kobs is halfway between
k'1 and k
, and a3 represents the slope of the linear portion of
the plot, when present. The parameters (a1,
a2, and a3) obtained for
the different amino acids are shown in Table
I.
View this table:
[in this window]
[in a new window]
|
Table I
Kinetic parameters of the reaction of the free amino acids with
peroxynitrite
Plots of the observed rate constant of peroxynitrite decomposition in
the presence of increasing concentrations of the different amino acids
obtained at pH = 7.40 ± 0.05 and 37 °C were fitted to
Equation 9.
|
|
pH dependence of the Reaction between Peroxynitrite and
HSA--
The pH profile of the apparent rate constant of peroxynitrite
decay in the presence of HSA is shown in Fig.
4.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
pH dependence of the kinetics of
peroxynitrite reaction with HSA and its sulfhydryl. Peroxynitrite
(0.3 mM) was mixed with 0.284 mM native HSA
(R = 0.598) ( ) or sulfhydryl-blocked HSA ( ) in
0.1 M phosphate buffer of varying pH values, 0.1 mM dtpa, at 37 °C. Data are the mean ± S.D.
(n 7) of representative results from three different
experiments. Inset, , the data pairs for each pH were
subtracted and divided by the sulfhydryl concentration to determine the
rate constant for the reaction with the sulfhydryl.
|
|
For sulfhydryl-blocked HSA (see Equation 10), then
|
(Eq. 10)
|
The first term represents the spontaneous decomposition of
peroxynitrous acid as first proposed by Keith and Powell (39). k1 and pKa1 at
37 °C were determined in a separate experiment to be 4.6 ± 0.1 s
1 and 6.8 ± 0.1, respectively, in agreement with
previous reports (1, 7). The second term in Equation 10 represents the
reaction with HSA, assuming peroxynitrous acid is the reacting species. The experimental data were fitted to Equation 10. The value of k2 determined from the fit was 14.4 ± 0.5 × 103 M
1
s
1 and pKa2, the apparent
pKa of peroxynitrous acid ionization in the presence
of HSA, was 7.45 ± 0.05.
For native HSA, assuming peroxynitrite anion is reacting with
hydronated sulfhydryl (see Equation 11), then
|
(Eq. 11)
|
In order to fit the experimental data to Equation 11, the values
of k2 and pKa2
used were those just determined for sulfhydryl-blocked HSA. Thus,
k3 was 5.0 ± 0.9 × 103
M
1 s
1 and the dissociation
constant of the sulfhydryl was pKa3 = 8.6 ± 0.4. To confirm these values, kobs
for sulfhydryl-blocked HSA were subtracted from
kobs for native HSA at each pH and divided by
the sulfhydryl concentration in order to fit to the following function
shown in Equation 12.
|
(Eq. 12)
|
Despite the relatively high degree of dispersion arising from data
processing, the plot of k'3 as a function of pH
fitted this bell-shaped function, with a maximum around pH 7.55 (Fig. 4, inset). The values of k3 and
pKa3 were calculated to be 5.5 ± 0.5 × 103 M
1
s
1and 8.3 ± 0.2, respectively, and are in good
agreement with those determined from the fit to Equation 11. With the
values of k3 and pKa3 determined from Fig. 4, it can be
calculated from Equation 12 that k'3 at pH = 7.4 would be 3.7 × 103 M
1
s
1, in accordance with the value of 3.8 ± 0.8 × 103 M
1 s
1
obtained experimentally at pH 7.4 in Fig. 2.
From the data reported in Fig. 4 it cannot be elucidated which the
reacting species are, since the pH dependence of the kinetics of
peroxynitrite anion reaction with hydronated sulfhydryl are homomorphic
with those of peroxynitrous acid reacting with the thiolate. Although
Equations 11 and 12 correspond to the former, it can be calculated that
for peroxynitrous acid reacting with the thiolate, the pH-independent
rate constant would be k3 = 335 ± 39 × 103 M
1 s
1.
Role of the Sulfhydryl in Protein Nitration in the Presence and
Absence of Carbon Dioxide--
Since the kinetic constant of
peroxynitrite reaction with the single sulfhydryl can account for about
39% of the reactivity of HSA, it could be expected that the sulfhydryl
would inhibit nitration of aromatic residues. HSA (0.270 mM, R = 0.526) was reacted with
peroxynitrite (1 mM) in the stopped-flow spectrophotometer, and time-dependent spectra were recorded throughout the
reaction period (Fig. 5). Exposure of
native HSA to peroxynitrite led to a minimal change in absorbance in
the 400-450 nm range. As expected, the time required for 50% decrease
in peroxynitrite absorbance (t1/2) diminished to
~46% in the presence of HSA compared with that of peroxynitrite
decomposition alone (0.76 ± 0.03 s, n = 4).
With sulfhydryl-blocked HSA, the t1/2 decreased
instead to ~55%, whereas the absorbance at 430 nm increased with a
comparable t1/2, suggesting aromatic residues were
being nitrated. To estimate the increase in nitration better,
absorbance at 430 nm was measured after alkalinization to pH
10 in a parallel experiment (Table II).
Blockage of the sulfhydryl increased nitration 2-fold. This increase
could not be attributed to mercury ions catalyzing the reaction, since
controls showed no increase in HSA nitration with mercury treatment as
opposed to NEM treatment (data not shown).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 5.
Time-dependent spectra obtained
for the reaction of HSA with peroxynitrite. Peroxynitrite (1 mM) was mixed with 0.270 mM HSA in phosphate
buffer, 0.1 M, pH 7.35 ± 0.02, 0.1 mM
dtpa, at 37 °C. A, native HSA (R = 0.526), no added sodium bicarbonate; B, sulfhydryl-blocked
HSA, no added sodium bicarbonate; C, native HSA
(R = 0.526), 10 mM added sodium
bicarbonate; D, sulfhydryl-blocked HSA, 10 mM
added sodium bicarbonate. The time span between consecutive spectra was
138 ms for A and B and 10 ms for C and
D.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Sulfhydryl oxidation and nitration of tyrosine residues by
peroxynitrite
Native or sulfhydryl-blocked HSA (0.270 mM,
R = 0.618) was exposed to peroxynitrite (1 mM) in phosphate buffer, 0.1 M, pH 7.3 ± 0.1, 0.1 mM dtpa, at 37 °C, in the presence or absence
of 10 mM added sodium bicarbonate. Remaining sulfhydryls
were quantified with DTNB, and nitration was measured through the
increase in absorbance at 430 nm after alkalinization to pH 10.
|
|
Carbon dioxide reacts with peroxynitrite at a rate of 5.8 × 104 M
1 s
1 at
37 °C (40, 41). The adduct formed
(ONOOCO2
) can react with target
molecules or recycle to carbon dioxide, thus redirecting peroxynitrite
reactivity (41-44). In the presence of 10 mM added sodium
bicarbonate, where the concentration of carbon dioxide in equilibrium
with it would be 0.48 mM (apparent pKa = 6.1), peroxynitrite decomposition was accelerated, and the
t1/2 was 0.028 ± 0.002 s. Carbon dioxide has
been reported to inhibit sulfhydryl oxidation and enhance nitration of
aromatics (19, 41, 45, 46). Indeed, when 10 mM sodium
bicarbonate was added, sulfhydryl oxidation was inhibited by 50%
whereas nitration increased 4-fold, with the t1/2 of
nitration ~0.017 s (Fig. 5 and Table II). In the presence of carbon
dioxide, aromatic nitration of sulfhydryl-blocked HSA also increased as
compared with native HSA, with nitration 25% higher when the
sulfhydryl was blocked (Fig. 5 and Table II).
 |
DISCUSSION |
In this paper, we report the kinetics of peroxynitrite reaction
with amino acids and human serum albumin.
For HSA, the second order rate constant for reaction with peroxynitrite
at pH 7.4 and 37 °C was 9.7 ± 1.1 × 103
M
1 s
1 (assuming
R = 1). With the single sulfhydryl the rate constant was 3.8 ± 0.8 × 103
M
1 s
1, which can be compared
with that found for BSA and those reported for cysteine (4.5 × 103 M
1 s
1) (1, 15)
and glutathione (1.35 × 103
M
1 s
1) (7). The plot of the
rate constant with the sulfhydryl versus pH was bell-shaped
(Fig. 4). The ionization constant of the sulfhydryl (pKa = 8.6 ± 0.4) can be compared with that of
cysteine (8.36) (47), glutathione (8.75) (48), and BSA (8.0-8.5) (1, 48). The reactivity of sulfhydryl-blocked HSA with peroxynitrite increased as pH decreased, suggesting peroxynitrous acid as the main
reactive species (Fig. 4). However, if peroxynitrous acid was the only
reacting species toward a single target, the expected pKa for the reaction would be 6.8, as for tryptophan (19). The increase in the pKa for peroxynitrite
reaction with sulfhydryl-blocked HSA to 7.45 ± 0.05 could be due
to the fact that either unhydronated HSA residues were reacting with peroxynitrous acid or that peroxynitrite anion was also reacting. In
addition, the pH dependence of the reactivity of HSA toward peroxynitrite may be affected by conformational changes of albumin (49), but spin trapping of thiyl radicals formed during BSA reaction
with peroxynitrite suggest that the environment of the cysteine is not
critically changed at pH lower than 9 (20).
The parallel study of peroxynitrite reaction kinetics with free amino
acids showed that the most reactive amino acids were Cys, Met, and Trp.
The other amino acids were 1-3 orders of magnitude less reactive, as
calculated from Equation 9 and Table I. The amino acids that reacted
slowly with peroxynitrite could possibly still have been modified, by
reaction with highly reactive species formed from peroxynitrite in
rate-limiting steps. Reaction of these species with tyrosine are likely
to lead to tyrosine nitration (17, 18).
We estimated the contribution of the individual amino acids to the
increase in peroxynitrite decomposition rate by HSA or BSA (Table
III). The rate constants predicted for
HSA and BSA from the sum of the individual contributions compared well
with the experimental values. In fact, they were 30-40% higher, since
it is likely that primary, secondary, and tertiary structure of
proteins will affect reactivity, for instance by diminishing the
accessibility of reactive amino acids. These observations indicate that
it is possible to make rough predictions about peroxynitrite reactivity toward a certain protein from its amino acidic composition. In addition
to the presence of prosthetic groups, these data reveal that the most
relevant factor in protein reactivity would be usually the amount of
Cys and Met. As for most proteins Trp is not a common amino acid, its
contribution to overall reactivity will be often minor. In HSA, the
sulfur-containing amino acids (seven residues in a total of 585)
accounted for 65% of the reactivity of HSA toward peroxynitrite (Table
III). Although the other amino acids were much less reactive, their
high content explains net contributions to HSA reactivity.
View this table:
[in this window]
[in a new window]
|
Table III
Contribution of the individual amino acids to peroxynitrite reaction
with HSA and BSA
The contribution of the individual amino acids in 0.4 mM
HSA and BSA to the increase in peroxynitrite decomposition rate was
calculated from the protein composition and the data in Table I
according to the following equation:
To estimate the apparent second order rate constant
(M 1 s 1) with native
(k'2 + k'3, R = 1) and sulfhydryl-blocked (k'2) HSA or BSA, the sum
of the amino acid contributions was divided by the concentration of
protein (0.4 mM).
|
|
The critical role of the single free cysteine was evident in the fact
that nitration increased in sulfhydryl-blocked HSA. This increase may
in part be due to simple kinetic competition of the sulfhydryl for
reaction with peroxynitrite. Computer simulation showed, however, that
kinetic competition was not sufficient to explain the increase in
nitration,3 suggesting that
additional mechanisms may be operative, such as the sulfhydryl acting
as a radical sink (50).
In the presence of carbon dioxide nitration was enhanced due to
ONOOCO2
or its secondary intermediates
being good nitrating species (Fig. 5). As tyrosine nitration by
peroxynitrite in HSA is a rather low yield process (Table II), the
presence of CO2 in biological systems provides a way to
promote nitration processes that reflect peroxynitrite formation and reactions.
Remarkably, under conditions that most peroxynitrite (~90%) will be
forming the adduct with carbon dioxide, nitration was further enhanced
in sulfhydryl-blocked HSA (Fig. 5C), suggesting that the
adduct or its secondary products are reacting with the sulfhydryl, as
proposed previously (41, 46), with this scavenging reaction preventing
aromatic nitration.
Herein, we have established a link between peroxynitrite reactivity
with HSA and its constituent amino acids. In addition, since HSA is the
principally oxidizable protein of plasma, and peroxynitrite is produced
by vascular cells at increased rates during inflammation (51), it is
likely that this work will also provide insights into the molecular
mechanisms of vascular injury.
 |
ACKNOWLEDGEMENTS |
We thank Celia Quijano, Héctor Musto,
and José Tort for helpful discussions and Wim Koppenol for
fruitful exchange on the issues discussed in the "Note Added in Proof."
 |
Note Added in Proof |
Recent observations in our laboratory
with lower peroxynitrite concentrations than reported herein revealed
that the parameter a1 in Table I was often decreased. This
affirms that Cys, having an a1 = 0, will be an even more
significant component of protein reactivity with peroxynitrite.
 |
FOOTNOTES |
*
This work was supported in part by grants from Consejo
Nacional de Investigaciones Científicas y Técnicas
(Uruguay), the Swedish Agency for Research and Cooperation (to R. R.),
and National Institutes of Health Grant RO3-TW0099 (to R. R. and
B. A. F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported in part by a scholarship from PEDECIBA and CONICYT.

To whom correspondence should be sent: Dept. de
Bioquímica, Facultad de Medicina, Av. General Flores 2125, 11800 Montevideo, Uruguay. Fax: 5982-9249563; E-mail:
rradi{at}lobbm.fmed.edu.uy.
The abbreviations used are:
HSA, human serum
albumin; BSA, bovine serum albumin; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); dtpa, diethylenetriaminepentaacetic acid; NEM, N-ethylmaleimide; R, sulfhydryl to albumin molar ratio.
1
The term peroxynitrite is used to refer to both
peroxynitrite anion (ONOO
) and peroxynitrous acid
(ONOOH). IUPAC recommended names are oxoperoxonitrate (1
) and
hydrogen oxoperoxonitrate, respectively.
3
B. Alvarez, G. Ferrer-Sueta, B. A. Freeman,
and R. Radi, unpublished results.
 |
REFERENCES |
-
Radi, R.,
Beckman, J. S.,
Bush, K. M.,
and Freeman, B. A.
(1991)
J. Biol. Chem.
266,
4244-4250[Abstract/Free Full Text]
-
Huie, R. E.,
and Padmaja, S.
(1993)
Free Radical Res.
18,
195-199
-
Ischiropoulos, H.,
Zhu, L.,
and Beckman, J. S.
(1992)
Arch. Biochem. Biophys.
298,
446-451[Medline]
[Order article via Infotrieve]
-
Castro, L.,
Rodríguez, M.,
and Radi, R.
(1994)
J. Biol. Chem.
269,
29409-29415[Abstract/Free Full Text]
-
Rubbo, H.,
Radi, R.,
Trujillo, M.,
Telleri, R.,
Kalyanaraman, B.,
Barnes, S.,
Kirk, M.,
and Freeman, B. A.
(1994)
J. Biol. Chem.
269,
26066-26075[Abstract/Free Full Text]
-
MacMillan-Crow, L. A.,
Crow, J. P.,
Kerby, J. D.,
Beckman, J. S.,
and Thompson, J. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
93,
11853-11858[Abstract/Free Full Text]
-
Koppenol, W. H.,
Moreno, J. J.,
Pryor, W. A.,
Ischiropoulos, H.,
and Beckman, J. S.
(1992)
Chem. Res. Toxicol.
5,
834-842[Medline]
[Order article via Infotrieve]
-
Beckman, J. S.,
Beckman, T. W.,
Chen, J.,
Marshall, P. A.,
and Freeman, B. A.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
1620-1624[Abstract]
-
Augusto, O.,
Gatti, R. M.,
and Radi, R.
(1994)
Arch. Biochem. Biophys.
310,
118-125[CrossRef][Medline]
[Order article via Infotrieve]
-
Pryor, W. A.,
and Squadrito, G. L.
(1995)
Am. J. Physiol.
268,
L699-L722[Abstract/Free Full Text]
-
Stadtman, E. R.
(1990)
Free Radical Biol. & Med.
9,
315-325[CrossRef][Medline]
[Order article via Infotrieve]
-
Dean, R. T.,
Fu, S.,
and Davies, M. J.
(1997)
Biochem. J.
324,
1-18[Medline]
[Order article via Infotrieve]
-
Berlett, B. S.,
and Stadtman, E. R.
(1997)
J. Biol. Chem.
272,
20313-20316[Free Full Text]
-
Beckman, J. S.,
Ye, Y. Z.,
Anderson, P. G.,
Chen, J.,
Accavitti, M. A.,
Tarpey, M. M.,
and White, C. R.
(1994)
Biol. Chem. Hoppe-Seyler
375,
81-88[Medline]
[Order article via Infotrieve]
-
Quijano, C.,
Alvarez, B.,
Gatti, R. M.,
Augusto, O.,
and Radi, R.
(1997)
Biochem. J.
322,
167-173[Medline]
[Order article via Infotrieve]
-
Pryor, W. A.,
Jin, X.,
and Squadrito, G. L.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11173-11177[Abstract/Free Full Text]
-
Beckman, J. S.,
Ischiropoulos, H.,
Zhu, L.,
van der Woerd, M.,
Smith, C.,
Chen, J.,
Harrison, J.,
Martin, J. C.,
and Tsai, M.
(1992)
Arch. Biochem. Biophys.
298,
438-445[Medline]
[Order article via Infotrieve]
-
Ramezanian, M. S.,
Padmaja, S.,
and Koppenol, W. H.
(1996)
Chem. Res. Toxicol.
9,
160-169
-
Alvarez, B.,
Rubbo, H.,
Kirk, M.,
Barnes, S.,
Freeman, B. A.,
and Radi, R.
(1996)
Chem. Res. Toxicol.
9,
390-396[CrossRef][Medline]
[Order article via Infotrieve]
-
Gatti, R. M.,
Radi, R.,
and Augusto, O.
(1994)
FEBS Lett.
348,
287-290[CrossRef][Medline]
[Order article via Infotrieve]
-
Pietraforte, D.,
and Minetti, M.
(1997)
Biochem. J.
321,
743-750[Medline]
[Order article via Infotrieve]
-
Pietraforte, D.,
and Minetti, M.
(1997)
Biochem. J.
325,
675-684[Medline]
[Order article via Infotrieve]
-
Radi, R.
(1996)
Chem. Res. Toxicol.
9,
828-835[CrossRef][Medline]
[Order article via Infotrieve]
-
Thomson, L.,
Trujillo, M.,
Telleri, R.,
and Radi, R.
(1995)
Arch. Biochem. Biophys.
319,
491-497[CrossRef][Medline]
[Order article via Infotrieve]
-
Crow, J. P.,
Beckman, J. S.,
and McCord, J. M.
(1995)
Biochemistry
34,
3544-3552[Medline]
[Order article via Infotrieve]
-
Hughes, M. N., and Nicklin, H. G. (1968) J. Chem.
Soc. 450-456
-
Chen, R. F.
(1967)
J. Biol. Chem.
242,
173-181[Abstract/Free Full Text]
-
Meucci, E.,
Mordente, A.,
and Martorana, G. E.
(1991)
J. Biol. Chem.
266,
4692-4699[Abstract/Free Full Text]
-
Peters, T., Jr.
(1975)
in
The Plasma Proteins (Putman, F. W., ed), Vol. 1, pp. 133-181, Academic Press, New York
-
Bunk, D. M.
(1996)
Anal. Chem.
69,
2457-2463[CrossRef]
-
Wada, Y.
(1996)
J. Mass Spectrom.
31,
263-266[CrossRef][Medline]
[Order article via Infotrieve]
-
Ellman, G.,
and Lysko, H.
(1979)
Anal. Biochem.
93,
98-102[Medline]
[Order article via Infotrieve]
-
Radi, R.,
Denicola, A.,
and Freeman, B. A.
(1998)
Methods Enzymol.
301,
353-367[CrossRef]
-
Mendes, P.
(1993)
Comput. Appl. Biosci.
9,
563-571[Abstract]
-
Mendes, P.
(1997)
Trends Biochem. Sci.
22,
361-363[CrossRef][Medline]
[Order article via Infotrieve]
-
Radi, R.
(1996)
Methods Enzymol.
269,
354-366[Medline]
[Order article via Infotrieve]
-
Alvarez, B.,
Denicola, A.,
and Radi, R.
(1995)
Chem. Res. Toxicol.
8,
859-869[Medline]
[Order article via Infotrieve]
-
Alvarez, B.,
Ferrer-Sueta, G.,
and Radi, R.
(1998)
Free Radical Biol. & Med.
24,
1331-1337[CrossRef][Medline]
[Order article via Infotrieve]
-
Keith, W. G., and Powell, R. E. (1969) J. Chem.
Soc. 90
-
Lymar, S. V.,
and Hurst, J. K.
(1995)
J. Am. Chem. Soc.
117,
8867-8868
-
Denicola, A.,
Freeman, B. A.,
Trujillo, M.,
and Radi, R.
(1996)
Arch. Biochem. Biophys.
333,
49-58[CrossRef][Medline]
[Order article via Infotrieve]
-
Lymar, S. V.,
Jiang, Q.,
and Hurst, J. K.
(1996)
Biochemistry
35,
7855-7861[CrossRef][Medline]
[Order article via Infotrieve]
-
Pryor, W. A.,
Lemercier, J.-N.,
Zhang, H.,
Uppu, R.,
and Squadrito, G. L.
(1997)
Free Radical Biol. & Med.
23,
331-338[CrossRef][Medline]
[Order article via Infotrieve]
-
Lymar, S. V.,
and Hurst, J. K.
(1998)
Inorg. Chem.
37,
294-301[CrossRef]
-
Gow, A.,
Duran, D.,
Thom, S. R.,
and Ischiropoulos, H.
(1996)
Arch. Biochem. Biophys.
333,
42-48[CrossRef][Medline]
[Order article via Infotrieve]
-
Zhang, H.,
Squadrito, G. L.,
Uppu, R. M.,
Lemercier, J.-N.,
Cueto, R.,
and Pryor, W. A.
(1997)
Arch. Biochem. Biophys.
339,
183-189[CrossRef][Medline]
[Order article via Infotrieve]
-
Torchinsky, Y. M.
(1981)
Sulfur in Proteins, pp. 3-10, Pergamon Press Ltd., Oxford
-
Wilson, J. M.,
Wu, D.,
Motiu-DeGrood, R.,
and Jupe, D. J.
(1980)
J. Am. Chem. Soc.
102,
359-363
-
Peters, T.
(1996)
All About Albumin: Biochemistry, Genetics and Medical Applications, pp. 57-78, Academic Press, San Diego
-
Davies, M. J.,
Gilbert, B. C.,
and Haywood, R. M.
(1993)
Free Radical Res. Commun.
18,
353-367[Medline]
[Order article via Infotrieve]
-
Kooy, N. W.,
and Royall, J. A.
(1994)
Arch. Biochem. Biophys.
310,
352-359[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.