Kinetics of S-nitrosation of thiols in nitric oxide solutions

Article abstract of DOI:10.1021/tx960036y

The S-nitroso adducts of nitric oxide (NO) may serve as carriers of NO and play a role in cell signaling and/or cytotoxicity. A quantitative understanding of the kinetics of S-nitrosothiol formation in solutions containing NO and O₂ is important for understanding these roles of S- nitroso compounds in vivo. Rates of S-nitrosation in aqueous solutions were investigated for three thiols: glutathione, N-acetylcysteine, and N- acetylpenicillamine. Nitrous anhydride (N₂O₃), an intermediate in the formation of nitrite from NO and O₂, is the most likely NO donor for N- nitrosation of amines as well as for S-nitrosation of thiols, at physiological pH. This motivated the use of a competitive kinetics approach, in which the rates of thiol nitrosation were compared with that of a secondary amine, morpholine. The kinetic studies were carried out with known amounts of NO and O₂ in solutions containing one thiol (400 μM) and morpholine (200-5700 μM) in 0.01 M phosphate buffer at pH 7.4 and 23 °C. It was found that disulfide formation, transnitrosation reactions, and decomposition of the S-nitrosothiol products were all negligible under these conditions. The rate of formation of S-nitrosothiols was expressed as k₇[N₂O₃][RSH], where RSH represents the thiol. The rate constant for S- nitrosation relative to that for N₂O₃ hydrolysis (k₄) was found to be k₇/k₄ = (4.15 ± 0.28) x 10⁴, (2.11 ± 0.11) x 10⁴, and (0.48 ± 0.04) x 10⁴ M^-1 for glutathione, N-acetylcysteine, and N-acetylpenicillamine, respectively. The overall (observed) rates of nitrosothiol formation reflect the fact that [N₂O₃] ∞ [NO]²[O₂] and that [N₂O³] also depends on [RSH] and the concentration of phosphate. Using a detailed kinetic model to account for these effects, the present results could be reconciled with apparently dissimilar findings reported previously by others.

Full text of DOI:10.1021/tx960036y

988
Chem. Res. Toxicol. 1996, 9, 988-993
Kin etics of S-Nitr osa tion of Th iols in Nitr ic Oxid e
Solu tion s
Manish Keshive,† Sukhjeet Singh,‡ J ohn S. Wishnok,‡
Steven R. Tannenbaum,‡,§ and William M. Deen*,†
Department of Chemical Engineering, Division of Toxicology, and Department of Chemistry,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received February 27, 1996X
The S-nitroso adducts of nitric oxide (NO) may serve as carriers of NO and play a role in
cell signaling and/or cytotoxicity. A quantitative understanding of the kinetics of S-nitrosothiol
formation in solutions containing NO and O2 is important for understanding these roles of
S-nitroso compounds in vivo. Rates of S-nitrosation in aqueous solutions were investigated
for three thiols: glutathione, N-acetylcysteine, and N-acetylpenicillamine. Nitrous anhydride
(N2O3), an intermediate in the formation of nitrite from NO and O2, is the most likely NO
donor for N-nitrosation of amines as well as for S-nitrosation of thiols, at physiological pH.
This motivated the use of a competitive kinetics approach, in which the rates of thiol nitrosation
were compared with that of a secondary amine, morpholine. The kinetic studies were carried
out with known amounts of NO and O2 in solutions containing one thiol (400 µM) and
morpholine (200-5700 µM) in 0.01 M phosphate buffer at pH 7.4 and 23 °C. It was found
that disulfide formation, transnitrosation reactions, and decomposition of the S-nitrosothiol
products were all negligible under these conditions. The rate of formation of S-nitrosothiols
was expressed as k7[N2O3][RSH], where RSH represents the thiol. The rate constant for
S-nitrosation relative to that for N2O3 hydrolysis (k4) was found to be k7/k4 ) (4.15 ( 0.28) ×
104, (2.11 ( 0.11) × 104, and (0.48 ( 0.04) × 104 M-1 for glutathione, N-acetylcysteine, and
N-acetylpenicillamine, respectively. The overall (observed) rates of nitrosothiol formation reflect
the fact that [N2O3] [NO]2[O2] and that [N2O3] also depends on [RSH] and the concentration
of phosphate. Using a detailed kinetic model to account for these effects, the present results
could be reconciled with apparently dissimilar findings reported previously by others.
stabilized by reactions with carrier molecules in vivo that
In tr od u ction
prolong its half-life (6-8).
Nitric oxide (NO) is a biological messenger molecule
synthesized in mammals via the oxidation of L-arginine
to citrulline by the enzyme NO synthase (1). Nitric oxide
synthesis is observed in a wide variety of cell types,
including macrophages, vascular endothelial cells, neu-
rons, epithelial cells, and neutrophils. The actions of NO
in the body are paradoxical, in that NO serves as a
regulatory agent as well as a cytotoxic or mutagenic
agent. Regulatory actions of NO include inhibition of
platelet aggregation, vasodilation (blood pressure regula-
tion), and neurotransmission (2). The mutagenic effects
of NO are attributed to the intermediates formed during
the reactions of NO with molecular oxygen and super-
oxide (O2-). The reaction of NO with O2, which proceeds
ultimately to nitrite (NO2-), forms as an intermediate
nitrous anhydride (N2O3), which can lead to cytotoxic
effects. Peroxynitrite (ONOO-), an intermediate in the
reaction of NO with O2- to form nitrate (NO3-), can exert
mutagenic effects via the oxidation of DNA (3). Nitric
oxide itself can react with heme (4) as well as non-heme
iron (5). The high reactivity of NO with iron compounds,
combined with their abundance in extracellular and
intracellular fluids, has led to the argument that NO is
It has been demonstrated that the intermediates
generated during the reaction of NO with O2 at physi-
ological pH readily react with secondary amines (9) and
thiols (6-8, 10) to form N-nitrosamines and S-nitrosothi-
ols, respectively. The nitrosation of morpholine (Mor),1
a secondary amine, proceeds via N2O3 at a rate which
has been examined in detail by Lewis et al. (9). There
is, however, relatively little information in the literature
regarding the kinetics of the reactions of thiols with NO.
Stamler et al. (6) suggested that NO generated in
biological systems reacts in the presence of protein thiols
to form S-nitrosoprotein derivatives. They also demon-
strated that the concentrations of S-nitrosothiols in
human plasma are about 3 orders of magnitude greater
than that of free NO. This suggests that NO circulates
in mammalian plasma complexed in S-nitrosothiol spe-
cies. The S-nitroso adducts may serve as carriers of NO
and/or may have cytotoxic effects. A quantitative un-
derstanding of the kinetics of S-nitrosothiol formation is
thus essential in determining the role of S-nitroso
compounds in vivo.
The reaction kinetics for the nitrosation of cysteine
(Cys) and glutathione (GSH) in aerobic NO solutions at
* Address correspondence to Dr. William M. Deen, Department of
Chemical Engineering, Room 66-509, Massachusetts Institute of
Technology, Cambridge, MA 02139. Telephone: (617) 253-4535; FAX:
(617) 258-8224; E-mail: wmdeen@mit.edu.
1
Abbreviations: Mor, morpholine; NMor, N-nitrosomorpholine;
GSH, reduced glutathione; GSSG, glutathione disulfide; GSNO, S-
nitrosoglutathione; Cys, cysteine; N-Ac-Cys, N-acetylcysteine; N-Ac-
CysNO, S-nitroso-N-acetylcysteine; N-Ac-Pen, N-acetylpenicillamine;
SNAP, S-nitroso-N-acetylpenicillamine; ESI-MS, electrospray ioniza-
tion-mass spectrometry.
† Department of Chemical Engineering.
‡ Division of Toxicology.
§ Department of Chemistry.
X Abstract published in Advance ACS Abstracts, J uly 15, 1996.
S0893-228x(96)00036-7 CCC: $12.00 © 1996 American Chemical Society

Kinetics of Nitrosothiol Formation
Chem. Res. Toxicol., Vol. 9, No. 6, 1996 989
Nitr ic Oxid e An a lysis. The external side of the composite
membrane was exposed to the high vacuum of a chemilumines-
cence detector (Thermedics Detection Inc., Woburn, MA, Model
TEA-502) for monitoring of NO. The response time was ∼2 s,
and the output of the detector was linear up to NO concentra-
tions of at least 40 µM. The minimum aqueous NO concentra-
tion measurable by the chemiluminescence detector was esti-
mated at ∼ 0.01 µM, based on a signal-to-noise ratio of 3 (14).
neutral pH have been studied, and a rate law for the
formation of the S-nitroso adducts has been proposed by
Wink et al. (10). The rate of formation of S-nitrosothiols
was found to be second order in NO, first order in O2,
and independent of the initial concentration of the thiol.
The independence from the substrate (thiol) concentra-
tion, in particular, makes the form of this rate law
unusual, but no explanation was offered for this finding.
Thiols are readily oxidized to the disulfide form (11, 12),
but it was not mentioned whether the disulfide was
observed during the reactions. Very recently, Kharitonov
et al. (13) investigated the kinetics of the nitrosation of
thiols by NO in the presence of O2 and proposed rate laws
for both high and low concentrations of thiols.
In view of the biological importance of the kinetics of
S-nitrosothiol formation, we carried out competitive
kinetic studies to determine the rate constant for the
reaction of various thiols with N2O3. In this approach,
which differs from that employed in the previous studies
of S-nitrosation cited above, the rate constant was
estimated from the rate of nitrosothiol formation in the
presence of morpholine. As will be discussed, this
competitive kinetics approach avoided complications due
to the effects of phosphate on the nitrosation of thiols,
which we think must have been prominent in one of the
previous studies (13). The reactor configuration devel-
oped by Lewis et al. (14) was used in these studies. The
thiols examined were GSH, N-acetylcysteine (N-Ac-Cys),
and N-acetylpenicillamine (N-Ac-Pen). All are biologi-
cally relevant: the level of GSH in cells can be as high
as 10 mM (15), N-Ac-Cys provides an analog for the
binding of NO to cysteine in serum albumin, and N-Ac-
Pen reacts with NO to form S-nitroso-N-acetylpenicil-
lamine (SNAP), an important NO donor compound.
N-Nitr osom or p h olin e a n d S-Nitr osoth iol An a lysis. The
reactor had a flow loop consisting of 1/8 in. tubing (volume ∼8
mL). The aqueous solution in the reactor was continuously
circulated by a pulseless pump (Cole Parmer, Chicago, IL,
Models 000-305, 184-000) through this flow loop, into a 10 mm
spectrophotometer flow cell (Shimadzu Model UV 160u). The
concentrations of S-nitrosoglutathione (GSNO) and S-nitroso-
N-acetylcysteine (N-Ac-CysNO) were measured at 335 nm and
that of SNAP at 340 nm. The concentration of NMor was
determined at 250 nm, with no interference from thiols, S-
nitrosothiols, or thiol disulfides. In each case the absorbance
was linear in concentration.
Com p etitive Kin etic Stu d ies. Morpholine (200-5700 µM)
and the thiol (400 µM) were added to 150 mL of 0.01 M
phosphate buffer solution, and the pH was adjusted to ∼7.4
using NH4OH and H3PO4. The solution was added to the
reactor, stirring initiated at 100 rpm, and recirculation begun
through the spectrophotometer flow loop. Argon at a flow rate
of 350 sccm was bubbled for 40 min through the solution to
remove O2. A mixture of NO and Ar was then bubbled into the
solution for at least 25 min to obtain the desired aqueous NO
concentration. After the NO reached a steady-state aqueous
concentration, the bubbling of the NO/Ar mixture was stopped,
and the residual NO in the head space was removed by
introducing Ar via the gas inlet for 1.5 min. A 21% O2, balance
N2, mixture was then introduced through the gas inlet at a flow
rate of 350 sccm. Diffusion of O2 into the aqueous phase
initiated the oxidation of NO, producing the reactive intermedi-
ates responsible for the N- and S-nitrosations. The initial NO
concentration in the solution (upon introduction of O2) was ∼30
µM. The reaction was allowed to proceed for at least 25 min,
during which the NO and NMor concentrations were monitored
continuously. The reaction protocol was then repeated, the only
difference being that the concentrations of NO and S-nitrosothiol
were monitored. The solution pH was measured at the end of
each reaction. For each thiol, a control study was performed
by carrying out the reaction protocol with 400 µM of the thiol
in the buffer but no Mor, and monitoring the absorbance at 250
nm. This was done to check for any interference in the
measurement of NMor absorbance at 250 nm.
Ma ter ia ls a n d Meth od s
Sa fety Con sid er a tion s (Ca u tion ). All experiments were
performed in
a certified hood, due to the toxicity of NO.
Precautions were also taken to avoid any skin contact with the
potentially toxic nitrosothiols.
Rea gen ts. Nitric oxide was passed through
a column
containing 10 M NaOH to remove NOx impurities. Argon, after
passing through an oxygen trap, was mixed with NO using
controlled gas flowmeters (Porter Instrument Co., Hatfield, PA)
to obtain the desired NO gas concentration. Buffer solutions
at 0.01 M ionic strength and pH 7.4 were prepared from the
acids and sodium salts of phosphate. Morpholine was obtained
from Aldrich Chemical Co. (Milwaukee, WI). Nitrosomorpho-
line, glutathione, S-nitrosoglutathione, glutathione disulfide,
N-acetylcysteine, S-nitroso-N-acetylcysteine, N-acetylpenicil-
lamine, and S-nitroso-N-acetylpenicillamine were obtained from
Sigma Chemical Co. (St. Louis, MO). All of the solutions were
prepared using deionized water.
Rea ctor . The reactor, a modified 200 mL stirred ultrafil-
tration cell (Amicon, Danvers, MA, Model 8200), was the same
as that used previously for NO oxidation and nitrosation studies
(9, 14). Briefly, a composite membrane (6.2 cm diameter)
consisting of a 1 mm thick Teflon sheet laminated with a 0.13
mm thick polydimethylsiloxane sheet (Mempro, Troy, NY) was
fitted to the base of the stirred cell for NO detection. Gas inlet
and outlet ports, two ports for a flow loop connected to a
spectrophotometer (for NMor and S-nitrosothiol measurements),
and a thermometer were added. A 20 gauge hypodermic needle
was inserted into the gas outlet port for gas purging and NO
introduction and removal during the reaction. All of the
samples were withdrawn from the reactor using a gas-tight
hypodermic syringe. The studies were performed at room
temperature (23 ( 1 °C).
Electr osp r a y Ion iza tion -Ma ss Sp ectr om etr ic (ESI-MS)
An a lysis. These experiments were done on a Hewlett Packard
Model 5989B mass spectrophotometer in the negative ion mode.
The instrument was tuned with special attention paid to the
spray capillary voltages and offsets to prevent arcing. Kinetic
experiments were carried out exactly as described above except
that ammonium acetate (5 mM; pH approximately 7.4) was the
buffer. To test for the formation of thiol disulfide, and to test
for the constancy of the thiol concentration, samples were
withdrawn from the reactor before and after the addition of O2.
In
a typical experiment, 100 µL of reaction solution was
withdrawn with a gas-tight syringe and mixed with 100 µL of
electrospray solvent and 20 µL of internal standard (1.9 mM
succinic acid). Aliquots of the resulting solution were introduced
into the mass spectrometer by flow injection into a stream of
50:50 water/methanol containing 1% acetic acid at a flow rate
of 20 µL/min from a Harvard syringe pump. This solvent is
more typical for positive-ion electrospray, but was used here
because the analytes were more stable under these conditions.
Spectra were taken over a 3-min period and averaged over the
maximum abundance for the sample bolus, and the peak areas
for the internal standard, the thiol, and the disulfide were
recorded after background subtraction.
Kin etic Mod el a n d Rea ction Sch em e. The oxidation
kinetics of NO in aqueous solutions can be represented by the

990 Chem. Res. Toxicol., Vol. 9, No. 6, 1996
Keshive et al.
following reaction scheme (14, 16-18):
where ∆[RSNO] and ∆[NMor] represent the changes in the
respective concentrations during the reaction (i.e., after O2
addition). The second form of eq 13 is preferred because the
rate of N-nitrosation of Mor relative to the rate of N2O3
hydrolysis (k6/k4) is known more precisely than are the absolute
rate constants for either reaction (9). Accordingly, although
estimates are given also for the absolute value of k7, the rate
constants for thiol nitrosation are reported here primarily as
the ratio, k7/k4.
k1
2NO + O2
98 2NO2
(1)
(2)
k2
NO +
\
k3
k4
9
N2O3 + H2O
8 2NO2- + 2H+
(3)
The other reactions needed to interpret the rates of nitrosation
are
Resu lts
Th iol Con cen t r a t ion a n d Disu lfid e F or m a t ion .
The reaction scheme and kinetic analysis given above
assume that the thiol concentration remained essentially
constant during the course of the reaction and that
disulfide formation was negligible. To check these as-
sumptions, samples were withdrawn from the reactor
before and after O2 addition during each experiment. The
analysis of these samples by ESI-MS showed insignifi-
cant changes in the concentration of each thiol. Disul-
fides were detectable in small amounts, but the concen-
trations were always similar in the pairs of samples. This
indicates that the disulfide was present as an impurity
in the thiol, as opposed to being formed during the
reaction period.
k5
9
-
N2O3 + Pi
8 PiNO + NO2
(4)
(5)
(6)
k6
N2O3 + Mor
N2O3 + RSH
9
8 NMor + NO2- + H+
k7
9
8 RSNO + NO2- + H+
Equation 4 represents the reaction of N2O3 with the inorganic
phosphate (Pi) in the buffer. (The active form of phosphate could
-
be H2PO4 and/or HPO42-; all phosphate concentrations are
expressed here as total phosphate at pH 7.4.) This reaction has
the effect of enhancing the hydrolysis of N2O3, represented
otherwise by eq 3 (9). Equations 5 and 6 represent the
nitrosation reactions of Mor and the thiol (RSH), respectively.
All of the rate constants in eqs 1-6 are known (at least
approximately) except for k7, the rate constant for the reaction
of N2O3 with the thiol.
The following kinetic analysis, applicable to a solution
containing Mor and one thiol, was used to estimate the unknown
rate constant, k7. Order-of-magnitude estimates of the rate
constants k1 through k4 (19, 20) suggest that NO2 and N2O3 were
present in the system in very small amounts. This justifies the
use of pseudo-steady-state approximations for those species,
Mor p h olin e Con cen tr a tion a n d p H. The concen-
tration of unprotonated morpholine, the substrate for
nitrosation, is related to the total morpholine concentra-
tion by
[Mor]
1 + 10pK-pH
[Mor0] )
(14)
where the pK at 25 °C is 8.5 for morpholine (9). The total
morpholine concentration did not change appreciably in
any experiment, because negligible amounts were con-
verted to NMor (<0.7%). The pH was also constant
during a given experiment, so that [Mor0] was indepen-
dent of time, as assumed in the kinetic analysis.
d[NO2]
) 2k1[NO]2[O2] - k2[NO][NO2] + k3[N2O3] = 0 (7)
dt
d[N2O3]
) k2[NO][NO2] - (k3 + k4)[N2O3] - (k5[Pi] +
dt
Tr a n sn itr osa tion Rea ction a n d Sta bility of S-
Nitr osoth iols. The kinetic analysis assumes also that
there was no direct transfer of NO from the nitroso
compounds to the substrates and that the S-nitrosothiol
was a stable end product. This was tested for each thiol
by letting a 0.01 M phosphate buffer solution, containing
known amounts of Mor and the S-nitrosothiol, sit for a
period of at least 1 h. These solutions showed no change
in absorbance at 250 nm, the absorbance maximum for
NMor. This demonstrates that no NMor formation
resulted from the direct transnitrosation reaction be-
tween Mor and the S-nitrosothiols. Similarly, 0.01 M
phosphate buffer solutions containing known amounts of
the S-nitrosothiols showed no change in absorbance at
335 nm for a period of at least 1 h. Thus, the S-
nitrosothiols were stable over the time scale used in the
kinetic experiments. These findings confirm that it is
valid to omit the transnitrosation and nitrosothiol de-
composition reactions from the kinetic scheme.
k6[Mor0] + k7[RSH])[N2O3] = 0 (8)
where [Mor0] is the concentration of neutral (unprotonated) Mor.
Using eqs 7 and 8, the pseudo-steady-state concentration of N2O3
is
2k1[NO]2[O2]
k4 + k5[Pi] + k6[Mor0] + k7[RSH]
[N2O3] )
(9)
The rates of formation of NMor and RSNO are
d[NMor]
) k6[Mor0][N2O3]
(10)
(11)
dt
d[RSNO]
) k7[RSH][N2O3]
dt
where eq 9 is used to evaluate [N2O3]. Combining eqs 10 and
11 we obtain
Nitr ic Oxid e, N-Nitr osom or p h olin e, a n d S-Nitr o-
soth iol An a lyses. Experiments carried out in neutral
phosphate buffer solutions, in the absence of O2, showed
no formation of either NMor or S-nitrosothiol species,
consistent with earlier studies (10, 11), indicating that
one of the intermediates generated during the reaction
of NO with O2 is responsible for the nitrosation of amines
and thiols. In aerobic phosphate buffer solutions, with
a steady-state NO concentration of 30 µM, addition of
k7
k6
d[RSNO]
d[NMor]
[RSH]
[Mor0]
)
(12)
Integration of eq 12, with the assumption that [RSH] and [Mor0]
are essentially constant during the reaction, gives
∆[RSNO] [Mor0]
6 ∆[NMor] [RSH]
∆[RSNO] [Mor0]
∆[NMor] [RSH]
k7 k6
k7 ) k
or
)
(13)
k4 k4

Kinetics of Nitrosothiol Formation
Chem. Res. Toxicol., Vol. 9, No. 6, 1996 991
Ta ble 1. Ra te Con sta n ts for th e Rea ction of N2O3 w ith
Th iolsa
thiol
glutathione
N-acetylcysteine
N-acetylpenicillamine
k7/ k4 (104 M-1
)
4.15 ( 0.28 (5)
2.11 ( 0.11 (5)
0.48 ( 0.04 (4)
a
The values for k7/k4 were calculated from eq 13, using k6/ k4
) 4 × 104 M-1 at 23 °C (9). The results are given as mean (
standard error for the number of experiments shown in paren-
theses.
was increased, the amount of NMor formed increased and
the amount of GSNO formed decreased. This is consis-
tent with our hypothesis of a competition for N2O3 by Mor
and GSH. Again, it is seen that the amounts of NMor
and GSNO formed were a very small fraction of the initial
concentrations of Mor and GSH, respectively. Similar
trends were observed for N-Ac-Cys and N-Ac-Pen (data
not shown).
F igu r e 1. Typical changes in NO, NMor, and GSNO concen-
trations with time during the reaction of NO with O2 at 23 °C.
The initial concentrations of Mor and GSH in this experiment
were 2850 and 400 µM, respectively.
Ra te Con sta n ts. The rate constants for the reaction
of N2O3 with thiols at 23 °C and a pH of 7.4 are shown
in Table 1. For the reasons mentioned earlier, the values
shown are k7/k4, the ratio of the rate constant for the
N2O3-thiol reaction to the rate constant for N2O3 hy-
drolysis. As can be seen, all of the thiols competed
effectively for N2O3, GSH being the most reactive and
N-Ac-Pen being the least. Using an estimated k4 value
of 1600 s-1 (21), the values of k7 for GSH, N-Ac-Cys, and
N-Ac-Pen are (6.6 ( 0.4) × 107, (3.4 ( 0.2) × 107, and
(0.8 ( 0.1) × 107 M-1 s-1, respectively. There is ap-
proximately a 4-fold range of reported values for k4, as
discussed previously (14), so that the values just given
for k7 must be viewed with caution. The utility of the
various rate constants in understanding the kinetics of
S-nitrosothiol formation is discussed below.
F igu r e 2. The effect of morpholine concentration on the
amounts of NMor and GSNO formation. The initial concentra-
tion of total morpholine (Mor) was varied from 950 to 5700 µM
to give the concentrations shown for free morpholine (Mor0). The
initial concentrations of GSH and NO in each case were 400
and 30 µM, respectively. The pH in the experiments ranged from
7.38 to 7.45.
Discu ssion
The rate constant for the reaction of N2O3 with selected
thiols, k7, was determined using a competitive kinetics
approach. Morpholine was used as the competing sub-
strate, because its rate of reaction with N2O3 in aqueous
solutions at room temperature is well-established (9).
With the values of k7 estimated for the three thiols
studied, the rate of S-nitrosothiol formation can be
determined from eq 11. Using the pseudo-steady-state
concentration for N2O3 given in eq 9, the rate of S-
nitrosothiol formation in a system containing only one
thiol, and no morpholine or phosphate, is given by
O2 caused a rapid increase in absorbance at 250 and 335
nm, indicating the formation of NMor and S-nitrosothiol,
respectively. Figure 1 shows the concentrations of NO,
NMor, and GSNO measured during a representative
experiment, in which the initial Mor concentration was
2850 µM and the initial GSH concentration was 400 µM.
The concentration of NO declined nearly to zero after ∼20
min, the result mainly of conversion of NO to NO2- and
-
physical losses of NO into the head space (14). No NO3
was detected in the system. As shown also in Figure 1,
the concentrations of NMor and GSNO were relatively
small, consistent with the assumption that the concen-
trations of Mor and GSH were essentially constant
during the reaction period. The same was true for the
other thiols, N-Ac-Cys and N-Ac-Pen. It should be
mentioned that, in all of the kinetic experiments, some
NMor and nitrosothiol was present in the solution prior
to the deliberate introduction of O2. This was due to a
small air leak in the spectrophotometer, which could not
be eliminated (see ref 14 for a detailed analysis). Ac-
cordingly, the concentrations of NMor and GSNO in
Figure 1 represent increments above the initial values.
d[RSNO]
) k7[RSH][N2O3] )
dt
2k1[NO]2[O2]
k7[RSH]
(15)
(
)
k4 + k7[RSH]
Thus, in general, the rate of S-nitrosothiol formation is
expected to be second order in NO and first order in O2,
and to depend also on the thiol concentration. A recent
study by Wink et al. (10) reported the rate of S-
nitrosothiol formation to be
Figure 2 shows the results of five experiments carried
out at initial Mor concentrations of 950-5700 µM and
an initial GSH concentration of 400 µM. The amounts
of NMor and GSNO formed during each experiment are
plotted as a function of the free (unprotonated) morpho-
line concentration. The pH in these experiments ranged
from 7.38 to 7.45. As the free morpholine concentration
d[RSNO]
) kSNO[NO]2[O2]
(16)
dt
where kSNO was measured as (6 ( 2) × 106 M-2 s-1 for
both GSH and Cys. Equation 16 seems at first to be at
variance with eq 15. However, if the thiol concentration
is such that the rate of thiol nitrosation is much faster

992 Chem. Res. Toxicol., Vol. 9, No. 6, 1996
Keshive et al.
than the rate of N2O3 hydrolysis (i.e., if k7[RSH] . k4),
then eq 15 reduces to
or
k7[RSH]
k4 + k5[Pi] + k7[RSH]
d[RSNO]/dt
4k1[NO]2[O2]
1
2
m )
)
(20)
(
)
d[RSNO]
) 2k1[NO]2[O2]
(17)
dt
where m (similar to Kharitonov et al.) represents the
fraction of NO converted to S-nitrosothiol. (The fraction
This is the same as eq 16, provided that kSNO ) 2k1. With
k1 ) 2.1 × 106 M-2 s-1 (14), the predicted value of kSNO is
4.2 × 106 M-2 s-1, which equals (within experimental
uncertainty) the value quoted above. In their experi-
ments, Wink et al. (10) used GSH and Cys concentrations
of 1-10 mM. Based on the present data, k7[GSH]/k4 )
41.5 with [GSH] ) 1 mM, consistent with the hypothesis
that thiol nitrosation was much faster than N2O3 hy-
drolysis under their experimental conditions.
-
of NO converted to NO2 is 1 - m.) Equation 20 is
rearranged to give
k7
k4
k
2m
)
1 + 5[Pi]
(21)
{
}
k4
(1 - 2m)[RSH]
Ignoring phosphate has the effect of setting the last term
in braces equal to unity. The value of k5/k4 at 25 °C is
400 M-1 (9). For a phosphate concentration of 0.1 M,
k5[Pi]/k4 ) 40, so that the correction factor is estimated
as 1 + 40 ) 41. Multiplying the rate constants of
Kharitonov et al. (13) by this factor, the corrected values
of k7 are 1.2 × 107 M-1 s-1 and 0.6 × 107 M-1 s-1 for GSH
and N-Ac-Cys, respectively. These values are of the same
order of magnitude as those of the present study.
Further support for this interpretation is given by the
data plotted in Figure 4 of Kharitonov et al. (13), which
show that, for [GSH] >5 mM, the rate of GSNO formation
is independent of [GSH]. As discussed earlier, this will
be true only if k7[GSH] . k4. This relationship holds at
[GSH] ) 5 mM for the corrected value of k7, but not for
their reported value. Using the corrected value, k7[GSH]
) 6 × 104 . 1.6 × 103 ) k4. In summary, it appears
that our data and those of Kharitonov et al. (13) are
basically consistent and that the large discrepancy in the
calculated rate constants is due mainly to the omission
of the phosphate effect from their kinetic analysis.
The kinetic analysis used here assumes that the
S-nitrosation reaction involves the protonated (un-
charged) thiol, rather than the dissociated (anionic)
species. Thus, as has been done in studies of S-nitrosa-
tion at pH 2-3 (22, 23), the rate expressions are written
in terms of [RSH] rather than [RS-]. There is essentially
no dissociated thiol under those acidic conditions, and
also very little at physiological pH. For example, the pK
of GSH at 25 °C is 8.75 (24), so that at pH 7.4 only 4% is
in the dissociated form. Nonetheless, participation of the
dissociated forms of the thiols at physiological pH cannot
be excluded, and it would be necessary to examine the
kinetics over a range of pH to more definitely establish
the identity of the reactants. It is inappropriate to
compare the present rate constants with the acid-
catalyzed values for GSH (23) and N-Ac-Pen (22), because
the nitrosating agent under those conditions is thought
to be NO+ (or the equivalent) rather than N2O3.
Using a kinetic analysis similar to that used here to
derive eq 17, Kharitonov et al. (13) also proposed a rate
law for S-nitrosation at high thiol concentrations (>5
mM), namely:
d[RSNO]
) k[NO]2[O2]
(18)
dt
where k ) (7.0 ( 0.1) × 106 M-2 s-1. (What is shown
here as k is given as k4 in their eq 5.) Their derivation
contained an algebraic error, however; the correct coef-
ficient in eq 18 should be k/2, or 3.5 × 106 M-2 s-1. This
corrected value is essentially the same as our value of
4.2 × 106 M-2 s-1
.
To summarize the situation for high thiol concentra-
tions, the present results are in agreement with those of
previous studies (10, 13) not only on the form of the rate
law but also (essentially) on the numerical value of the
rate coefficient. It seems clear that, for high thiol
concentrations, the rate of nitrosothiol formation is
controlled entirely by the availability of NO and O2.
Consequently, measurements of nitrosothiol formation at
thiol concentrations exceeding several millimolar will not
ordinarily yield rate data specific to individual thiols.
The values of the N2O3-thiol rate constant (k7) ob-
tained from the present data are 6.6 × 107 M-1 s-1 for
GSH and 3.4 × 107 M-1 s-1 for N-Ac-Cys. These values
are 2 orders of magnitude larger than those reported by
Kharitonov et al. (13) for the same compounds, 2.9 × 105
M-1 s-1 and 1.6 × 105 M-1 s-1 for GSH and N-Ac-Cys,
respectively. This discrepancy is explained by the fact
that those authors used a 0.1 M phosphate buffer in their
nitrosation experiments, but their kinetic analysis did
not account for the reactivity of phosphate with N2O3.
In other words, they did not consider the reaction given
here as eq 4. As shown by Lewis et al. (9), phosphate
has a scavenging effect on N2O3; similar effects were seen
with Cl-, but not with several other anions tested.
Consequently, neglecting the effect of phosphate in the
kinetic analysis for nitrosothiol formation will overesti-
mate the concentration of N2O3, and therefore underes-
timate k7.
Several reactions that were omitted from the present
kinetic scheme may be relevant under other conditions.
The decomposition reaction of S-nitrosothiols to release
NO and form disulfides can be important if experiments
are carried out under basic conditions (11, 24). Under
sufficiently acidic conditions, HNO2 becomes a prominent
species, the scavenging of N2O3 by phosphate and chloride
is suppressed, and certain anions are able to catalyze
N-nitrosation (9, 21). Transnitrosation reactions between
S-nitrosothiols and other thiols (25) may play an impor-
tant role in regulating the concentrations of these species
in intracellular and extracellular fluids. The relative
amounts of various thiols and S-nitrosothiols present in
a given milieu will dictate the equilibrium concentrations
The expected magnitude of the error in the rate
constant is calculated by examining the rate of S-
nitrosothiol formation in the presence of phosphate, but
without morpholine. Proceeding as before, the rate is
predicted to be
2k1[NO]2[O2]
d[RSNO]
) k7[RSH]
(19)
(
)
dt
k4 + k5[Pi] + k7[RSH]

Kinetics of Nitrosothiol Formation
Chem. Res. Toxicol., Vol. 9, No. 6, 1996 993
utility and toxicological implications. J . Org. Chem. 47, 156-159.
and eventually the regulatory or cytotoxic effects of NO
or the nitroso adducts. Additional studies are thus
required to completely understand the eventual fate of
the S-nitrosothiol species formed in the body.
(12) Park, J . W., Billman, G. E., and Means, G. E. (1993) Transnit-
rosation as a predominant mechanism in the hypotensive effect
of S-nitrosoglutathione. Biochem. Mol. Biol. Int. 5, 885-891.
(13) Kharitonov, V. G., Sundquist, A. R., and Sharma, V. S. (1995)
Kinetics of nitrosation of thiols by nitric oxide in the presence of
oxygen. J . Biol. Chem. 270, 28158-28164.
Ack n ow led gm en t. This work was supported by a
grant from the National Cancer Institute (PO1-CA26731).
(14) Lewis, R. S., and Deen, W. M. (1994) Kinetics of the reaction of
nitric oxide with oxygen in aqueous solutions. Chem. Res. Toxicol.
7, 568-574.
(15) Sies, H., Brigelius, R., and Akerboom, T. P. M. (1983) Intrahepatic
glutathione status. In Functions of Glutathione: Biochemical,
Physiological, Toxicological, and Clinical Aspects (Larsson, A. L.,
Orrenius, S., Holmgren, A., and Mannervik, B., Eds.) pp 51-64,
Raven Press, New York.
Refer en ces
(1) Marletta, M. A. (1993) Nitric oxide synthase structure and
mechanism. J . Biol. Chem. 268, 12231-12234.
(2) Moncada, S., Palmer, R. M. J ., and Higgs, E. A. (1991) Nitric
oxide: physiology, pathophysiology, and pharmacology. Pharma-
col. Rev. 43, 109-142.
(3) J uedes, M. J ., and Wogan, G. N. (1996) Peroxynitrite-induced
mutation spectra of pSP189 following replication in bacteria and
in human cells. Mutat. Res. 349, 51-61.
(4) Palmer, R. M., Ferrige, A. G., and Moncada, S. (1987) Nitric oxide
release accounts for the biological activity of endothelium-derived
relaxing factor. Nature (London) 327, 524-526.
(16) Wink, D. A., Darbyshire, J . F., Nims, R. W., Saavedra, J . E., and
Ford, P. C. (1993) Reactions of the bioregulatory agent nitric oxide
in oxygenated aqueous media: determination of the kinetics for
oxidation and nitrosation by intermediates generated in the NO/
O2 reaction. Chem. Res. Toxicol. 6, 23-27.
(17) Awad, H. H., and Stanbury, D. M. (1993) Autoxidation of NO in
aqueous solution. Int. J . Chem. Kinet. 25, 375-381.
(5) Lancaster, J . R., J r., and Hibbs, J . B., J r. (1990) EPR demonstra-
tion of iron-nitrosyl complex formation by cytotoxic activated
macrophages. Proc. Natl. Acad. Sci. U.S.A. 87, 1223-1227.
(6) Stamler, J . S., J araki, O., Osborne, J ., Simon, D. I., Keaney, J .,
Vita, J ., Singel, D., Valeri, C. R., and Loscalzo, J . (1992) Nitric
oxide circulates in mammalian plasma primarily as an S-nitroso
adduct of serum albumin. Proc. Natl. Acad. Sci. U.S.A. 89, 7674-
7677.
(7) Keaney, J . F., J r., Simon, D. I., Stamler, J . S., J araki, O.,
Scharfstein, J ., Vita, J . A., and Loscalzo, J . (1993) NO forms an
adduct with serum albumin that has endothelium-derived relax-
ing factor-like properties. J . Clin. Invest. 91, 1582-1589.
(8) Stamler, J . S., Simon, D. I., Osborne, J . A., Mullins, M. E., J araki,
O., Michel, T., Singel, D. J ., and Loscalzo, J . (1992) S-nitrosylation
of proteins with nitric oxide: synthesis and characterization of
biologically active compounds. Proc. Natl. Acad. Sci. U.S.A. 89,
444-448.
(9) Lewis, R. S., Tannenbaum, S. R., and Deen, W. M. (1995) Kinetics
of N-nitrosation in oxygenated nitric oxide solutions at physi-
ological pH: role of nitrous anhydride and effects of phosphate
and chloride. J . Am. Chem. Soc. 117, 3933-3939.
(10) Wink, D. A., Nims, R. W., Darbyshire, J . F., Christodoulou, D.,
Hanbauer, I., Cox, G. W., Laval, F., Laval, J ., Cook, J . A., Krishna,
M. C., DeGraff, W. G., and Mitchell, J . B. (1994) Reaction kinetics
for nitrosation of cysteine and glutathione in aerobic nitric oxide
solutions at neutral pH. Insights into the fate and physiological
effects of intermediates generated in the NO/O2 reaction. Chem.
Res. Toxicol. 7, 519-525.
(18) Ignarro, L. J ., Fukuto, J . M., Griscavage, J . M., Rogers, N. E.,
and Byrns, R. E. (1993) Oxidation of nitric oxide in aqueous
solution to nitrite but not nitrate: comparison with enzymatically
formed nitric oxide from L-arginine. Proc. Natl. Acad. Sci. U.S.A.
90, 8103-8107.
(19) Pogrebnaya, V. L., Usov, A. P., Baranov, A. V., Nesterenko, A. I.,
and Bez’yazychnyi, P. I. (1975) Liquid-phase oxidation of nitric
oxide by oxygen. Zh. Prikl. Khim. 48, 954-958.
(20) Schwartz, S. E. (1983) Trace atmospheric constituents: properties,
transformations, and fates. In Advances in Environmental Science
and Technology (Nriagu, J . O., Ed.) p 91, Wiley, New York.
(21) Licht, W. R., Tannenbaum, S. R., and Deen, W. M. (1988) Use of
ascorbic acid to inhibit nitrosation: kinetic and mass transfer
considerations for an in vitro system. Carcinogenesis 9, 365-372.
(22) Aldred, S. E., Williams, D. L. H., and Garley, M. (1982) Kinetics
and mechanism of the nitrosation of alcohols, carbohydrates, and
a thiol. J . Chem. Soc., Perkin Trans. 2 777-782.
(23) Williams, D. L. H. (1985) S-nitrosation and the reactions of
S-nitroso compounds. Chem. Soc. Rev. 14, 171-196.
(24) Arnelle, D. R., and Stamler, J . S. (1995) NO+, NO•, and NO-
donation by S-nitrosothiols: implications for regulation of physi-
ological functions by S-nitrosylation and acceleration of disulfide
formation. Arch. Biochem. Biophy. 318, 279-285.
(25) Pietraforte, D., Mallozzi, C., Scorza, G., and Minetti, M. (1995)
Role of thiols in the targeting of S-nitroso thiols to red blood cells.
Biochemistry 34, 7177-7185.
(11) Pryor, W. A., Church, D. F., Govindan, C. K., and Crank, G. (1982)
Oxidation of thiols by nitric oxide and nitrogen dioxide: synthetic
TX960036Y