(Received for publication, May 4, 1995; and in revised form, July 21, 1995 )
From the
Nitrosothiols are powerful vasodilators. They act by releasing
nitric oxide, which activates the heme protein guanylate cyclase. We
have studied the kinetics of nitrosothiol formation of glutathione,
cysteine, N-acetylcysteine, human serum albumin, and bovine
serum albumin upon reaction with nitric oxide (NO) in the presence of
oxygen. These studies have been made at low pH as well as at
physiological pH. At pH 7.0, contrary to published reports, nitric
oxide by itself does not react with thiols to yield nitrosothiol.
However, formation of nitrosothiols is observed in the presence of
oxygen. For all thiols studied, the rates of nitrosothiol formation
were first order in O concentration and second order in NO
concentration and at lower concentrations (<5 mM thiol)
also depended on thiol concentrations. Analysis of the kinetic data
indicated that the rate-limiting step was the reaction of NO with
oxygen. Analysis of the reaction products suggest that the main
nitrosating species is N
O
: RSH +
N
O
RSNO +
NO
+ H
. Rate
constants for this reaction for glutathione and several other low
molecular weight thiols are in the range of 3-1.5
10
M
s
, and
for human and bovine serum albumins 0.3
10
M
s
and 0.06
10
M
s
,
respectively. The data further indicate that the reaction rate of the
nitrosating species N
O
with thiols is
competitive with its rate of hydrolysis. At physiological
concentrations nitrosoglutathione formation represents a significant
metabolic fate of N
O
, and at glutathione
concentrations of 5 mM or higher almost all of
N
O
formed is consumed in nitrosation of
glutathione. Implications of these results for in vivo nitrosation of thiols are discussed.
S-Nitrosothiols are potent vasodilators and inhibitors of platelet aggregation (Ignarro et al., 1981; Stamler et al., 1992; Butler and Williams, 1993). This activity is almost certainly dependent on the ability of the nitrosothiol to yield NO, a bioregulatory molecule of considerable current interest (Moncada et al., 1991). Although the mechanism of NO release is not certain, the overall reaction seems to be as follows:
On-line formulae not verified for accuracy
Nitric oxide released in can then react with the heme group of guanylate cyclase (GC) and activate the enzyme:
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Combination reactions of NO with five coordinate ferro heme proteins are fast, and the NO dissociation reaction is slow (Moore and Gibson, 1976). GC(heme-NO)* represents the NO-activated form of the enzyme.
Stamler et al.(1992) have reported that naturally
produced nitric oxide circulates in plasma primarily complexed as S-nitrosothiol species, principal among which is S-nitroso-serum albumin. Although nitrosothiols (both low M and protein) have endothelium-derived relaxation
factor or NO-like properties, the mechanism by which circulating or
cellular RSNO is synthesized has not been described. At pH 7.0,
contrary to the published reports, nitric oxide, by itself, does not
react with thiols to yield nitrosothiol. However, formation of
nitrosothiol observed in the presence of oxygen suggested that
nitrosation proceeds via the formation of oxides of nitrogen
(N
O
, N
O
, etc.).
In
view of the fact that in aqueous solutions the rates of hydrolysis of
these oxides are high, it is not known how much nitrosation of low M thiols such as glutathione and protein thiols
such as HSA (
)can be expected to proceed via this mechanism.
The potential reactivity of NO with the sulfhydryl group is also of
interest when one considers that during its diffusion from endothelial
cells to the target molecule guanylate cyclase in neighboring muscle
cells, NO will be exposed in the cytoplasm to mM concentrations of the tripeptide glutathione (Meister and
Anderson, 1983). One wonders how NO is able to avoid reaction with such
a large excess of an intracellular thiol. To answer some of these
questions would require knowing the mechanism of nitrosation of thiols
in aqueous solutions in the presence of oxygen and the relevant rate
constants. In this article we present the results of our kinetic
studies on the reaction of nitric oxide with several thiols in the
presence of oxygen. In agreement with earlier studies it was observed
that NO reacts with thiols via the rate-limiting step of nitric
oxide's reaction with oxygen and formation of the nitrosating
species N
O
(Wink et al., 1994). The
reaction of N
O
with thiols is competitive with
its reaction with water. The novel finding of the present study is that
physiologically attainable concentrations (5 mM or higher) of
glutathione compete successfully with water for reaction with
N
O
. Nitrosation of HSA, on the other hand, is
less significant by this reaction path.
Reduced glutathione, cysteine-HCl, N-acetylcysteine, bis-Tris, Tris, sodium nitrite, N-ethylmaleimide, and human and bovine serum albumins (99%, fatty acid and globin free) were from Sigma. Since nitrosation of thiols can be sensitive to the presence of metal ions, deionized and glass-distilled water was used for washing all glassware and for the preparation of solutions. Spectral analysis and kinetic experiments were made in solutions both with and without 1 mM EDTA. Analytical grade sodium phosphate from Fisher was used in the present study. Like most other brands of sodium phosphate, it contained iron as a contaminant. In 0.1 M phosphate buffers used in the present study, iron contamination ranged between 0.6 and 1 µM. Therefore, all buffers were made in 1 mM in EDTA. In the time domain of the kinetic experiments made in this study, only in the case of cysteine inclusion of EDTA affected the kinetics of the reaction.
Nitric oxide (99.0% pure) was purchased from Matheson. Two methods were used to purify NO: passing it through a column filled with fresh KOH pellets or bubbling it through a solution of 0.5 M NaOH. After extended periods of use, KOH pellets lost their effectiveness, probably due to surface accumulation of nitrite. Two source tanks of NO were used in experiments: a tank that had been received from the manufacturer within one year of use and one that had been received roughly a decade earlier; NO from both behaved the same.
Solutions of NO, O, RSH, and RSH + O
were prepared in deoxygenated buffers (0.1 M sodium
phosphate, pH 7.0) in gas-tight syringes (Hamilton). To prepare
anaerobic albumin solutions, a tube containing a weighed quantity of
solid albumin was flushed with argon for 15 min and then filled with a
required amount of deoxygenated buffer. Deoxygenated buffers in
gas-tight syringes were prepared by bubbling high purity argon for
>40 min.
Kinetic measurements were made either on a Kontron 860
spectrophotometer in 4-ml matched quartz cuvettes fitted with septa at
29 °C or on a stopped-flow spectrophotometer with a thermostatted
(20 °C) cell. Kinetic data were obtained in the form of 200
electronically digitized absorbance versus time data points.
Spectra were recorded with the Kontron spectrophotometer using
1-cm-pathlength cuvettes. Only freshly prepared solutions were used in
all experiments.
HPLC studies were conducted using an anion exchange
column (Aquapor AX-300, 4.6 250 mm; Perkin-Elmer/ABI). The
gradient was obtained with eluent A (10 mM sodium phosphate,
pH 6.0) and eluent B (100 mM sodium sulfate in eluent A).
After applying the sample (5 µl), a linear change from 100% eluent
A to 100% eluent B over 30 min was used. The flow rate was 1 ml per
min, and detection was at 210 nm. N-Ethylmaleimide, a
sulfhydryl-specific reagent, was added (6 mM) to the solutions
prior to HPLC analysis to block excess GSH and enable measurement of
GSNO (Jocelyn, 1972).
When NO was used without alkali treatment, nitrosothiol formation was readily observed upon bubbling NO directly from the gas tank into the deoxygenated thiol solution. S-Nitrosoglutathione formation is demonstrated as an increase in the absorbance band at 335 nm in Fig. 1. Similarly, GSNO formation was readily observed when purified NO was bubbled through a solution of glutathione in the presence of oxygen. However, nitrosothiol formation was not observed when a solution of unpurified NO was prepared in deaerated phosphate buffer and added to the solution of thiol. Similarly, no RSNO formation was seen when solutions of unpurified NO were prepared in 0.1 M Tris, pH 7.0, prior to reaction with thiols. These results indicate that an oxidation product of NO present as a contaminant in unpurified NO is the nitrosating species and that it decomposes when aqueous solutions of NO are prepared prior to reaction with thiols.
Figure 1: Effect of NO purity on product formation from NO and GSH. Spectra were recorded of a deaerated solution of GSH (5 mM) before (A) and after (B) bubbling with purified NO for 10 min. In a second experiment, a deaerated GSH solution was bubbled with unpurified NO (C) for 2 min, 4 min (D), and 6 min (E). Solutions of GSH were prepared in 0.1 M sodium phosphate, pH 7.0.
Results for glutathione are
presented in Fig. 1. Curve A is for deoxygenated
solution of GSH. After bubbling NO through it for 10 min, curve B is obtained; curve E is for solutions in which unpurified
NO was bubbled through GSH solution for 6 min. A small absorbance
change (curve B) around 350 nm, which is about 3 percent of
A for curve E, is most likely due to RSNO formation from
slow leakage of O
or incomplete deoxygenation of the
starting solution. Because GSH is the preponderant low M
thiol in cells and because its nitroso derivative, GSNO, is very
stable in solution (Park et al., 1993), the results of
experiments with GSH are highlighted below. Similar results were
obtained with N-acetylcysteine. Cysteine, however, behaved
differently and is discussed separately.
Three sets of experiments were
performed to determine the order of the reaction with respect to each
of the reactants. 1) A solution of constant GSH (10.8 mM) and
O concentrations (0.72 mM) was mixed with nitric
oxide solutions of varying concentrations (0.064-0.44
mM). The slope of
ln(d[GSNO]/dt)
versus ln[NO] (Fig. 2) plot was 2, which demonstrates a
second order concentration dependence of reaction rates on NO
concentration. 2) Solutions with constant GSH concentrations (10.4
mM) and varying concentrations of O
(0.24-1.2 mM) were mixed with a solution of
constant nitric oxide concentration (0.2 mM). These data
yielded first order dependence of reaction rates on O
concentration (Fig. 3). 3) Solutions of varying GSH
concentrations (0.6-60 mM) and constant O
concentration (0.72 mM) were mixed with a solution of constant
NO concentration (0.2 mM). In Fig. 4, ln R
is plotted as a function of ln[GSH]. It is clear that
at lower GSH concentrations initial rates depend on GSH concentration.
At higher GSH concentrations (
5 mM) initial rates become
independent of GSH concentration.
Figure 2:
Second
order dependence on [NO] for the S-nitrosation
reaction of GSH. Anaerobic solutions of varied [NO] but
constant [O] and [GSH] in 0.1 M sodium phosphate, pH. 7.0, were mixed, and measurements of GSNO
formation as an increase in absorbance at 335 nm were made at 20 °C
using a stopped-flow spectrophotometer in the single mixing mode (see
``Materials and Methods'' for details). Reactant
concentrations after mixing were [GSH] = 5.4 mM and [O
] = 0.31 mM;
[NO] varied from 0.032 to 0.22
mM.
Figure 3:
First order dependence on
[O] for the S-nitrosation reaction of
GSH. After mixing, reactant concentrations were [NO] =
0.1 mM and [GSH] = 5.2 mM;
[O
] varied from 0.12 to 0.60 mM. Other
conditions are as described in Fig. 2.
Figure 4:
Dependence of initial rates on GSH
concentrations. After mixing, reactant concentrations were
[NO] = 0.1 mM and [O]
= 0.36 mM; [GSH] varied from 0.3 to 20.0
mM. Other conditions are as for Fig. 2.
Similar concentration dependence
with respect to NO, O, and thiol was observed for N-acetylcysteine.
On the basis of these kinetic observations and earlier studies by Wink et al.(1993, 1994), we can tentatively write the following reaction mechanism:
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Justification for the individual steps is provided under ``Discussion.''
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
The approximate value of k was 1.5
s
. However, it has not been possible to obtain
quantitatively consistent results over a large enough concentration
range of the reactants to give some confidence to the rate law and a
reaction mechanism that may be postulated. Observations such as the
effect of buffers on the reaction rates are difficult to explain even
qualitatively. McAninly et al.(1993) made similar observations
in their study of nitrosothiol decomposition and attributed these
effects to the presence of metal ion contaminants. The effect
disappeared on inclusion of EDTA in the reaction mixture. The reaction
of cysteine with NO in the presence of O
therefore was
studied in detail only in the presence of 50 mM EDTA in 0.1 M phosphate buffer. Under these conditions the reaction
follows the same kinetics as those observed for the formation of GSNO
and N-acetylcysteine-NO.
Figure 5:
Dependence of nitrosothiol yield on
initial NO concentration. A, the low M
thiols in 0.1 M phosphate, pH 7.0, were mixed with NO in the
presence of O
, and the yield of nitrosothiol was calculated
based on the total increase in A
using published
extinction coefficients for each nitrosothiol. Measurements were made
in a stopped-flow spectrophotometer. Data for albumins are included for
comparison; details are given in B. All concentrations are in
mM. B, same as A for human and bovine serum
albumin.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
The coefficient m represents the fraction of total NO used in nitrosothiol formation. The following can be shown:
On-line formulae not verified for accuracy
Figure 6:
HPLC chromatogram of solutions of the
following compositions. A, GSH + O + NO; B, control, 5 mM GSH + 6 mM NEM; C, buffer blank (1 mM NEM); D, GSNO +
GSSG + NaNO
+ NaNO
(0.3 mM each). NEM was added at 6 mM to samples to block excess
GSH and yield the GS adduct of NEM. Peak identification: 1,
NEM; 2, GS-NEM product; 3, GSNO; 4, GSSG; 5, nitrite; 6, nitrate. For other details see the
text. Absorbance is at 210 nm in arbitrary
units.
On-line formulae not verified for accuracy
At GSH concentrations 5 mM or higher, the rates of GSNO
formation are independent of GSH concentration, indicating that k[GSH]
k
[H
O]. Under these
conditions the rate simplifies to the following:
On-line formulae not verified for accuracy
Using and the stoichiometric relationship
¼[NO] =
[O
], k
can be calculated from a reaction time course
using the algorithm described earlier (Kharitonov et al.,
1994). Over the entire range of concentrations of NO, O
,
and thiols used in the present study, least squares analysis of
reaction time courses (each consisting of more than 200 data points)
yielded k
= 7.0 ± 1
10
(S.D.) M
s
. This
is a global average of k
for the three thiols used
in this study. Fig. 7shows typical observed and calculated
reaction time courses for glutathione. This value of k
should be compared with the value of k
= 6.3
10
M
s
reported by us earlier for the auto-oxidation of
NO (Kharitonov et al., 1994).
Figure 7:
Third
order fit for reaction time courses of S-nitrosation of
glutathione by NO in the presence of O. All concentrations
are after mixing; Fraction = fraction of the reaction completed;
time is shown in seconds. [GSH] = 5.4 mM;
[NO] = 0.23 mM; [O
]
= 0.31 mM.
On-line formulae not verified for accuracy
explain
all qualitative and quantitative observations described under
``Results.'' First and second order dependence of reaction
rates with respect to NO and O is explained by and substantiated by data in Fig. 2and Fig. 3. Dependence of reaction rates
(d[GSNO]/dt) on thiol concentration arises from
competition between and . This should be
obvious from the rate and is made further clear by Fig. 8, in which initial reaction rates corrected for hydrolysis
of N
O
by are plotted against
thiol concentration (i.e. initial reaction rates are divided
by the factor m). Rate predicts that 1) at high
thiol concentrations the reaction rates should be independent of thiol
concentration and 2) at low thiol concentrations the reaction rates
should approach first order dependence on thiol concentration. Both of
these predictions are consistent with the data in Fig. 4. These
observations indicate that the rate-limiting step in is .
Figure 8:
Initial reaction rates corrected for
hydrolysis of NO
plotted against GSH
concentrations. Other conditions are as for Fig. 4. After
mixing, concentrations were [NO] = 0.1 mM and
[O
] = 0.36 mM; [GSH]
varied from 0.5 to 30 mM.
In there are
two potential nitrosating agents: NO
, a dimer
of NO
, and N
O
. For electrophilic
nitrosation, these two reagents should act as sources of
NO
. In the case of N
O
, this
would lead to the formation of NO
.
Nitrosation via N
O
would form
NO
. The HPLC data support the latter
possibility.
In Table 2are listed the calculated values of k. These depend on the value of k
used for calculating k
from and
data in Fig. 5. Two values of k
differing
by a factor of three have been reported (k
[H
O] = 5.3
10
s
, Grätzel et
al.(1970); and 1.6
10
s
,
Licht et al.(1988)). We have used the more recent value
reported by Licht et al.(1988). These calculations indicate
that the second order rate constant k
is about
four orders of magnitude greater than k
for the
hydrolysis of N
O
. For the two protein thiols k
is much lower than for the low M
thiols. This is most likely due to the inaccessibility of the SH
group in the protein matrix.
A similar trend is present in the rate
constants for nitrosation at low pH. As compared to low M thiols for both HSA and BSA the rate constant (k
) is lower by a factor of more than 100. The
rate constants listed in Table 1are overall third order
constants. Simple calculations show that nitrosation by this reaction
path at pH values of physiological interest should be insignificant.
Glutathione is the most important intracellular thiol. Its
intracellular concentration in mammalian cells varies over a wide range
(0.5-10 mM, Meister and Anderson(1983)). Blood plasma
concentrations are in the micromolar range. From the data in Fig. 4, it is clear that in cells with GSH concentration 5
mM or more almost all of NO
formed
from the reaction of naturally occurring NO will be used up for the
nitrosation of intracellular glutathione. However, this discussion
should not lead to the conclusion that all of NO formed in vivo will be consumed to form RSNO. Nitric oxide can react with various
molecules found in biological systems. Those most often considered in
this regard are oxygen, thiols, amines, tyrosine, free radicals, and
ferric and ferro heme proteins. Kinetic studies of Wink et
al.(1993, 1994) indicate that the third order reaction of nitric
oxide with oxygen is slow and acts as the rate-limiting step in
nitrosation reactions via the formation of N
O
.
Reactions of NO with O
and ferro and
ferric heme proteins, on the other hand, are very fast (Padmaja and
Huie, 1993; Beckman and Crow, 1993; Cassoly and Gibson, 1975; Sharma et al., 1983, 1987). Therefore, in vivo only a small
fraction of naturally produced NO will be able to react with oxygen to
produce N
O
. The novel finding of the present
study is that almost all of N
O
thus formed is
consumed in GSNO formation in cells in which GSH concentrations are 5
mM or higher. At lower concentration of GSH, nitrosation of
GSH by N
O
will become progressively less
significant.
In the case of the two protein thiols HSA and BSA, the
situation is quite different. The rate constant for nitrosation is
about of that for the low molecular weight thiols, and their plasma
concentration rarely exceeds 1 mM. Therefore, it seems that
nitrosation of these two proteins via reaction with
NO
may not be as significant. For protein
thiols, alternative reaction mechanisms of nitrosation may be of more
physiological significance than the reaction with
N
O
. Keaney et al.(1993) have suggested
the possibility of HSA nitrosation by reaction with the nitrosonium
ion, NO
. Metal-catalyzed formation of NO
observed by us is consistent with this proposition. We have also
observed that the reaction of NO with ferric heme-proteins can also be
a potential source of nitrosonium ion formation as shown:
On-line formulae not verified for accuracy
Brackman and Smit(1965) have reported nitrosation via the
mediation of copper salts and NO formation.
Nitrosation of thiols and other reactive groups by metal ion-mediated
formation of NO
is likely to be considerably faster
than via the reactions of NO with oxygen.
To summarize, in aqueous
solutions of thiols, NO and O nitrosation of the thiol
proceeds via the rate-limiting step of formation of oxides of nitrogen.
The nitrosating intermediate seems to be N
O
.
Its reaction with thiols is competitive with the rate of its
hydrolysis. Considerations based on the rate constants and in vivo concentrations of glutathione and HSA suggest that in the case of
glutathione, significant nitrosation can take place by its reaction
with N
O
particularly in cells with high GSH
concentration (
5 mM). This reaction path seems to be less
significant for the nitrosation of HSA.