Mechanism of the nitrosation of thiols and amines by oxygenated .NO solutions: The nature of the nitrosating intermediates

Article abstract of DOI:10.1021/ja9536680

The nitrosation of various thiols and morpholine by oxygenated ^.NO solutions at physiological pH was investigated. The formation rates and the yields of the nitroso compounds were determined using the stopped-flow technique. The stoichiometry of this process has been determined, and is given by 4^.NO + O₂ + 2RSH/2RR'NH → 2RSNO/2RR'NNO + 2NO₂^- + 2H⁺. Kinetic studies show that the rate law is -d[O₂]/dt = k₁ [^.NO]²[O₂] with k₁ = (2.54 + 0.26) x 10⁶ M^-2 s^-1 and -d[^.NO]/dt = 4k₁ [^.NO]²[O₂] with 4k₁ = (1.17 + 0.12) x 10⁷ M^-2 s^-1, independent of the kind of substrate present. The kinetic results are identical to those obtained for the autoxidation of ^.NO, indicating that the rate of the autoxidation of ^.NO is unaffected by the presence of thiols and amines. The nitrosation by ^.NO takes place only in the presence of oxygen, and therefore the rate of the formation of S-nitrosothiols from thiols and oxygenated ^.NO solution is relatively slow in biological systems. Under physiological conditions where [^.NO] < 1 μM and [O₂] < 200 μM, the half-life of the nitrosation process exceeds 7 min. Therefore, this is an unlikely biosynthetic pathway for the formation of S-nitrosothiols. As such, S-nitrosothiols cannot serve as carrier molecules of ^.NO in vivo. The rate-determining step of the nitrosation of thiols and amines by oxygenated ^.NO solution is the formation of ONOONO (or ONONO₂ or O₂NNO₂), which is the precursor of ^.NO₂ and N₂O₃. The stoichiometry of the nitrosation process suggests that ^.NO₂ and/or N₂O₃ are the reactive species. We have demonstrated that ^.NO₂ initiates the nitrosation process unless it is scavenged faster by ^.NO to form N₂O₃. The latter entity is also capable of directly nitrosating thiols and amines with rate constants exceeding 6 x 10⁷ M^-1 s^-1.

Full text of DOI:10.1021/ja9536680

J. Am. Chem. Soc. 1996, 118, 3419-3425
3419
Mechanism of the Nitrosation of Thiols and Amines by
•
Oxygenated NO Solutions: the Nature of the Nitrosating
Intermediates
Sara Goldstein* and Gidon Czapski
Contribution from the Department of Physical Chemistry, The Hebrew UniVersity of Jerusalem,
Jerusalem 91904, Israel
ReceiVed October 31, 1995X
•
Abstract: The nitrosation of various thiols and morpholine by oxygenated NO solutions at physiological pH was
investigated. The formation rates and the yields of the nitroso compounds were determined using the stopped-flow
technique. The stoichiometry of this process has been determined, and is given by 4•NO + O2 + 2RSH/2RR′NH
-
f 2RSNO/2RR'NNO + 2NO2 + 2H+. Kinetic studies show that the rate law is -d[O2]/dt ) k1[•NO]2[O2] with
k1 ) (2.54 ( 0.26) × 106 M-2 s-1 and -d[•NO]/dt ) 4k1[•NO]2[O2] with 4k1 ) (1.17 ( 0.12) × 107 M-2 s-1
,
independent of the kind of substrate present. The kinetic results are identical to those obtained for the autoxidation
•
of NO, indicating that the rate of the autoxidation of •NO is unaffected by the presence of thiols and amines. The
nitrosation by •NO takes place only in the presence of oxygen, and therefore the rate of the formation of S-nitrosothiols
•
from thiols and oxygenated NO solution is relatively slow in biological systems. Under physiological conditions
where [•NO] < 1 µM and [O2] < 200 µM, the half-life of the nitrosation process exceeds 7 min. Therefore, this is
an unlikely biosynthetic pathway for the formation of S-nitrosothiols. As such, S-nitrosothiols cannot serve as carrier
•
•
molecules of NO in ViVo. The rate-determining step of the nitrosation of thiols and amines by oxygenated NO
•
solution is the formation of ONOONO (or ONONO2 or O2NNO2), which is the precursor of NO2 and N2O3. The
stoichiometry of the nitrosation process suggests that •NO2 and/or N2O3 are the reactive species. We have demonstrated
that •NO2 initiates the nitrosation process unless it is scavenged faster by •NO to form N2O3. The latter entity is also
capable of directly nitrosating thiols and amines with rate constants exceeding 6 × 107 M-1 s-1
.
•
Introduction
compounds are more stable than NO,6-8,10,11,14 and certain
S-nitrosothiols are potent vasodilators and platelet inhibitors,6-8,14
similar to endothelium-derived relaxing factor (EDRF),15 which
Nitric oxide (•NO) is formed enzymatically from L-arginine
by many types of cells and is an important mediator in several
physiological processes.1 In neuronal and endothelial cells, •NO
is an intercellular messenger, effecting signal transduction by
stimulation of heme-containing soluble guanylate cyclase.1 •NO
is unusual as a biochemical messenger and effector because of
its small size, high diffusibility, and high chemical reactivity
as a free radical. It reacts with superoxide,2 oxygen,3 and heme
and non-heme iron,4,5 which are all present in the medium.
has been identified as NO.16,17 A recent comparison of the
•
physiological properties of S-nitrosothiols to those of •NO and
EDRF has shown that they differ in their stability and reactivity
with oxyhemoglobin.18 However, there is currently no convinc-
ing experimental evidence to indicate that EDRF is S-nitroso-
•
thiol rather than NO.
•
Although NO is a major participant in a large number of
physiological processes,1 excess production of NO can be
•
•
Therefore, it has been argued that NO is stabilized by the
toxic.10,11,18-23 The cytotoxic effects of NO have been at-
•
reaction with a carrier molecule that prolongs its half-life and
preserves its biological activity.6-9 Important classes of po-
tential carrier molecules are those containing sulfhydryl (RSH)
and amine (RR′NH) functional groups. It has been demon-
strated that oxygenated •NO solutions nitrosate thiols and amines
at physiological pH to form S-nitrosothiols (RSNO) and
N-nitrosoamines (RR′NNO), respectively.10-13 These nitroso
(10) Wink, D. A.; Kasprzak, K. S.; Maragos, C. M.; Elespuru, R. K.;
Misra, M.; Dunams, T. M.; Cebula, T. A.; Koch, W. H.; Andrews, A. W.;
Allen, J. S.; Keefer, L. K. Science 1991, 254, 1001.
(11) Nguyen, T.; Crespi, C. L.; Penman, B. W.; Wishnok, J. S.;
Tannenbaum, S. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 3030.
(12) 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., Mitchell, J. B. Chem. Res. Toxicol. 1994, 7, 519.
(13) Lewis, R. S.; Tannenbaum, S. R.; Deen, W. M. J. Am. Chem. Soc.
1995, 117, 3933.
(14) Ignarro, L. J.; Lipton, H.; Edwards, J. C.; Baricos, W. H.; Hyman,
A. L.; Kadowitz, P. J.; Gruetter, C. A. J. Pharmacol. Exp. Ther. 1981,
218, 739.
(15) Furchgott, R. F.; Zawadski, J. V. Nature (London) 1980, 288, 373
(16) Palmer, R. M. J.; Ferrige, A. J.; Moncada, S. Nature 1987, 327,
524.
* To whom correspondence should be addressed. Phone: 972-2-
6586478. Fax: 972-2-586925. E-mail: sarag@vms.huji.ac.il.
X Abstract published in AdVance ACS Abstracts, March 15, 1996.
(1) Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Pharmacol. ReV. 1991,
43, 109.
(2) Goldstein, S.; Czapski, G. Free Radical Biol. Med. 1995, 19, 505.
(3) Awad, H. H.; Stanbury, D. M. Int. J. Chem. Kinet. 1993, 25, 375.
(4) Reddy, D.; Lanaster, J. R.; Cornforth, D. P. Science 1983, 221, 769.
(5) Tsai, A.-L. FEBS Lett. 1994, 341, 141.
(17) Ignarro, L. J.; Buga, J. M.; Wood, K. S.; Byrns, R. E.; Chaudhuri,
G. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 9265.
(6) Ignarro, J. L. Annu. ReV. Pharmacol. Toxicol. 1990, 30, 535.
(7) Stamler, J. S.; Simon, D. L.; Osborne, J. A.; Mullins, M. E.; Jarkai,
O.; Michel, T.; Singel, D. J.; Loscalzo, J. Proc. Natl. Acad. U.S.A. 1992,
89, 444.
(18) Feelisch, M.; te Poel, M.; Zamora, R.; Deussen, A.; Moncada, S.
Nature 1994, 368, 62.
(19) Hibbs, J. B., Jr.; Taintor, R. R.; Vavrin, Z.; Rachlin, E. M. Biochem.
Biophys. Res. Commun. 1988, 157, 87.
(8) Stamler, J. S.; Jaraki, O.; Osborne, J.; Simon, D. I.; Keaney, J.; Vita,
J.; Singel, D.; Valeri, C. R.; Loscalzo, J. Proc. Natl. Acad. U.S.A. 1992,
89, 7674.
(20) Beckman, J. S.; Beckman, T. W.; Chen, J.; Marshall, P. A.; Freeman,
B. A. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1620.
(21) Nowicki, J. P.; Duval, D.; Poignet, H.; Scatton, B. FEBS Lett. 1991,
204, 339.
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0002-7863/96/1518-3419$12.00/0 © 1996 American Chemical Society

3420 J. Am. Chem. Soc., Vol. 118, No. 14, 1996
Goldstein and Czapski
tributed partially to the formation of oxidizing and nitrosating
•
•-
agents that are formed via the reaction of NO with O2 and
O2. Thus, the formation of NO adducts may have several roles
•
in ViVo, either as carrier molecules of NO or as effective
scavengers that may further lead to toxicity, e.g., deamination
of DNA10,11 and S-nitrosation of enzymes.22
•
The autoxidation of NO in aqueous solution is a very
complex process.3,9,24,25 It has been suggested by several groups
that the nitrosating species in the •NO/O2 system is N2O3,10,11,13,24
whereas Wink et al.12 have suggested that the nitrosating species
is an unidentified NOx species. The rates of the formation of
the nitroso compounds by •NO/O2 and the nature of the
nitrosating intermediates are of great importance from the
biological point of view. The identification of these species is
crucial for understanding the various biological roles of •NO in
ViVo.
In this study we will demonstrate that the rate of the
•
autoxidation of NO is unaffected by the presence of various
thiols and amines. We will show that the nitrosation process
•
•
is initiated by NO2 unless NO2 is scavenged more rapidly by
•NO to form N2O3, which is capable of directly nitrosating these
compounds.
Figure 1. Observed first-order rate constant of the formation of RSNO
and MorNNO as a function of [•NO]2 in the presence of 20.4-102
µM O2 at pH 7.4 ( 0.1 (1-2 mM phosphate buffer), and the following
substrates: ([) GSH; (×) CysSH; (2) NAPenSH; (+) DTT; (b)
MorNH. [•NO] was taken as [•NO]o - 2[O2]o.
Experimental Section
Materials. All chemicals were of analytical grade and were used
as received. Nitric oxide, C.P., was purchased from Matheson Gas
Products. Solutions were prepared with deionized water that was
distilled and purified using a Milli-Q water purification system.
Glutathione (GSH), cysteine (CysSH), penicillamine (PenSH), N-
acetylpenicillamine (NAPenSH), captopril (CapSH), dithiothreitol
(DTT), morpholine (MorNH), N-nitrosomorpholine (MorNNO), S-
nitrosoglutathione (GSNO), and S-nitroso-N-acetylpenicillamine (SNAP)
were purchased from Sigma.
M-1 cm-1, and when [DTT]o g [NO2-]o, ꢀ332 ) 825 ( 25 M-1 cm-1
and ꢀ544 ) 13.8 ( 1.4 M-1 cm-1, which indicates the formation of two
nitroso groups in the presence of excess nitrite over DTT. The
absorption maxima of GSNO, SNAP, and MorNNO were also
determined by dissolving commercial materials at pH 7.4 (5 mM
phosphate buffer) and are at 338 nm (ꢀ ) 760 ( 20 M-1 cm-1), 340
nm (ꢀ ) 930 ( 30 M-1 cm-1) and 340 nm (ꢀ ) 100 ( 1 M-1 cm-1),
respectively.
Methods. Stopped-flow kinetic measurements were carried out
using the Bio SX-17MV sequential stopped-flow apparatus from
Applied Photophysics. A xenon lamp (Osram XBO 150W) produced
the analyzing light, and a Hammamatsu R928 photomultiplier was used
for measurements in the near UV and visible regions. The formation
of the nitroso compounds was followed at 333-340 nm. The optical
path length was 1 cm. All measurements were carried out at 22 °C.
Each value given is an average of at least five measurements.
Oxygen-saturated solutions (1.12 mM at 25 °C and 690 mmHg,
which is the barometric pressure in Jerusalem)26 were prepared by
bubbling gas-tight syringes with oxygen for 30 min. The •NO gas was
purified by passing it through a series of scrubbing bottles containing
50% NaOH and distilled water in this order. The solutions in the traps
were first deaerated by purging them with helium for 1 h. Nitric oxide
solutions were prepared in gas-tight syringes by purging first 1 mM
phosphate buffer solutions (pH 7.4) with helium to remove O2, followed
•
•
by bubbling for 30 min with NO. The NO-saturated solutions (1.72
mM at 25 °C and 690 mmHg)26 were stored in syringes and
subsequently diluted with helium-saturated solutions to the desired
concentrations by the syringe technique. Stock solutions of thiols were
daily prepared and were used after neutralization. Acetate, phosphate,
and borate buffers were used for pH 4-5, 6-8, and 9-10, respectively.
Results
When aerated solutions of RSH (GSH, CysSH, PenSH,
NAPenSH, CapSH, DTT) or MorNH at pH 7.4 ( 0.1 were
mixed with •NO-saturated solution to yield final concentrations
of 0.57-1.56 mM •NO, 20.4-102 µM O2, 1-2 mM phosphate
buffer, and 0.2-40 mM RSH or 0.5-150 mM MorNH, a rapid
formation of an absorption with a maximum at 332-340 nm
was observed, which was attributed to the formation of RSNO
or MorNNO. Under these conditions the rate of the formation
of RSNO and MorNNO was first-order. The observed first-
order rate constant was linearly dependent on [•NO]2, yielding
a slope of (2.54 ( 0.26) × 106 M-2 s-1 for all compounds
studied (Figure 1). This third-order rate constant is within
experimental error identical to that determined for the autoxi-
dation of •NO and for the oxidation of ferrocyanide and ABTS
S-Nitrosothiols were prepared by acid-catalyzed S-nitrosation of the
thiol with sodium nitrite at 2 °C as previously described.27 Equimolar
concentrations of NaNO2 and thiol in 0.1 N sulfuric acid were mixed
and allowed to react for a few minutes. Dilutions were made with 0.1
M phosphate buffer at pH 7.4. S-Nitrosothiols have two absorption
maxima at 330-340 and 544 nm, except SNAP, which absorbs at 590
nm. The extinction coefficients are as follows: (for GSNO) ꢀ336
)
770 ( 30 M-1 cm-1 and ꢀ544 ) 15.1 ( 0.6 M-1 cm-1; (for SNAP) ꢀ340
) 815 ( 50 M-1 cm-1 and ꢀ590 ) 11.6 ( 0.3 M-1 cm-1; (for CapSNO)
ꢀ332 ) 940 ( 40 M-1 cm-1 and ꢀ544 ) 16.7 ( 0.5 M-1 cm-1 28
When
.
[NO2-]o g 2[DTT]o, ꢀ336 ) 1685 ( 60 M-1 cm-1 and ꢀ544 ) 30 ( 1.6
•
by the NO/O2 under limiting concentrations of O2.25 No
(22) Molina y Vedia, L.; McDonald, B.; Reep, B.; Brune, B.; Di Silvio,
M.; Billiar, T. R.; Lapetina, E. G. J. Biol. Chem. 1992, 267, 24929.
(23) Dawson, T. M.; Snyder, S. H. J. Neurosci. 1994, 14, 5147.
(24) Pires, M.; Rossi, M. J.; Ross, D. S. Int. J. Chem. Kinet. 1994, 26,
1207.
(25) Goldstein, S.; Czapski, G. J. Am. Chem. Soc. 1995, 117, 12078.
(26) Lange’s Handbook of Chemistry, 13th ed.; Dean, J. A., Ed.;
McGraw-Hill: New York, 1985; pp 10-15.
(27) Loscalzo, J.; Smick, D.; Andon, N.; Cooke, J. J. Pharmacol. Exp.
Ther. 1989, 249, 726.
formation of absorbance at 332-340 nm was observed when
(28) It has been reported that the absorption spectrum of CapSNO consists
of four absorption maxima: 333 nm (ꢀ ) 1890 M-1 cm-1), 404 nm (ꢀ )
339 M-1 cm-1), 512 nm (ꢀ ) 7.9 M-1 cm-1), and 545 nm (ꢀ ) 13.3 M-1
cm-1).27 However, we did not observe any absorption peak at 404 nm and
determined ꢀ333 ) 940 M-1 cm-1, which differs from than that determined
in ref 27, but is similar to those of all S-nitrosothiols that have been measured
so far as well as to those of many other thionitrites in nonaqueous solutions
(Oae; et al. J. Chem. Soc., Perkin Trans. 1 1978, 913).

Nitrosation of Thiols and Amines
J. Am. Chem. Soc., Vol. 118, No. 14, 1996 3421
Figure 3. Observed second-order rate constant of the formation of
RSNO as a function of [O2]o at pH 7.4 ( 0.1: (+) [CysSH] ) 2 mM;
([) [GSH] ) 4 mM; (b) [NAPenSH] ) 15 mM.
Figure 2. Measured OD at the end of the formation of SNAP and
CysSO at 340 and 338 nm, respectively, in the presence of 1-4 mM
thiol at pH 7.4 ( 0.1 (1.2-1.9 mM phosphate buffer) and (a) under
limiting concentration of O2, ([) SNAP, (+) CysNO and (b) under
Table 2. Observed Second-Order and the Third-Order Rate
•
Constants of the Formation of RSNO and MorNNO under Limiting
limiting concentration of NO, (b) SNAP, (9) CysSNO.
•
Concentrations of NO
Table 1. Slopes of the Lines of the Maximun Absorbance of
k
obs/[O2] )
k, M-2 s-1
k, M-2 s-1
•
RSNO and MorNNO versus O2 and NO Concentrations at pH 7.4
substrate k/ꢀl, M-1 s-1 (ꢀ from Table 1) (ꢀ determined directly)
( 0.1 (1-2 mM Phosphate Buffer)a
GSH
CysSH
3.34 × 104
3.86 × 104
2.64 × 107
2.40 × 107
2.04 × 107
2.14 × 107
2.16 × 107
2.18 × 107
1.96 × 107
2.58 × 107
nd
slope a, M-1 cm-1 slope b, M-1 cm-1
substrate
[•NO]o > [O2]o
[O2]o > [•NO]o
slope a/slope b
NAPenSH 2.52 × 104
2.34 × 107
nd
GSH
1583
1245
1621
1625
1653
1530
182
387
323
425
423
350
391
46
4.09
3.85
3.82
3.84
4.72
3.91
3.96
PenSH
CapSH
DTT
2.62 × 104
2.62 × 104
2.80 × 104
2.13 × 105
2.46 × 107
2.32 × 107
2.14 × 107
CysSH
NAPenSH
PenSH
CapSH
DTT
MorNH
all the nitroso products under limiting concentrations of •NO is
(2.34 ( 0.24) × 107 M-2 s-1, which is about 8 times the value
determined under limiting concentrations of O2.
MorNH
a Slope a ) The slope of the line as in Figure 2a, obtained under
limiting concentrations of O2. b Slope b )The slope of the line as in
•
Maximum nitrosation yields were obtained when [RSH]o g
[•NO]o/2 in the case of CysSH, GSH, PenSH, and DTT, which
decreased when [RSH]o exceeded 4 mM. In the case of
NAPenSH, CapSH, and MorNH, maximum product yields were
obtained for [RSH]o > 15, 1, and 2 mM, respectively. The
maximum yields of RSNO and MorNNO were linearly depend-
ent on [•NO]o (Figure 2b). The slopes obtained for the various
substrates from plots similar to those given in Figure 2b are
summarized in Table 1. The ratio of the slopes obtained under
limiting concentrations of O2 to those obtained under limiting
Figure 2b, obtained under limiting concentrations of NO.
•NO-saturated solutions were mixed with deaerated solutions
containing the various thiols or morpholine at pH 7.4, indicating
that O2 is essential for the nitrosation process as previously
reported.12,29
Maximum nitrosation yields were obtained when [RSH]o g
2[O2]o in the case of CysSH, GSH, PenSH, and DTT, but
decreased when [RSH]o exceeded 4 mM. In the case of
NAPenSH, CapSH, and MorNH, maximum yields were obtained
for [RSH]o > 15, 1, and 2 mM, respectively. The yields of
RSNO and MorNNO were linearly dependent on [O2]o (Figure
2a). The slopes obtained for the various substrates from plots
similar to those given in Figure 2a are summarized in Table 1.
When oxygen-saturated solutions containing RSH and MorNH
were mixed with •NO solutions to yield final concentrations of
0.3-1.02 mM O2, 39-287 µM •NO, 1-2 mM phosphate buffer
(pH 7.4 ( 0.1), and 0.2-100 mM RSH or 0.5-480 mM
MorNH, the rate of the formation of RSNO and MorNNO was
second-order. The observed second-order rate constant was
linearly dependent on [O2]o (Figure 3). The third-order rate
constants obtained for the formation of RSNO and MorNNO
from the dependence of kobs on [O2]o and the known or measured
ꢀmax, are summarized in Table 2. The rate of the formation of
•
concentrations of NO is 4.0 ( 0.2 (Table 1).
The nitrosation yields decreased with the increase in phos-
phate buffer concentrations and with the decrease in pH (Table
•
3). The effect of phosphate on the product yields in the NO/
O2 system has already been observed in the presence of
MorNH,13 ferrocyanide,25 and ABTS.25
Discussion
The rate of the nitrosation of thiols and amines by oxygenated
•NO solution is second-order in [•NO] and first-order in [O2].
The third-order rate constant of the nitrosation is identical to
that of the autoxidation of •NO, indicating that the rate-
determining step of the nitrosation by oxygenated •NO solution
is the same as that of the autoxidation of •NO. The ratio of the
nitrosation yields under limiting concentrations of O2 to those
(29) Pryor, W. A.; Curch, D. F.; Govindan, C. K.; Crank, J. J. Org.
Chem. 1982, 47, 156.
•
under limiting concentrations of NO is 4. Therefore, the

3422 J. Am. Chem. Soc., Vol. 118, No. 14, 1996
Goldstein and Czapski
Table 3. Effect of Phosphate Buffer and pH on the Nitrosation Yields
substrate
[RSH]o, mM
[NO]o, mM
[O2]o, mM
[Pi], mM
pH
[RSNO], µM
[RSNO]/([RSNO]max - [RSNO])
PenSH
PenSH
PenSH
PenSH
PenSH
PenSH
PenSH
GSH
GSH
GSH
GSH
GSH
0.56
0.56
0.56
0.56
0.56
0.56
0.56
2
2
2
2
2
1.37
1.37
1.37
1.37
1.37
1.37
1.37
0.156
0.156
0.156
0.156
0.156
0.156
0.0363
0.0363
0.0363
0.0363
0.0363
0.0363
0.0363
1
1
1
1
1
1
1.6
40
80
160
1.6
-
-
1.6
16
160
480
10
7.4
7.4
7.4
7.4
6.5
4.1
71
48
40
34
60
15
82
74
61
43
29
49
72
2.09
1.29
0.92
10
7.4
7.4
7.4
7.4
6.2
4.7
1.38
0.64
GSH
2
-
10
stoichiometry of the whole nitrosation process is given by eq
1, whereas that of the autoxidation of •NO is given by eq 2.3,24,25
limiting concentrations of •NO, each •NO yields 0.5RSNO, and
therefore rate equation 9b is obtained for the formation of
RSNO. Thus, irrespective of the detailed mechanism of the
4•NO + O2 + 2RSH/2RR′NH f
d[RSNO]/dt ) 2k1[•NO]2[O2] ) 2k1[O2][2RSNO]2 )
8k1[O2][RSNO]2 (9b)
2RSNO/2RR′NNO + 2NO2- + 2H+ (1)
4•NO + O2 + 2H2O f 4NO2- + 4H+
(2)
nitrosation process, the rate constant determined by following
the formation of RSNO under limiting concentrations of O2 is
We have recently shown that the rate-determining step of
the autoxidation of •NO is the formation of ONOONO, and the
whole process can be described by reactions 3-7.25 According
•
k1 (Figure 1), and under limiting concentrations of NO 8k1
(Figure 3, Table 2), and hence k1 ) (2.73 ( 0.19) × 106 M-2
s-1. This value is in excellent agreement with the value of k1
) (2.9 ( 0.1) × 106 M-2 s-1, which has been determined for
•NO + O2 h ONOO• (or NO‚‚‚O2)30
(3)
the autoxidation of NO.25
•
•
Since the rate of the autoxidation of NO is identical to that
O2NO• (or NO‚‚‚O2) + •NO f
of the nitrosation of thiols and amines by •NO/O2, the nitrosating
•
ONOONO (or ONONO2 or O2NNO2) (4)
species must be ONOONO (or ONONO2 or O2NNO2), NO2,
or N2O3. If ONOONO is the nitrosating agent, one expects
that reactions 10-13 will take place. The direct oxidation of
ONOONO f 2•NO2
(5)
ONOONO + RS- f RSNO + ONOO-
ONOO- + RSH f RS• + •NO2 + OH-
(10)
(11)
•NO2 + •NO h N2O3 k6 ) 1.1 × 109 M-1 s-1;
k-6 ) 8.1 × 104 s-1 33 (6)
N2O3 + H2O f 2NO2- + 2H+ k7 ) 530 s-1 33 (7)
-
•NO2 + RS- f RS• + NO2
(12)
(13)
to this mechanism, rate equation 8 is obtained, where k-3
>
RS• + •NO f RSNO
k4[•NO] and k1 ) k3k4/k-3 ) (2-2.9) × 106 M-2 s-1 3,24,25,32
.
cysteine to cystine by peroxynitrite in aerated solution has been
demonstrated by Radi et al.,33 the stoichiometry being 2.2 mol
of cysteine/mol of peroxynitrite. This process is mediated by
one-electron transfer with the formation of thiyl radical and most
probably •NO2.34 In this case reactions 11 and 12 are followed
by reactions 14-16, where O2•- dismutates faster than it reacts
d[O2] k3k4[•NO]2[O2]
k-3 + k4[•NO]
dt
d[•NO]
1
4
-
) -
)
) k1[•NO]2[O2]
dt
(8)
The stoichiometry of the nitrosation process is given by eq
1. Thus, rate equation 9 is obtained irrespective of the detailed
mechanism. The rate of the nitrosation process was determined
RS• + RS- h RSSR•-
RS• + RS• f RSSR
(14)
(15)
(16)
d[•NO]
d[RSNO]
d[O2]
1
4
1
2
-
) -
)
) k1[•NO]2[O2] (9)
dt
dt
dt
•-
RSSR•- + O2 f RSSR + O2
by following the formation of RSNO/RR′NNO. Under limiting
concentrations of O2, each O2 yields 2RSNO, and therefore rate
equation 9a is obtained for the formation of RSNO. Under
with the thiol.35 The addition of molecular oxygen to RS. does
not compete efficiently with reactions 14 and 16,36 and can be
•
ignored. We assume that, in the NO/O2 system, reaction 13
d[RSNO]/dt ) 2k1[•NO]2[O2] ) 2k1[•NO]2[0.5RSNO] )
k1[•NO]2[RSNO] (9a)
(33) Radi, R.; Beckman, J. S.; Bush, K. M.; Freeman, B. A. J. Biol.
Chem. 1991, 266, 4244.
(34) Gatti, R. M.; Radi, R.; Augusto, O. FEBS Lett. 1994, 348, 287.
(35) Bielski, B. H. J.; Shiue, G. G. Oxygen Free Radicals and Tissue
Damage; Ciba Foundation Symposium 65 (new series); Excerpta Medica:
New York, 1979, p. 43-56.
(36) Monig, J.; Asmus, K.-D.; Forni, L. G.; Willson, R. L. Int. J. Radiat.
Biol. 1987, 52, 589.
(30) McKee, M. L. J. Am. Chem. Soc. 1995, 117, 1629.
(31) Gratzel, Von, M.; Taniguchi, S.; Henglein, A. Ber. Bunsen-Ges.
Phys. Chem. 1970, 74, 488.
(32) Ford, A. C.; Wink, D. A.; Stanbury, D. M. FEBS Lett. 1993, 326,
1.

Nitrosation of Thiols and Amines
J. Am. Chem. Soc., Vol. 118, No. 14, 1996 3423
competes efficiently with reactions 14-16,37,38 and therefore,
the stoichiometry of the nitrosation process will be given by eq
17, which differs from the stoichiometry of eq 1. If, however,
1/[RSNO] as a function of 1/[RS-] should yield a straight line
with slope/intercept ) S/I ) k6k7[•NO]/(k-6 + k7)k12.
Pathway II. N2O3 is the only nitrosating species. This will
be the case if k12[RS-] , k6k7[NO]/(k-6 + k7). With this
mechanism, the nitrosation yields will be independent of [•NO],
and will be given by eq 22. A plot of 1/[RSNO] as a function
of 1/[RS-] should yield a straight line with S/I ) k7/k20.
4•NO + O2 + 3RSH/3RR′NH f
3RSNO/3RR′NNO + NO2- + H+ + H2O (17)
k20[RS-]
k20[RS-] + k7
peroxynitrite does not oxidize the substrate via reaction 11, but
decomposes into nitrate, or reaction 18 takes place, the net
[RSNO]
)
(22)
[RSNO]max
-
RS- + ONONO2 (or O2NNO2) f RSNO + NO3 (18)
Pathway III. •NO2 and N2O3 are both responsible for the
nitrosation process. In this case k12[RS-] does not exceed
nitrosation process will be given by eq 19, which also differs
from the stoichiometry of eq 1. If ONOONO (or ONONO2 or
k6k7[•NO]/(k-6 + k7), and the ratio k12[RS-]/k6k7[•NO]/(k-6
+
•
k7) determines the amount of the nitrosation by NO2. The
nitrosation yield will be given by eq 23. When k12[RS-] >
2•NO + O2 + RSH/RR′NH f
RSNO/RR′NNO + NO3- + H+ (19)
[RSNO]
[RS-]
)
k12 +
k12[RS-] + k6k7[•NO]/(k-6 + k7)
{
[RSNO]max
O2NNO2) is not the nitrosating species, one should consider
both NO2 and N2O3 as the active intermediates. •NO2 may
•
k20k6k7[NO]/(k-6 + k7)
k20[RS-] + k7
(23)
nitrosate thiols and amines via reactions 12 and 13, which are
both feasible.37,39-42 The nitrosation of thiols and amines by
•NO2 has been demonstrated previously,29,42,43 even at physi-
ological pH.43 However, if reaction 12 does not compete
efficiently with reaction 6, N2O3 will be formed. This latter
species is a strong nitrosating agent that can directly nitrosate
thiols and amines via reaction 20. The stoichiometry of the
}
k6k7[NO]/(k-6 + k7), eq 23 reduces to eq 21, and when k12[RS-]
< k6k7[NO]/(k-6 + k7), eq 23 reduces to eq 22.
The kinetics and the stoichiometry of the nitrosation by •NO/
•
O2 are the same whether NO2 (pathway I) or N2O3 (pathway
II) is the active intermediates. However, according to pathway
I, the nitrosation yield decreases with the increase in [•NO] (eq
21), whereas according to pathway II, it is independent of [•NO]
(eq 22).
-
N2O3 + RS- f RSNO + NO2
(20)
nitrosation process via •NO2 or N2O3 as the active intermediate
is in agreement with our experimental results (eq 1).
The reduction potentials of RS•/RS- and RS•,H+/RSH are
The only known values of k12 are for CysS- (k12 ) 2.4 ×
108 M-1 s-1 at pH 9.2,39 k12 > 5 × 108 M-1 s-1 at pH 7.9-
9.540), DTT (k12 ) 4.6 × 108 M-1 s-1 at pH 9),41 and PenS-
(k12 ) 2.8 × 108 M-1 s-1 at pH 10).46 Though there is some
discrepancy between the values of k12 for CysS-, it is clear that
reaction 12 is very fast at pH 7.4 as the pK values of CysSH
and PenSH are 8.18 and 7.93, respectively,47 and •NO2 is capable
of oxidizing both the protonated and unprotonated forms of
DTT.
-
about 0.73 and 1.35 V, respectively, whereas that of •NO2/NO2
is 1.04 V.44 The only exception is DTT, which has an unusually
low reduction potential of -0.33 V at pH 7.45 Therefore, from
the thermodynamic point of view, •NO2 and most probably N2O3
(E°(NO+/•NO) ) 1.21 V)44 are capable of oxidizing/nitrosating
the unprotonated forms of thiols. In the case of DTT the
protonated form is also available for oxidation/nitrosation by
•NO2 and N2O3. Thus, of great importance is the concentration
of the unprotonated form, which at a given pH is [RS-] )
[RSH]o/(1 + 10pK-pH).13,42,43 The alternative nitrosation path-
ways that yield eq 1 are as follows:
•
Under limiting concentrations of NO or O2 at pH 7.4, and
in the presence of CysSH, GSH, PenSH, and DTT, full
nitrosation yields were obtained for [RSH]o g [•NO]o/2 or
[RSH]o g 2[O2]o, respectively. Under these conditions k12[RS-]
> k6k7[•NO]/(k-6 + k7), and therefore •NO2 is the only species
responsible for the nitrosation process in the presence of these
thiols (pathway I). In these cases, the maximum nitrosation
yields decrease at relatively high concentrations of RSH, most
probably due to the competition of reactions 14-16 with
reaction 13.
Pathway I. The nitrosation will take place exclusively by
•NO2. This will be the case when reaction 12 competes
efficiently with reactions 6 and 7 (k12[RS-] . k6k7[•NO]/(k-6
+ k7)). If this condition is not fulfilled, the nitrosation yield
will be given by eq 21, provided that k7 >k20[RS-]. A plot of
k12[RS-]
The nitrosation yields decreased with the increase in phos-
phate buffer concentrations (Table 3). This effect has also been
reported in the presence of MorNH,13 ABTS,25 and ferrocya-
nide.25 It has been suggested that phosphate reacts directly with
[RSNO]
)
(21)
k12[RS-] + k6k7[•NO]/(k-6 + k7)
[RSNO]max
(37) Williams, D. H. L. Nitrosation; Cambridge University Press:
Cambridge, 1988.
-
N2O3,13 or that HPO4 catalyzes the hydrolysis of N2O3.25
(38) Eiserich, J. P.; Butler, J.; vander Vliet, A.; Cross, C. E.; Halliwell,
B. Biochem. J. 1995, 310, 745.
(39) Prutz, W. A.; Monig, H.; Butler, J.; Land, E. J. Arch. Biochem.
Biophys. 1985, 243, 125.
(40) Forni, L. G.; Mora-Arellano, V. O.; Packer, J. E.; Willson, R. L. J.
Chem. Soc., Perin Trans. 2 1986, 1.
(41) Elliot, A. J.; Sopchyshyn, F. C. Radiat. Phys. Res. 1982, 19, 417.
(42) Challis, B. C.; Kyrtopoulos, S. A. Br. J. Cancer 1977, 35, 693.
(43) Cooney, R. V.; Ross, P. D.; Bartonili, G. L. Ramseyer, J. EnViron.
Sci. Technol. 1987, 21, 77.
According to both suggestions, k7 ) (530 + kp[Pi]), and
(46) N2O-saturated solutions containing nitrite and penicillamine were
pulse-irradiated at pH 7.6 (2 mM phosphate buffer) and 10. The formation
of the absorbance at 330 nm was followed, and k12 ) (2.8 ( 0.3) × 108
M-1 s-1 was obtained from a fit to a complex mechanism which includes
reactions 12, 14, and 15 for which k14 ) 2.8 × 109 M-1 s-1, k-14 ) 2.9 ×
106 s-1, and k15 ) 2.3 × 109 M-1 s-1 (Hoffman, H. Z.; Hayon, E. J. Phys.
Chem. 1973, 77, 990). No reaction was observed at pH 6.
(47) Stability Constants of Metal Ion Complexes: Part B, Organic
Ligands; Perrin, D. D., Ed.; IUPAC Chemical Data Series No. 22; Pergamon
Press: Oxford-New York-Toronto-Sydney-Paris-Frankfurt, 1979.
(44) Stanbury, D. M. AdV. Inorg. Chem. 1989, 33, 69.
(45) Cleland, W. W. Biochemistry 1964, 3, 480.

3424 J. Am. Chem. Soc., Vol. 118, No. 14, 1996
Goldstein and Czapski
Figure 5. Double reciprocal plot of the absorbance of MorNNO with
varying concentrations of MorNH in the presence of (+) 0.286 mM
Figure 4. Double reciprocal plot of the absorbance of SNAP with
varying concentrations of NAPenSH in the presence of (+) 0.86 mM
•
•NO and 0.933 mM O2 ([) 1.43 mM NO and 0.187 mM O2. All
•
•NO and 0.112 mM O2 and ([) 1.43 mM NO and 37.3 µM O2. All
solutions contained 2 mM phosphate buffer at pH 7.4 ( 0.1.
solutions contained 1.9 mM phosphate buffer at pH 7.4 ( 0.1.
respectively. Therefore, even if k12 for all thiols is within the
same order of magnitude, the concentrations of NAPenS- and
CapS- are considerably lower than those of the other thiols at
pH 7.4. Thus, the rate of reaction 12 in the case of NAPenSH
and CapSH is considerably slower as compared to that of the
other thiols, and reaction 6 competes efficiently with reaction
12. The pK of MorNH is 8.5, and therefore we have to assume
that k12 for MorNH is more than an order of magnitude lower
than k12 for thiols.
phosphate shifts the whole nitrosation/oxidation process toward
nitrite formation. When kp[Pi] > 530 s-1, eq 24 is obtained
k12[RS-]
k6[NO]
k-6
[RSNO]
)
1 +
(24)
{
}
[RSNO]max - [RSNO]
kp[Pi]
from eq 21. From the data given in Table 3, we determined kp
) 6.7 × 105 and 9.4 × 105 M-1 s-1 in the presence of PenSH
and GSH, respectively. These values are in good agreement
with the value of 6.4 × 105 M-1 s-1, which has been determined
by Lewis et al.13 We have demonstrated25 that HPO42- catalyzes
the hydrolysis of N2O3, and hence k(N2O3 + HPO42-) ∼ 1 ×
106 M-1 s-1 at pH 7.4.
Wink et al.12 have also studied the nitrosation of GSH and
•
CysSH by NO/O2 at pH 7.4 (10 mM phosphate buffer), and
concluded that the nitrosating species is an unidentified NOx
species. However, this conclusion was based on several kinetic
errors and miscalculation of the extinction coefficients of RSNO
(see the Appendix).
In the presence of NAPenSH, CapSH, and MorNH, maximum
nitrosation yields were obtained at substrate concentrations
higher than 15, 1, and 2 mM, respectively. Plots of 1/OD340 as
a function of 1/[NAPenSH]o (Figure 4), 1/[MorNH]o (Figure
5), and 1/[CapSH]o (results not shown) yield straight lines with
S/I ) 0.94 ( 0.16, 0.096 ( 0.009, and 0.30 ( 0.06 mM,
respectively, independent of •NO concentrations. These results
indicate that N2O3 is the main nitrosating species at pH 7.4 in
the presence of NAPenSH, MorNH, and CapSH (pathway II).
The nitrosation yields decreased substantially with the decrease
in pH, indicating that the unprotonated forms of these com-
pounds are the main species which are available for nitrosation.
From the values of S/I, one calculates that k20/(1 + 10pK-pH) )
(5.63 ( 0.95) × 105, (1.78 ( 0.36) × 106, and (5.52 ( 0.52)
× 106 M-1 s-1 for NAPenSH, CapSH, and MorNH, respec-
tively. The pK values of NAPenSH, CapSH, and MorNH are
10,47 9.7,48 and 8.5,49 and therefore k20 ) 1.8 × 108, 3.5 × 108,
and 7.5 × 107 M-1 s-1, respectively. The latter value is in
good agreement with the value of 6.4 × 107 M-1 s-1, which
has been determined by Lewis et al.13
Piers et al.24 measured the formation of 4-nitrophenol from
phenol by •NO/O2 at pH 12. They found that the net nitrosation
process is described by eq 1, and concluded that the nitrosating
•
species is N2O3. However, NO2 reacts with C6H5O- to form
C6H5O. with k12 ) 8.6 × 106 M-1 s-1 50
.
The latter may react
with •NO to yield nitrophenol, and therefore •NO2 may initiate
the nitrosation of phenol. The rate of the hydrolysis of N2O3
is pH dependent (k7 ) 2000 + 108[OH-]),51 and therefore,
according to eq 21, the maximum nitrosation yield at pH 12 is
expected if k12[phenol] > k6[•NO], regardless of whether k7 )
530 or 2000 s-1 at neutral pH. Piers et al.24 found that
maximum nitrosation yields were obtained at [phenol] > 0.2
M in the presence of [•NO]o ) 0.6 mM and [O2]o ) 0.4 mM at
pH 12. This result is in accord with •NO2 being the main species
responsible for the nitrosation of phenol since 8.6 × 106 × 0.2
> 1.1 × 109[•NO] ([•NO] < 0.6 mM).
Conclusions
•
The rate of the autoxidation of NO is unaffected by the
Our results demonstrate that the rates of the oxidation of
NAPenSH, CapSH, and MorNH by •NO2 at pH 7.4 are too slow
to compete effectively with reaction 6. The pK values of
NAPenSH, and CapSH are 10 and 9.7, respectively, whereas
those of CysSH, PenSH, and GSH are 8.18, 7.93, and 8.75,
presence of various thiols and morpholine. The stoichiometry
of this process is identical for all thiols and morpholine and is
given by eq 1. The kinetic studies show that the rate laws
-d[O2]/dt ) k1[•NO]2[O2] with k1 ) (2.54 ( 0.26) × 106 M-2
s-1 and -d[•NO]/dt ) 4k1[•NO]2[O2] with 4k1 ) (1.17 ( 0.12)
× 107 M-2 s-1 are independent of the kind of substrate present.
(48) Hughes, H. A.; Smith, G. L., Williams, D. R. Inorg. Chim Acta
1985, 107, 247.
(49) Hetzer, H. B.; Bates, R. G.; Robinson, R. A. J. Phys. Chem. 1966,
70, 2869.
(50) Huie, R. E.; Neta, P. J. Phys. Chem. 1986, 90, 1193.
(51) Treinin, A.; Hayon, E. J. Am. Chem. Soc. 1970, 92, 5821.

Nitrosation of Thiols and Amines
J. Am. Chem. Soc., Vol. 118, No. 14, 1996 3425
•
The rate-determining step of the nitrosation by NO/O2 is the
formation of ONOONO (or ONONO2 or O2NNO2), which is
corrected values are 2.6 × 106 and 2.9 × 106 M-2 s-1 for CysSH
and GSH, respectively, in agreement with our results. Further-
more, they stated that, under limiting concentrations of O2, the
formation of RSNO was first-order, but the traces in their paper
under these conditions follow second-order kinetics.
•
the precursor of NO2 and N2O3. The stoichiometry of this
process suggests that NO2 and/or N2O3 are the only reactive
species. We have demonstrated that NO2 initiates the nitro-
•
•
sation process unless it is scavenged by •NO to form N2O3. The
latter species is also capable of directly nitrosating thiols and
amines with rate constants that exceed 6 × 107 M-1 s-1. Full
nitrosation yields are obtained either when •NO2 rapidly oxidizes
the substrate or when the nitrosation by N2O3 competes
efficiently with the hydrolysis of N2O3 to nitrite.
Wink et al.12 measured the absorbance of RSNO as a function
of [•NO] in aerated solutions, and determined ꢀ338(max) ) 1400
( 200 and 1300 ( 200 M-1 cm-1 (per molar •NO) for GSNO
and CysSNO, respectively. However, from the values given
in their figures, one calculates that ꢀ338 ) 960 and 970 M-1
•
cm-1 (per molar NO) for GSNO and CysSNO, respectively.
Thus, using [•NO]real, the corrected values of ꢀ338 are 575 and
It has been suggested that S-nitrosylation of proteins is a
favorable reaction under physiologic conditions that prolongs
the half-life of •NO and preserves its biological activity.
581 M-1 cm-1, respectively. We have demonstrated that 1 mol
•
of NO forms 0.5 mol of RSNO, and therefore according to
•
However, NO is incapable of nitrosating thiols and amines in
Wink’s results, correcting the stoichiometry and the concentra-
tion, the real values of ꢀ338 are 1150 and 1162 M-1 cm-1 for
GSNO and CysSNO, respectively. These values are higher than
those determined directly. We suspect that their method of
mixing the solutions is less accurate than the stopped-flow
the absence of oxygen at physiological pH, and the rate of the
•
formation of S-nitrosothiols from thiols and oxygenated NO
solution is relatively slow in biological systems. Under physi-
ological conditions where [•NO] < 1 µM and [O2] < 200 µM,
the half-life of the nitrosation process exceeds 7 min (t1/2
)
•
technique as they added NO solutions via a gas-tight syringe
1/(1.17 × 107[O2][•NO]) > 7 min). Thus, the formation of
to aerated stirred solutions containing the thiol at pH 7.4.
•
S-nitrosothiols from thiols and oxygenated NO solution is an
Their conclusion that NOx is the nitrosating species was based
on competitive kinetic studies. They measured the nitrosation
yield as a function of [RSH] at constant [•NO] and [O2] and as
a function of [azide] at constant [RSH], [•NO], and [O2].
However, as noted above, their yields differ from those
measured using the stopped-flow technique, and do not agree
with the known extinction coefficients of RSNO and with the
stoichiometry of the whole nitrosation process. Therefore, their
proposal of an unknown NOx has no justified basis.
unlikely biosynthetic pathway. We conclude that S-nitrosothiols
cannot serve as carrier molecules of •NO as •NO will react with
the various substrates present in the medium (O2•-, hemoglobin,
etc.) before it will nitrosate thiols.
Acknowledgment. This research was supported by The
Council for Tobacco Research and by The Israel Science
Foundation.
Wink et al.12 noted that, in the presence of 1 mM O2, 0.09
Appendix
•
mM NO, and 1 mM GSH or CysSH, k ) 5 × 103 M-1 s-1
Wink at al.12 found that, under limiting concentrations of O2,
the observed first-order rate constant of the formation of RSNO
was linearly dependent on [•NO]2, and determined k1 ) 7.3 ×
106 and 8.1 × 106 M-2 s-1 for CysSH and GSH, respectively.
However, under limiting concentrations of O2, the third-order
rate constant cannot exceed k1, and should be (2-2.9) × 106
assuming ꢀ338 ) 1400 M-1 cm-1, and hence the third-order rate
constant is 5 × 106 M-2 s-1. If we use the correct value of
ꢀ338 ) 760 M-1 cm-1, the third-order rate constant will be 2.7
× 106 M-2 s-1, which is about an order of magnitude lower
than our values (Table 2), and an order of magnitude lower
•
M-2 s-1 3,9,13,25,32
independent of the kind of substrate present.
,
than the rate of the autoxidation of NO. In the same study,
Wink et al.12 reported that the rate of the decay of •NO was not
affected by the addition of 1 mM thiol, which supports our
results and mechanism, and contradicts their experimental value.
Wink et al.12 did not calculate the concentration of •NO in their
•
stock solutions from the known solubility of NO in aqueous
solution, but determined it with the use of ABTS, assuming
that this reaction gives a 1:1 ratio between ABTS+ and NO.
•
Note Added in Proof. After submission of this paper, a
related paper appeared: Kharitonov, V. G.; Sundquist, A. R.;
Sharma, V. S. J. Biol. Chem. 1995, 270 (47), 28158-28164.
However, we have recently shown that this ratio is only 0.6:
1,25 and hence the concentration of •NO in their stock solutions
is 1.67 times higher ([•NO]real ) 1.67[•NO]wink). As a result,
their values for k1 should be divided by (1.67)2 ) 2.79, and the
JA9536680