Equilibrium and kinetics studies of transnitrosation between S-nitrosothiols and thiols

Article abstract of DOI:10.1016/S0960-894X(00)00688-0

Using UV-vis spectrometrical measurements, equilibrium constants for NO transfer between S-nitroso-N-acetyl-penicillamine (SNAP) and different thiols as well as kinetic data for NO transfer from S-nitroso bovine serum albumin (BSANO) to thiols have been obtained. NO transfer from SNAP to other primary/secondary thiols are thermodynamically favorable, whereas other S-nitrosothiols exhibit similar NO transfer potential. The obtained Gibbs free energy, enthalpy and entropy data indicated that NO transfer reactions from SNAP to four thiols are exothermic with entropy loss. The kinetic behavior of BSANO/RSH transfer can be related to both the acidity of sulfhydryl group and the electronic structure in thiol.

Full text of DOI:10.1016/S0960-894X(00)00688-0

Bioorganic & Medicinal Chemistry Letters 11 (2001) 433±436
Equilibrium and Kinetics Studies of Transnitrosation Between
S-Nitrosothiols and Thiols
Kun Wang,a,b Zhong Wen,a Wei Zhang,a Ming Xian,a,b Jin-Pei Chengb
and Peng George Wanga,*
aDepartment of Chemistry, Wayne State University, Detroit, MI 48202, USA
bDepartment of Chemistry, Nankai University, Tianjin 300071, China
Received 26 September 2000; accepted 30 November 2000
AbstractÐUsing UV±vis spectrometrical measurements, equilibrium constants for NO transfer between S-nitroso-N-acetyl-peni-
cillamine (SNAP) and di€erent thiols as well as kinetic data for NO transfer from S-nitroso bovine serum albumin (BSANO) to
thiols have been obtained. NO transfer from SNAP to other primary/secondary thiols are thermodynamically favorable, whereas
other S-nitrosothiols exhibit similar NO transfer potential. The obtained Gibbs free energy, enthalpy and entropy data indicated
that NO transfer reactions from SNAP to four thiols are exothermic with entropy loss. The kinetic behavior of BSANO/RSH
transfer can be related to both the acidity of sulfhydryl group and the electronic structure in thiol. # 2001 Elsevier Science Ltd. All
rights reserved.
In recent years, tremendous attention has been focused
on the chemistry and biology of nitric oxide (NO) due
to the discovery that NO plays a key role in a wide
variety of human physiological processes.1 It has been
suggested that S-nitrosothiols (RSNOs) may be directly
involved in many of the biological functions of nitric
oxide.2 4 RSNOs have been detected in human airway
lining ¯uid plasma, platelets and neutrophils,5 therefore
they can act as an NO carrier in the form of free thiol or
cysteine containing protein in biological systems.6 These
endogenous S-nitrosothiols may play an important role
in NO storage, transport, and delivery. Some RSNO
compounds, such as S-nitroso-N-acetyl-penicillamine
(SNAP)7 and S-nitrosocaptopril,8 can indeed be used as
therapeutic drugs for treatment of angina and other
circulation problems, their functions also rely on their
ability to generate NO in vivo.9
reported previously by Williams,10 Meyer,11 Rossi,12
and Means,13 using UV±vis spectroscopic methods.
Recently using HPLC method, Hogg14 reported equili-
brium and kinetics study on transnitrosation. As an
e€ort to o€er a thermodynamic view of NO transfer
both in vitro and in vivo, we reported herein our inves-
tigation on the equilibrium of the transnitrosation
between SNAP and thiols as well as kinetic study on the
transnitrosation between S-nitroso bovine serum
albumin (BSANO) and thiols.
Equilibrium Study of NO Exchange Between SNAP
and Thiols
In the present study, SNAP was selected as the NO
donor since it has a maximum absorption at 590 nm
while lmax for primary S-nitrosothiols is around 540
nm, transnitrosation between SNAP and primary thiols
can be readily analyzed using UV±vis spectrometric
measurement (Fig. 1). UV±vis spectra of reaction mix-
tures were obtained by adding an equimolar amount of
thiol to a cuvette containing RSNO. After the equili-
brium was reached (Fig. 2), the concentrations of the
reactants and products can be calculated from the
change of the absorption of SNAP at 590 nm according
to the Beer±Bouger±Lambert law. Equilibrium constant
(Keq) of NO transfer between SNAP and di€erent thiols
can be determined from the calculated concentrations as
Fully understanding the biological roles of NO requires
fundamental knowledge of the kinetics and thermo-
dynamics of the NO-related reactions. NO transfer from
one NO donor, especially S-nitrosothiol, to another
thiol has been a focus of interest due to their role in NO
transfer in vivo. Mechanistic and kinetics studies on
transnitrosation from RSNOs to thiols have been
*Corresponding author. Tel.: +1-313-577-6759; fax: +1-313-577-
2554; e-mail: pwang@chem.wayne.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00688-0

434
K. Wang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 433±436
shown in eq (1). Equilibrium constants for the reverse
reactions (Keq1) re¯ect the NO donating potential of the
corresponding RSNO. The results were summarized in
Table 1.
Transnitrosation between SNAP and four thiols was
also carried out at di€erent temperatures. Gibb free
energy (ÁGꢀ), enthalpy (ÁHꢀ), and entropy (ÁSꢀ) were
thus calculated. The results are summarized in Table 2.
Table 2 showed that ÁHꢀs for these reactions are nega-
tive, again indicating that the NO transfer from SNAP
to these thiols is thermodynamically favorable. While
ÁHs of reactions 1, 2 and 3 are similar, ranging from
14.6 to 21.6 kJ/mol, the ÁHꢀ for reaction 4 is fairly
k2
*
SNAP ‡ RSH ) NAP ‡ RSNO
k
2
‰NAPŠ‰RSNOŠ
keq ˆ k2=k
ˆ
ꢀ1†
2
‰SNAPŠ‰RSHŠ
high, accompanied by signi®cant entropy loss ( 208
1
J mol
K
1) for NO transfer from SNAP to cysteine-
amine. This may be related to the di€erence between
S-nitrosocysteineamine and other RSNO compounds in
terms of structure and thermal stability. The S-nitroso-
cysteineamine exists largely as the protonated form
(H3N+CH2CH2SNO) at pH 7.4, and there exists sig-
ni®cant intramolecular electrostatic interaction between
the ammonium group and nitroso oxygen atom, which
leads to an energetically preferred cyclic form and sig-
ni®cant entropy loss.15 On the other hand, intramole-
cular H-bonding between sulfhydryl and COO in
other thiols may lower both enthalpy and entropy, and
this may eventually lead to smaller ÁHꢀ and ÁSꢀ after
S-nitrosation.
As can be seen from Table 1, overall, there exists a ten-
dency of NO transfer from SNAP to all other thiols
which is either primary or secondary (Keq>1). This can
largely be attributed to the steric repulsion between two
methyl groups and SNO moiety in tertiary SNAP, as
suggested by our ab initio density functional theory
calculation.15
Among the eight reactions, Keq1 for the reaction between
SNAP and N-acetyl cysteine is the largest, indicating
that S-nitroso-N-acetyl cysteine has the highest NO
donating ability. The reaction with 2-mercaptoethanol
1
has the smallest Keq of 0.12, suggesting that S-nitroso-
2-mercaptoethanol is less capable of transferring NO to
another thiol. All other S-nitrosothiol compounds have
similar NO transferability with Keq1s ranging from 0.2 to
0.3. It is worth noting that N-acetylation of cysteine
resulted in an increase of NO donating ability (Keq
from 0.28 to 0.68).
Table 1. Equilibrium constants of SNAP±thiol exchange reactions at
pH 7.4
1
1
Thiol
Keq
Keq
2-Mercaptoethanol
3-Mercaptoproprionic acid
2-Propethiol
l-Cysteine ethyl ester
Cysteineamine
l-Cysteine
Glutathione
N-Acetyl-cysteine
8.44
5.04
5.04
4.99
3.89
3.63
3.34a
1.47
0.12
0.20
0.20
0.20
0.26
0.28
0.30
0.68
aK=3.75±5.32 reported by Hogg.14
Table 2. Thermodynamic parameters for reaction between SNAP
and thiols
ÁGꢀ
(kJ mol
ÁHꢀ
(kJ mol
ÁSꢀ
a
b
Thiol
Temp Keq
(C)
1
1
1
)
)
(J mol 1K
)
Glutathione
25
30
33
37
3.69
3.27
3.00
2.63
3.23
2.99
2.80
2.49
21.6
61
59
Figure 1. UV absorption of SNAP and CysNO.
l-Cysteine
20
25
30
33
4.18
3.66
3.35
2.58
3.38
3.21
3.05
2.44
20.9
14.6
66.4
l-Cysteine
ethyl ester
25
30
33
37
4.56
4.15
3.92
3.60
3.76
3.59
3.48
3.32
36
Cysteineamine
25
30
33
37
3.89
3.22
2.56
1.33
3.37
2.94
2.39
0.73
208
aObtained from ÁGꢀ= RT lnKeq (3).
blnKeq
ˆ
ÁHꢀ=ꢁRT† ‡ ÁSꢀ=R (4). The ÁHꢀ and ÁSꢀ values can be
Figure 2. Kinetics of NO transfer between SNAP and l-cysteine.
obtained from a plot of lnKeq versus 1=T.

K. Wang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 433±436
435
Kinetics Study of NO Transfer Between BSANO and
Thiols
In summary, an equilibrium study of NO transfer
between SNAP and di€erent thiols shows that this
transfer is thermodynamically favorable, and the di€er-
ence between thiols was generally insigni®cant. This is
further supported by the observed enthalpy change for
NO transfer reaction. Entropy loss was found for NO
transfer from SNAP to four biologically relevant thiols.
These results can be rationalized in terms of stereo-
electronic factors. Kinetics of the transnitrosation
between BSANO and low-molecular-weight thiols can
be directly related to the pKa of the parent thiols. It is
suggested that electrostatic interaction may also con-
tribute to the observed relative NO transfer kinetics
behavior.
It should be noted that other reactions, mainly S-thio-
lation of RSNO compounds in the presence of thiols
which gives disul®des, would compete with the investi-
gated transnitrosation process.16 Nevertheless it has
been reported that S-thiolation rates are generally sev-
eral orders of magnitude smaller than S-nitrosation and
therefore the equilibrium and kinetics study reported
here would not be interfered.17 To probe the kinetics of
NO transfer between S-nitrosylated proteins and thiols,
we also examined transnitrosation between BSANO and
several thiol compounds (Table 3).
It has been established that the transnitrosation occurs
as nitroso-nitrogen atom is attacked by the thiolate
nucleophile.10 Therefore, the reaction rate is dependent
on the reactivity of the thiol, which is directly correlated
with pKa of the sulfhydryl group and stereoelectronic
factors in thiol. From Table 3 one can ®nd that trans-
nitrosation rate constant increases with increased acid-
ity of -SH, and a linear relationship exists between pKa
and lgk2 as shown in Figure 3. The slope of the corre-
lation is less than unity, indicating that other factors
may also a€ect the reactivity of thiolate. Noted is that
the thiols exist as the ionized form at pH 7.4: cysteine
ethyl ester (protonated); cysteine and penicillamine
(zwitterionic); entries 4±6 (deprotonated). This exhibits
roughly an increase in negative charge from entry 1
through 6. Thus it appears that negative charge neigh-
boring thiolate favors nucleophilic attack, and therefore
this partly compensates for the loss in acidity of sulfhy-
dryl. Since the pKa's of protein thiols are greatly
a€ected by their environment, it is conceivable that NO
transfer in vivo may be particularly ecient from
S-nitrosothiols such as GSNO or BSANO to certain
proteins.
Experimental
General
All the reagents were purchased from commercial sup-
pliers and were used as received. Thin-layer chromato-
graphy was conducted on Baker Si250f silica gel TLC
plates with a ¯uorescent indicator. Bovine serum albu-
min was purchased from Sigma Co. Dialysis was per-
formed against deionized water using dialysis tubing
(8000 MW cuto€). 1H and 13C NMR spectra were
recorded on a Varian Unity 500 MHz NMR instru-
ment. UV±visible measurements were carried out on a
HP 8453 UV±visible spectrometer (Hewlett Packard
Co.).
S-Nitrosopenicillamine (SNAP) preparation.19 To a
solution of N-acetyl-dl-penicillamine (10 mmol) in
MeOH (20 mL), 1 N HCl (20 mL), and concentrated
H2SO4 (2 mL) was added NaNO2 (20 mmol) in water
(20 mL) in a period of 20 min with vigorous stirring at
0 ꢀC. Stirring was continued for another 15 min, the
liquid was separated and the solid was washed with cold
water and dried in vacuum. The product SNAP was
obtained as deep green crystals.
Table 3. Kinetic data for reaction between BSANO and thiols
a
1
1
Entry
RSH
pKa
k2 (M
s )
1
BSANO Preparation.14 BSA was dissolved in PBS buf-
fer to 150 mg/mL followed by addition of 10 mM
dithiothreithiol and 10 mM EDTA. After 1 h at 20 ꢀC,
dithiothreitol and EDTA were removed by dialysis
against PBS bu€er. The sulfhydryl-to-protein ratio of
BSA is 0.6. S-Nitrosylation of the BSA was then
achieved by incubation with a 5-fold molar excess of
isobutyl nitrite at 37 ꢀC until no further increase in A335
was observed. BSANO concentration was determined
by the UV spectrometer (e335=3869 M 1.cm 1).20
1
2
3
4
5
6
l-Cysteine ethyl ester
l-Cysteine
d-Penicillamine
Glutathione
N-Acetyl cysteine
N-Acetyl-d-penicillamine
6.50
8.30
8.53
8.75
9.52
9.90
6.45Â10
6.12Â10
3.01Â10
1.68Â10
8.41Â10
9.90Â10
2
2
2
3
3
aRef 18.
Equilibrium study of transnitrosation between SNAP and
thiols. The equilibrium constants (Keq) for transnitrosa-
tion were determined by reacting equimolar amounts of
thiols and SNAP. Brie¯y, S-nitrosothiol (29 mM) in
phosphate bu€er (pH 7.4, 1 mM EDTA) was placed in a
quartz cell at di€erent temperatures. A330 370 or
A500 600 nm was monitored for 5 min to obtain the slow
decomposition rate. An equimolar amount of thiol was
added and the exchange reaction was monitored until
equilibrium was reached. Keq was calculated based on
Figure 3. Correlation of lgk2 and pKa(RSH).

436
K. Wang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 433±436
the change of the UV absorption. Experiments were
repeated at least once.
5. Rockett, K. A.; Auburn, M. M.; Cowden, W. B.; Clark,
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Kinetics study of transnitrosation between BSANO and
thiols. The NO transfer reactions were carried out at
25 ꢀC. BSANO (0.20 mM) in PBS bu€er (pH 7.4, 1 mM
EDTA) was placed in a quartz cell. Thiol (3.0 mM) was
added and the NO exchange reaction was monitored at
A335 for 10 min. The second order rates k2 can be derived
from the pseudo-®rst order rates ko€ as ko€/[RSH]0.
10. (a) Barnett, D. J.; McAninly, J.; Williams, D. L. H. J.
Chem. Soc., Perkin Trans. 2 1994, 1131. (b) Barnett, D. J.;
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This work is generously supported by research grants
from the NIH (GM 54074), National Science Founda-
tion of China (NSFC No. 29832040) and Ministry of
Education of China.
15. Wen, Z.; Schlegel, H. B.; Wang, P. G. Manuscript in pre-
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