Selective trapping of SNO-BSA and GSNO by benzenesulfinic acid sodium salt: Mechanistic study of thiosulfonate formation and feasibility as a protein S-nitrosothiol detection strategy

Article abstract of DOI:10.1016/j.tetlet.2013.08.011

The conversion of S-nitrosothiols to thiosulfonates by reaction with the sodium salt of benzenesulfinic acid (PhSO₂Na) has been examined in detail with the exemplary substrates S-nitrosoglutathione (GSNO) and S-nitrosylated bovine serum albumin (SNO-BSA). The reaction stoichiometry (2:1, PhSO₂Na:RSNO) and the rate law (first order in both PhSO ₂Na and RSNO) have been determined under mild acidic conditions (pH 4.0). The products have been identified as the corresponding thiosulfonates (GSSO₂Ph and BSA-SSO₂Ph) along with PhSO₂NHOH obtained in a 1:1 ratio. GSH, GSSG, and BSA were unreactive to PhSO ₂Na.

Full text of DOI:10.1016/j.tetlet.2013.08.011

Tetrahedron Letters 54 (2013) 5707–5710
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Selective trapping of SNO-BSA and GSNO by benzenesulfinic acid
sodium salt: mechanistic study of thiosulfonate formation and
feasibility as a protein S-nitrosothiol detection strategy
Benjamin D. Reeves, Jonathan K. Hilmer, Lisa Mellmann, Myra Hartzheim, Kevin Poffenberger,
⇑
Keith Johnson, Neelambari Joshi, David J. Singel, Paul A. Grieco
Department of Chemistry and Biochemistry, Montana State University, PO Box 173400, Bozeman, MT 59717-3400, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The conversion of S-nitrosothiols to thiosulfonates by reaction with the sodium salt of benzenesulfinic
acid (PhSO2Na) has been examined in detail with the exemplary substrates S-nitrosoglutathione (GSNO)
and S-nitrosylated bovine serum albumin (SNO-BSA). The reaction stoichiometry (2:1, PhSO2Na:RSNO)
and the rate law (first order in both PhSO2Na and RSNO) have been determined under mild acidic condi-
tions (pH 4.0). The products have been identified as the corresponding thiosulfonates (GSSO2Ph and BSA-
SSO2Ph) along with PhSO2NHOH obtained in a 1:1 ratio. GSH, GSSG, and BSA were unreactive to PhSO2Na.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 22 July 2013
Revised 31 July 2013
Accepted 3 August 2013
Available online 11 August 2013
Keywords:
S-Nitrosothiols
Benzenesulfinic acid
S-Phenylthiosulfonates
Protein labeling
Protein S-nitrosothiols have emerged as an important post-
translational modification involved in cellular signal transduction.
Both hypo- and hyper-S-nitrosylation have been implicated in dis-
ease states.1 The importance of this functional group in biology
motivates the development of analytical methods for identifying
and quantifying protein S-nitrosothiols. The most prominent tech-
nique used today is the biotin-switch technique,2 which has facil-
itated the identification of many protein S-nitrosothiols. The
complexity of experimental design, in particular the necessity of
multi-step chemical processing, has led to interest in the develop-
ment of probes that can simplify the procedure by reacting directly
and selectively with protein S-nitrosothiols.3
Recently, several triphenylphosphine based probes have been
studied that undergo Staudinger-like ligation of S-nitrosothiols to
form sulfenamides,4 which can be further manipulated to form
disulfides,5 thioethers,6 or dehydro-alanines.7 The requirement of
organic solvents limits the potential of triphenylphosphine based
probes. Water soluble triphenylphosphine probes derivatized with
sulfonic acids have been developed that convert S-nitrosothiols
into S-alkylphosphonium salts detectable by mass spectrometry.8
Over a quarter of a century ago a reaction (Scheme 1) was re-
ported in the literature9 which transformed S-nitrosoglutathione
(GSNO) into S-phenylsulphonylglutathione (GSSO2Ph) under
highly acidic conditions (pH 0.5) in the presence of benzenesulfinic
acid. This reaction has promising features as a viable detection
strategy for S-nitrosylated proteins. However, the highly acidic
pH conditions are not compatible with biological samples. The fun-
damental reaction properties that ultimately determine the utility
of this approach, namely the reaction mechanism and rate, pH
dependence, and utility toward protein S-nitrosothiol detection,
remain unknown. These issues are fully addressed for the first time
by studying the reaction of benzenesulfinic acid sodium salt with
GSNO and SNO-BSA, and characterizing the products.
As a point of departure, the effect of pH on reaction rate (at
ambient temperature) was assessed by measuring the disappear-
ance of GSNO by UV–vis spectroscopy (545 nm, 15 MÀ1 cmÀ1
,
Varian Cary 6000i) upon the addition of benzenesulfinic acid
sodium salt (Fig. 1). As a control, GSNO degradation was monitored
over the same time frame and found to be negligible. GSNO
⇑
Corresponding author. Tel.: +1 (406) 994 7127; fax: +1 (406) 994 3744.
E-mail address: grieco@chemistry.montana.edu (P.A. Grieco).
Scheme 1.
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.08.011

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B. D. Reeves et al. / Tetrahedron Letters 54 (2013) 5707–5710
Figure 1. Rate of disappearance of GSNO (545 nm, 15 MÀ1 cmÀ1) taken in the initial
2 min after mixing vs pH for reactions of PhSO2Na (20 mM) and GSNO (10 mM) in
buffer (pH 1.0–2.0 KCl/HCl, 200 mM and pH 2.5–5.0, potassium hydrogen phthalate,
100 mM). Reactions were performed in triplicate and the length of the error bar
represents the standard deviation.
(10 mM) was reacted with 2.0 equiv of PhSO2Na in aqueous solu-
tions with pH ranging from 1.0 to 5.0. The initial reaction rate
was observed to increase by 50% as the pH was varied from 1 to
2.5, where the rate reaches a maximum close to the reported pKa
of PhSO2H.10 The rate declines as the pH increases. These results
reveal that the reaction is feasible under modestly acidic condi-
tions and does not require the harsh acidic reaction conditions pre-
viously employed,9 which are incompatible with protein samples.
Since a mild pH is desirable when working with protein sam-
ples, further investigations were conducted at pH 4.0 rather than
at the optimum pH. Ammonium formate (100 mM) was chosen
as the buffer because it is compatible with mass spectrometry
analysis due to its volatility and low molecular weight ions.
Reversed phase HPLC in tandem with mass spectrometry was used
to monitor reaction progress and to identify all products (see Sup-
plementary Fig. SI-1). As a control, GSNO degradation was moni-
tored over the same time frame and found to be negligible. Two
products were identified by high resolution mass spectrometry
and ion fragmentation as GSSO2Ph and PhSO2NHOH (see Supple-
mentary Fig. SI-2) in a 1:1 ratio. It is important to note that the ni-
troxyl group on GSNO becomes incorporated into PhSO2NHOH,
otherwise known as Piloty’s acid, which is a poor HNO donor at
neutral pH.11 Furthermore, the reaction profiles show that
PhSO2NHOH is stable at pH 4.0 for ca. 2 h. These results stand in
stark contrast to O-nitrosoascorbate, the byproduct of the switch
assay, which is considerably less stable than S-nitrosothiols and
releases NO in minutes at physiological pH.12
Reaction profiles were obtained by comparing absorbance peak
areas to calibration curves made with authentic samples and sim-
ulated using Copasi integration software (Fig. 2).13 By varying ini-
tial concentrations of GSNO and PhSO2Na, the rate law and reaction
stoichiometry were determined. Evaluation of the kinetic parame-
ters within the framework of this model (cf. Table 1) reveals the
reaction to be first order in both GSNO and PhSO2Na; the first addi-
tion of PhSO2Na is the rate determining step with a rate constant of
0.041 MÀ1sÀ1
.
When GSNO was treated with a large excess of PhSO2Na (20:1)
at pH 4.0, the reaction is 85% complete in 1 h without the detection
of byproducts (Fig. 2D). The concentration of PhSO2NHOH and
GSSO2Ph remained constant over the next 7 h. This experiment
indicates that a large excess of PhSO2Na can be used to increase
the rate of the reaction without creating undesirable byproducts.
Figure 2. Reaction profiles obtained by comparing absorbance peak areas to
calibration curves for three different initial conditions (A) 5 mM PhSO2Na, 5 mM
GSNO, (B) 5 mM PhSO2Na, 10 mM GSNO, (C) 10 mM PhSO2Na, 5 mM GSNO, (D)
100 mM PhSO2Na, 5 mM GSNO. All reactions were run in 100 mM ammonium
formate, pH 4.0.

B. D. Reeves et al. / Tetrahedron Letters 54 (2013) 5707–5710
5709
Table 1
With an understanding of the mechanism and selectivity of the
reaction, the reactivity of PhSO2Na with a protein S-nitrosothiol
was probed. As a proof of concept, a test protein, bovine serum
albumin (BSA), was used because it contains a single reactive thiol
(Cys34) that undergoes S-nitrosylation.14 BSA, moreover, contains
17 protein disulfides and is typically obtained with mixed disul-
fides at Cys34.
In a preliminary study, BSA (10 mg/mL, ammonium formate
buffer, pH 4.0) with and without PhSO2Na (0.1 M) was examined
and the deconvoluted mass spectra were virtually the same
(Fig. 3A). The free thiol content of BSA (Sigma) was found to be
49% by the DTDP method.15 It should be noted that in addition to
BSA (66,430 amu), covalent adducts were detected including the
mixed disulfide BSA-Cys (66,549 amu), and glycosylation adducts
BSA(Glc) (66,592 amu) and BSA-Cys(Glc) (66,711 amu) which have
been observed by others.16
Rate constants calculated by Copasi modeling
k1
k2
(MÀ1 sÀ1
)
R1 GSNO+H+ = GSNOH+
R2 GSNOH++PhSO2Na ? B
R3 B+H+ = BH+
2.72E + 03 1.3À61sÀ1
0.041a
0 s
2.79E + 06 0 sÀ1
R4 BH++PhSO2Na = PhSO2NHOH+GSSO2Ph 2.86E + 02 1.26E + 02 MÀ1 sÀ1
Rate determining step.
a
In order to probe the viability of the reaction, BSA was nitrosy-
lated employing 5 equiv S-nitrosocysteine in HEN buffer (100 mM
Scheme 2.
The mechanism detailed in Scheme 2 is consistent with the
Copasi simulations. The oxygen of the S-nitrosothiol is protonated
in the first step, activating the addition by the benzenesulfinate
anion. The protonation of the resulting intermediate activates the
attack of the second benzenesulfinate thus furnishing the products
in ratio of 1:1.
To further probe the suitability of this reaction as
a
S-nitrosothiol labeling strategy, control reactions of PhSO2Na with
GSH and GSSG were assessed (Scheme 3). Reactions between GSH
(5 mM) and PhSO2Na (10 mM) and GSSG (5 mM) and PhSO2Na
(10 mM) were monitored using the same HPLC method in tandem
with MS. GSSO2Ph concentration was quantified by UV–vis and
remained undetectable over 3 h (see Supplementary Fig. SI-3).
The concentration of PhSO2Na was also quantified and remained
unchanged over the course of the reaction. The mass spectra were
also taken at each time point and were absent of byproducts. Since
the absorbance of GSH is low, the MS signal of GSH while mixed
with PhSO2Na was observed over time. The ratio of signal between
the reaction and a standard remained unchanged over 3 h without
the detection of byproducts (see Supplementary Fig. SI-3).
Figure 3. Deconvoluted mass spectra of (A) BSA (10 mg/mL) and BSA with PhSO2Na
(0.1 M), (B) SNO-BSA (100 lM total protein, 44% SNO) (C) SNO-BSA and PhSO2Na
(0.1 M) after 12 min and after 2.5 h. All reactions were run in pH 4.0 ammonium
Scheme 3.
formate buffer (100 mM).

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B. D. Reeves et al. / Tetrahedron Letters 54 (2013) 5707–5710
HEPES, 1.0 mM EDTA, and 0.1 mM neocuprine, pH 8.0) for 1 h in
the dark. The nitrosylated protein was purified by elution through
a Sephadex G25 column which also served to exchange the HEN
buffer with 100 mM ammonium formate (pH 4.0). The concentra-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.08.
011. These data include MOL files and InChiKeys of the most
important compounds described in this article.
tion of total protein was 100 lM (e ) and a
280 = 44,300 MÀ1 cmÀ1 17
Greiss/Saville assay18 determined the SNO content to be 44%, leav-
ing 5% free thiol content as determined by the DTDP method.15 The
mass spectrum of SNO-BSA is seen in Figure 3B. The S-nitrosothiol
adduct is seen as the major component (66,459 amu). BSA-Cys was
unreactive while BSA(Glc) underwent nitrosylation as seen by the
peak at 66,621 amu. The BSA starting material (66,430 amu) is
likely due to the fragmentation of the labile nitroxide and is not
from an incomplete reaction. Also, S-nitrosothiol fragmentation
of the NO group is well documented even when using soft ioniza-
tion methods such as ESI.19
References and notes
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In Figure 3C, the thiosulfonate adduct (66,570 amu) was domi-
nant 12 min after the addition of PhSO2Na (0.1 M final concentra-
tion). The deconvoluted spectrum remained unaltered after 2.5 h,
suggesting the reaction was near completion in the first 12 min.
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preclude recovery of the parent peak by fragmentation loss of
NO, thus the thiol peak is sharply diminished as compared to
Figure 3B.
In summary, the mechanism of thiosulfonate formation from
the reaction of S-nitrosothiols and PhSO2Na has been elucidated.
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side reactions. The reaction is selective for S-nitrosothiols and is
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disulfides, as seen for the reactions of SNO-BSA and BSA. The
byproduct, PhSO2NHOH, is stable over the duration of the labeling
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Acknowledgment
We thank the National Institute of Health (CoBRE
5P20RR024237-04) for funding this work.
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