Water-soluble triarylphosphines as biomarkers for protein S -nitrosation

Article abstract of DOI:10.1021/cb900302u

S-Nitrosothiols (RSNOs) represent an important class of post-translational modifications that preserve and amplify the actions of nitric oxide and regulate enzyme activity. Several regulatory proteins are now verified targets of cellular S-nitrosation, an

Full text of DOI:10.1021/cb900302u

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ARTICLE
Water-Soluble Triarylphosphines as
Biomarkers for Protein S-Nitrosation
Erika Bechtold†, Julie A. Reisz†, Chananat Klomsiri‡, Allen W. Tsang§, Marcus W. Wright†,
Leslie B. Poole‡, Cristina M. Furdui§, and S. Bruce King†,*
†Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109, ‡Center for Structural Biology,
Department of Biochemistry, §Department of Molecular Medicine, Wake Forest University School of Medicine, Winston-
Salem, North Carolina 27157
eactive oxygen and nitrogen species (ROS/RNS)
affect cellular signaling processes at concentra-
tions far below those required to inflict oxidative
ABSTRACT S-Nitrosothiols (RSNOs) represent an important class of post-
translational modifications that preserve and amplify the actions of nitric oxide
and regulate enzyme activity. Several regulatory proteins are now verified targets
of cellular S-nitrosation, and the direct detection of S-nitrosated residues in pro-
teins has become essential to better understand RSNO-mediated signaling. Cur-
rent RSNO detection depends on indirect assays that limit their overall specificity
and reliability. Herein, we report the reaction of S-nitrosated cysteine, glutathione,
and a mutated C165S alkyl hydroperoxide reductase with the water-soluble phos-
phine tris(4,6-dimethyl-3-sulfonatophenyl)phosphine trisodium salt hydrate (TX-
PTS). A combination of NMR and MS techniques reveals that these reactions pro-
duce covalent S-alkylphosphonium ion adducts (with SϪPϩ connectivity), TXPTS
oxide, and a TXPTS-derived aza-ylide. Mechanistically, this reaction may proceed
through an S-substituted aza-ylide or the direct displacement of nitroxyl from the
RSNO group. This work provides a new means for detecting and quantifying
S-nitrosated species in solution and suggests that phosphines may be useful
tools for understanding the complex physiological roles of S-nitrosation and its im-
plications in cell signaling and homeostasis.
R
damage (1−4). The overoxidation and metabolism of ni-
tric oxide (NO), an endogenous cell signaling agent, pro-
duces several RNSs including nitrogen dioxide and ni-
trite, which can generate competent nitrosation agents
(5, 6). Nitrosation of cysteine sites yields S-nitrosothiols
(RSNOs) that preserve, modulate, and amplify the ac-
tions of NO. This selective and reversible process repre-
sents an important post-translational modification in
cell signaling and regulation (3, 6−8). Several proteins
have been identified as targets of cellular S-nitrosation
including glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), papain, hemoglobin (Hb), and several
caspases (5, 9−12). Inflammatory stimuli, including
RNS, increase overall cellular RSNO formation, which fur-
ther mediates the inflammatory process (9, 13−17). Di-
rect detection and specific identification of S-nitrosated
residues in proteins is essential to better understand
RSNO-mediated signaling. Current techniques, such as
the biotin switch assay, Saville assay, and
chemiluminescence-based methods, are indirect meth-
ods of detection (18−23). Also, variability in these as-
says has challenged their accuracy and driven the
search for alternative detection systems (24, 25). Deci-
phering the role and importance of RSNOs in biology re-
quires direct and specific methods of quantification.
We report a new strategy to covalently label biological
RSNOs using the water-soluble phosphine tris(4,6-
dimethyl-3-sulfonatophenyl) phosphine trisodium salt
hydrate (TXPTS, Scheme 1) (26).
*Corresponding author,
kingsb@wfu.edu.
Received for review November 30, 2009
and accepted February 10, 2010.
Published online February 10, 2010
10.1021/cb900302u
Earlier work showed that the reaction of trityl-S-
nitrosothiol with triphenylphosphine yields an
S-substituted aza-ylide (1, Scheme 1) and provided in-
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S-substituted aza-ylides through their reaction with the
water-soluble triarylphosphine (TXPTS). Recent work by
Xian shows that a variety of S-nitrosothiols react with tri-
aryl and trialkylphosphines to give S-substiuted aza-
ylides, which undergo further reactions to yield a vari-
ety of different products depending on both RSNO and
phosphine structure (29, 30). In the presence of an elec-
trophilic trap, these S-substituted aza-ylide intermedi-
ates ligate to generate sulfenamides or rearrange to di-
sulfide iminophosphoranes (29, 30). In the absence of
an electrophilic trap, the intermediate S-substituted aza-
ylide eliminates to form dehydroalanine-containing
products (28). This work reveals rich reaction chemistry
between S-nitrosothiols and phosphines that may form
the basis of new labeling methods, but in general, these
processes have been limited to protected versions of
cysteine and glutathione in organic or organic/buffer
mixtures. Using a combination of NMR- and MS-based
techniques, we sought to critically evaluate the reaction
between TXPTS and several physiologically relevant
S-nitrosothiols. We report that the reactions between
S-nitrosothiols of cysteine, glutathione, and a mutant
of the bacterial peroxiredoxin, AhpC, with the water-
soluble triarylphosphine (TXPTS) yield a unique set of
products that include TXPTS oxide (3), the stable aza-
ylide of TXPTS (4), and an S-alkylphosphonium adduct
(RS-Pϩ connection, 5 and 6, Schemes 2 and 3).
SCHEME 1. Reactions of triarylphosphines
with S-nitrosothiols in organic and aqueous
systems
spiration for developing an SNO-specific label (27). Re-
cently, Xian revealed that the treatment of various or-
ganic SNOs with derivatized triarylphosphines provides
products arising from similar S-substituted aza-ylides
(28−30). In the presence of properly situated electro-
philes on the triarylphosphine, the S-substituted aza-
ylide intermediates undergo ligation to form stable
sulfenamides or disulfide iminophosphoranes (28−30).
Treatment of S-nitrosocysteine ester derivatives with
various phosphines yields the corresponding dehy-
droalanines through intramolecular elimination via a
similar S-substituted aza-ylide (28). These results en-
couraged our use of the water-soluble phosphine TX-
PTS as a potential RSNO trap. We report the reactions
of TXPTS with S-nitrosocysteine (Cys-SNO),
Reaction of Triphenylphosphine with Trityl
S-Nitrosothiol. Treatment of trityl S-nitrosothiol with 2
equiv of triphenylphosphine in benzene yields the
S-substituted aza-ylide (1, 92% yield, Scheme 1), which
corroborates previous results and provides the first
X-ray crystallographic and 31P NMR characterization of
this compound (see Supporting Information).
S-nitrosoglutathione (GSNO), and a mutated peroxire-
doxin, S-nitrosated C165S alkyl hydroperoxide reduc-
tase (C165S AhpC-SNO) to yield the covalent
Reaction of TXPTS and S-Nitrosoglutathione
(GSNO). The reaction of TXPTS and S-nitrosoglutathione
(GSNO, a commercially available or readily prepared
stable pink solid) offers an opportunity to evaluate the
formation of S-substituted aza-ylides in water or buffer.
UVϪvis spectroscopy shows the rapid loss of the
S-nitrosothiol functional group in GSNO upon addition
of TXPTS as evidenced by the decrease in absorbance at
545 nm (Supporting Information). 31P NMR spectros-
copy provides detailed information regarding this reac-
tion. A mixture of products results when freshly prepared
GSNO (1 equiv) and TXPTS (2 equiv) are combined in
buffer (50 mM HEPES, 1 mM DTPA, pH 7.1) in the dark.
S-alkylphosphonium salt (2, Scheme 1).
RESULTS AND DISCUSSION
Haake’s observation that the reaction of trityl
S-nitrosothiol and triphenylphosphine gives an
S-substituted aza-ylide (1, Scheme 1) reveals that or-
ganic S-nitrosothiols can act in a similar manner as the
organic azide in Staudinger-type reductions/ligations
(27, 31). On the basis of this work, we hypothesized that
protein and other biologically relevant S-nitrosothiols
could potentially be labeled as structurally unique
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ARTICLE
SCHEME 2. Reaction of TXPTS with S-nitrosoglutathione at pH 7
The peak for phosphine (␦ ϭ Ϫ28.8 ppm) decreases
with time, and three new peaks at ␦ ϭ 34.5, 39.7, and
47.2 ppm emerge over 30 min (Figure 1). No other
phosphorus-derived species are observed on this time
scale, and control experiments show that TXPTS does
not react with GSH and only to a small extent (ϳ3%)
with GSSG to form the same product with the resonance
at 47.2 ppm as judged by 31P NMR (Supporting Informa-
tion). Comparison to known standards allows identifica-
tion of the peak at 39.7 ppm as TXPTS oxide (3, Scheme
2) and the peak at 34.5 ppm as the TXPTS-derived ylide
(4, Scheme 2). Monitoring this reaction mixture over
time shows the immediate formation of the species at
47.2 ppm, whereas 3 and 4 emerge more gradually over
the course of 2 h (Figure 2). Integration of the 31P NMR
spectrum after 1 h indicates the formation of these
three products in roughly a 1:1:1 ratio, which gives an
estimated yield of 67% for 5 (based on starting GSNO).
The use of 15N-labeled GSNO results in an apparent cou-
pling (1JNϪP ϭ 23 Hz) in the peak at 34.5 ppm, which cor-
responds to 4, but not in any of the other peaks. This ob-
served splitting varies over time, suggesting some
dynamic changes of 4 under these conditions (possibly
water addition to the ylide). Treatment of this reaction
mixture with dithiothreitol regenerates TXPTS (␦ ϭ
Ϫ28.8 ppm) at the expense of the species at 47.2 ppm
with no change in the peaks that correspond to 3 and
4 (Supporting Information). The addition of a sodium hy-
droxide solution to the reaction mixture results in the
disappearance of the peaks at 34.5 and 47.2 ppm and
resonance at 47.5 ppm or definitive estimates of the to-
tal product yields. Gas chromatographic headspace
analysis and chemiluminescence nitric oxide detection
reveal that no nitrous oxide (evidence for HNO) and only
a trace amount of nitric oxide form during the reaction
of TXPTS and GSNO.
Liquid chromatographyϪmass spectrometry
(LCϪMS) further confirms the identity of these prod-
ucts and provides insight into the structure of the com-
pound responsible for the peak at 47.2 ppm in the 31
NMR spectrum. Analysis of the GSNO/TXPTS reaction
mixture validates the formation of 3 and 4 given their
comparison to known standards (Supporting Informa-
P
tion). The observed m/z of the final compound (892.1)
suggests an adduct of GSNO and TXPTS with the loss of
NO as opposed to the initially predicted -S-NAPR3 mo-
tif leading to the proposal of an S-alkylphosphonium
product (5, Scheme 2) (27−30). The use of 15N-labeled
GSNO results in the formation of 15N-labeled 4 indicat-
ing the transfer of the -S-NAO nitrogen atom to the
phosphorus atom of TXPTS (Supporting Information).
Similar LCϪMS experiments do not support the forma-
tion of GSH or GSSG during this reaction.
A series of 2-D NMR experiments confirm the pro-
posed SϪPϩ bonding motif of 5. A 1HϪ31P COSY indi-
SCHEME 3. Reaction of TXPTS with
S-nitrosocysteine at pH 7
an increase in phosphine oxide (3, 39.7 ppm). The 13
C
and 1H NMR spectra of this reaction show complicated
mixtures that do not allow for the clear identification of
the phosphorus-containing species responsible for the
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Figure 1. 31P NMR time course experiment of the GSNO and TXPTS reaction. 15N-GSNO (1 equiv) and TXPTS (2 equiv) were incubated in a HEPES/DTPA
buffered solution (pH 7.1) with 10% D2O, and the reaction was monitored using 31P NMR with 1 scan every 20 min. The peaks at 47.2, 39.7, and 34.5
ppm correspond to 5, 3, and 4, respectively, and TXPTS starting material at ؊28.8 ppm. All peaks were referenced to a 3% H3PO4 (␦ ؍ 0 ppm) external
standard, which was omitted for clarity.
cates that the 31P signal of 5 (47.2 ppm) correlates to
three protons in the 1H NMR spectrum (3.85, 3.42, and
3.01 ppm, presumably the -CH and diastereotopic -CH2
Figure 2, panel B), providing evidence for attachment of
the phosphine through an SϪPϩ linkage.
Reaction of TXPTS and S-Nitroso-L-cysteine
of the cysteine side chain of 5), in the reaction mixture (Cys-SNO). Similar 31P NMR experiments show the for-
(Figure 2, panel A). Using this obtained 1H correlation,
the 1HϪ13C HMQC of the same reaction mixture reveals mixture of freshly prepared Cys-SNO (1 equiv) with TX-
mation of three phosphorus-derived products from the
the proximity of the P atom of the SϪPϩ moiety to
nearby -CH2 and -CH carbons. Coupling constant analy-
sis shows splitting of the -CH2 carbon (2JPϪC ϭ 14.6 Hz,
PTS (2 equiv) in buffer (50 mM HEPES, 1 mM DTPA, pH
7.1) in the dark that correspond to TXPTS oxide (3, ␦ ϭ
39.7 ppm), the TXPTS-derived ylide (4, ␦ ϭ 34.5), and a
Figure 2. 2-D NMR of the GSNO and TXPTS reaction. a) 1H؊31P COSY of the GSNO/TXPTS reaction mixture in D2O. b) 1H؊13C HMQC of the GSNO/TXPTS
reaction mixture in D2O
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