Mechanistic investigations reveal that dibromobimane extrudes sulfur from biological sulfhydryl sources other than hydrogen sulfide

Article abstract of DOI:10.1039/c4sc01875c

Hydrogen sulfide (H₂S) has emerged as an important biological signaling molecule in the last decade. During the growth of this field, significant controversy has arisen centered on the physiological concentrations of H₂S. Recently, a monobromobimane (mBB) method has been developed for the quantification of different biologically-relevant sulfide pools. Based on the prevalence of the mBB method for sulfide quantification, we expand on this method to report the use of dibromobimane (dBB) for sulfide quantification. Reaction of H₂S with dBB results in formation of highly-fluorescent bimane thioether (BTE), which is readily quantifiable by HPLC. Additionally, the reaction of sulfide with dBB to form BTE is significantly faster than the reaction of sulfide with mBB to form sulfide dibimane. Using the dBB method, BTE levels as low as 0.6 pM can be detected. Upon use of the dBB method in wild-type and CSE^-/- mice, however, dBB reports significantly higher sulfide levels than those measured using mBB. Further investigation revealed that dBB is able to extract sulfur from other sulfhydryl sources including thiols. Based on mechanistic studies, we demonstrate that dBB extracts sulfur from thiols with α- or β-hydrogens, thus leading to higher BTE formation than from sulfide alone. Taken together, the dBB method is a highly sensitive method for H₂S but is not compatible for use in studies in which other thiols are present.

Full text of DOI:10.1039/c4sc01875c

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Mechanistic investigations reveal that
dibromobimane extrudes sulfur from biological
Cite this: DOI: 10.1039/c4sc01875c
sulfhydryl sources other than hydrogen sulfide†
a
b
a
b
Leticia A. Montoya,‡ Xinggui Shen,‡ James J. McDermott, Christopher G. Kevil*
and Michael D. Pluth*a
Hydrogen sulfide (H
the growth of this field, significant controversy has arisen centered on the physiological concentrations of
S. Recently, a monobromobimane (mBB) method has been developed for the quantification of different
2
S) has emerged as an important biological signaling molecule in the last decade. During
H
2
biologically-relevant sulfide pools. Based on the prevalence of the mBB method for sulfide quantification,
we expand on this method to report the use of dibromobimane (dBB) for sulfide quantification. Reaction of
2
H S with dBB results in formation of highly-fluorescent bimane thioether (BTE), which is readily quantifiable
by HPLC. Additionally, the reaction of sulfide with dBB to form BTE is significantly faster than the reaction of
sulfide with mBB to form sulfide dibimane. Using the dBB method, BTE levels as low as 0.6 pM can be
ꢀ
/ꢀ
detected. Upon use of the dBB method in wild-type and CSE
mice, however, dBB reports significantly
higher sulfide levels than those measured using mBB. Further investigation revealed that dBB is able to
extract sulfur from other sulfhydryl sources including thiols. Based on mechanistic studies, we
demonstrate that dBB extracts sulfur from thiols with a- or b-hydrogens, thus leading to higher BTE
Received 24th June 2014
Accepted 10th October 2014
DOI: 10.1039/c4sc01875c
2
formation than from sulfide alone. Taken together, the dBB method is a highly sensitive method for H S
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but is not compatible for use in studies in which other thiols are present.
different protonation states broadens its ability to diffuse across
Introduction
cell membranes, modulate its nucleophilic or reduction
8–11
Hydrogen sulde (H S) is now recognized as a ubiquitous potential, and modulate its reactivity with metal targets.
In
2
gaseous signaling molecule that plays important and diverse addition to its different protonation states, sulde can be stored
1
–3
roles in the endocrine, neuronal, and cardiovascular systems.
Misregulation of basal H
S levels has been demonstrated to be partially-oxidized sulfur pools including hydrodisuldes/per-
associated with various (patho) physiological diseases ranging suldes (RS-SH), hydropolysuldes (RS -SH), and polysuldes
These diverse protonation and storage states not
levels contribute to various disorders of mental deciency only complicate unravelling the multifaceted biological roles of
in acid-labile sources, such as iron–sulfur clusters, or in
2
x
1
12,13
from diabetes to hypertension. Additionally, abnormal H
2
S
(RS-S -SR).
x
4–6
including Alzheimer's disease and Down syndrome. Most
H
2
S, but also complicate H
Despite the widespread and accepted emergence of new
S, meaningful forward progress has
transferase (CAT) working in concert with 3-mercaptosulfur- been slowed in many cases by the dearth of appropriate
2
S detection or quantication.
biological H S is produced enzymatically from cystathionine-b-
2
synthase (CBS), cystathionine-g-lyase (CSE), and cysteine amino biological functions of H
2
transferase (3MST), although recent reports have documented methods of H
2
S detection and quantication. Although the last
H
3
2
S production for D-cys through a D-amino acid oxidase (DAO) few years have seen an impressive growth of new reaction-based
7
ꢀ
14–25
MST pathway. Once generated, H
2
S exists primarily as HS
methods for H
S detection,
2
few of these methods are suit-
under physiological conditions, however the accessibility of able for quantication of endogenous sulde levels. Most
uorescence-based probes exhibit low micromolar functional
detection limits in biological systems, which makes the accu-
S genesis an unmet chal-
Furthermore, although many of these systems show
S over other reactive sulydryl-containing

a
Department of Chemistry and Biochemistry, Institute of Molecular Biology, Materials rate measurement of real-time H
2
26–30
Science Institute, University of Oregon, Eugene, OR 97403, USA. E-mail: pluth@ lenge.
uoregon.edu
good selectivity for H
2
b
Department of Pathology, Louisiana State University Health Science Center,
Shreveport, LA 71130, USA. E-mail: ckevil@lsuhsc.edu
Electronic supplementary information (ESI) available: Experimental details, pH
stability data for BTE, NMR spectra. See DOI: 10.1039/c4sc01875c
These authors contributed equally to this work.
species, potential side- or competing-reactions oen produce
identical products to those generated upon reaction with H S,
2
†
thus precluding accurate H S quantication in complex
2
samples. This ambiguity, as well as whether such scaffolds
‡
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31
report on free, acid-labile, or total sulde remains a challenge two equiv. of mBB. We viewed that use of dibromobimane
in further understanding the multifaceted roles of H S. (dBB), which has two pendant electrophilies on the same uo-
2
Direct H S quantication has been maligned by similar rogenic platform, would serve as a viable strategy to improve the
2
challenges. For example, use of the methylene blue method, mBB assay. We report here a full study of mBB and dBB sulde
which was the measurement standard of the eld for many quantication, which provides unexpected results regarding the
years, requires sample acidication followed by treatment with sources from which dBB extracts sulfur in biological samples,
N,N-dimethyl-p-phenylenediamine and FeCl
3
to generate the and provides a detailed mechanistic analysis of the activity of
methylene blue dye. This method typically reported mid- both mBB and dBB in the presence of other thiol reagents.
32–34
micromolar levels of H
2
S in biological samples.
Because the
human nose is sensitive to aqueous solutions of 1 mM H S, such
results do not match well with qualitative observational data.
Additionally, the reaction conditions required for methylene
2
Results and discussion
Comparing the mBB and dBB sulde response
34
blue formation, especially treatment with strong acid, can result With the broad use of the mBB method as a sensitive and robust
in liberation of sulde from acid-labile sulfur sources, such as H S quantication method, modications to this system
2
35
iron–sulfur clusters. Furthermore, it has been shown that the allowing for faster sulde trapping and/or lower trapping agent
methylene blue method is insufficient to differentiate between loadings would provide a signicant benet. Because mBB
36
wild type and heterozygous CSE knock out mice, and has a reacts with any sulydryl-containing nucleophiles, high
revised detection limit of 2 mM, which is much less sensitive concentrations of mBB are required to effectively trap H S in the
2
than the initially indicated detection limit (ꢁ10 nM). Taking presence of endogenous thiols. Additionally, because reaction
these limitations into account, many of the measured levels of with H S initially generates bimane-SH, sufficient concentra-
2
2
H S have come under increased scrutiny as new, improved tions of mBB must be used such that each bimane-SH produced
methods for H
2
S measurement are developed.
is efficiently converted to SdB prior to HPLC quantication. To
One method that has helped to clarify actual biological H
2
S
overcome such limitations, we viewed dBB as an attractive
levels is the monobromobimane (mBB) quantication platform for enhanced sulde quantication. Specically, dBB
36–38
method.
2
In this method, the sample of interest is treated should react with H S in a 1 : 1 stoichiometry, thus not only
with mBB to trap sulde as sulde dibimane (SdB) (Fig. 1). One improving the reaction kinetics, but also subsequently lowering
key benet of the mBB method is that the analytical selectivity the overall trapping agent concentration required for effective
for H S over other thiols can be superimposed at the end of the H S quantication. Recently, we have noted that dibromobi-
2
2
44,45
experiment by chromatographic separation of the different mane has been used as a turn-on uorescent sensor for H S;
2
reaction products by HPLC. Additionally, the use of different however, this use is problematic because the reaction products
sample treatment workows allows for the separation and of dBB with thiols also generate uorescent bimane thioether
quantication of free, acid-labile, and total sulde thereby products, thus precluding uorogenic selectivity for sulde over
3
7
allowing for direct investigation of different sulde pools.
With a 2.0 nM detection limit, the mBB method is sensitive rescent components.
enough for most biological applications and has found wide
For both mBB and dBB, the initial attack of HS to generate
application ranging from clinical to experimental studies bimane-SH should be fast due to the higher acidity of H S by
thiols without prior chromatographic separation of the uo-
ꢀ
2
7,39–43
investigating sulde metabolism.
several limitations exist, including the high mBB loading undergo a second bimolecular reaction with mBB to form the
required to effectively trap all H S and sulydryl nucleophiles, SdB product. This reaction is inherently slower than the reac-
as well as the required trimolecular reaction between H
Despite this prevalence, comparison to thiols. For mBB, the generated bimane-SH must
2
2
S and tion with sulde due to the decreased nucleophilicity of the
ꢀ
bimane sulydryl group by comparison to HS . For dBB,
however, although the initial attack should proceed at the same
rate as for mBB, the subsequent attack of the pendant thiol is
now transformed into an intramolecular reaction, thus greatly
increasing the potential rate of reactivity. To conrm this design
hypothesis, we treated 3.3 mM solutions of mBB and dBB with
3
.3 mM H S under the conditions used for the mBB method and
2
compared the rates of reaction by uorescence spectroscopy
Fig. S1†). As expected, the growth of the uorescence signal of
(
the BTE product is faster than that of SdB, thus conrming the
importance of the intramolecular reaction manifold for maxi-
mizing the rate of sulde trapping.
Having demonstrated that dBB traps H S more quickly than
2
mBB, we next compared the photophysical properties of the SdB
and BTE products (Table 1, Fig. S2†). Treatment of either mBB
or dBB with NaSH in CH CN/buffer solutions followed by
3
2
Fig. 1 Reaction of mBB and dBB with H S forms the SdB and BTE
products, respectively. Both SdB and BTE can be quantified by fluo-
rescence HPLC.
purication afforded the SdB and BTE products in moderate
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a
Table 1 Comparison of the photophysical properties of SdB and BTE
2
quantication and also opens new avenues of H S detection in
which low H S levels are present. To the best of our knowledge,
the dBB method provides the most sensitive reaction-based
2
Absorption
Emission
Brightness
method of H S quantication reported to date.
l
max
3
2
ꢀ
1
ꢀ1
b
(nm)
(M cm
)
lem (nm)
F (%)
F ꢂ 3
2
Quantication of exogenous and endogenous H S
SdB
BTE
387
356
8800 ꢃ 100
4800 ꢃ 100
478
484
8.3 ꢃ 0.3
62 ꢃ 2
730
3000
To further evaluate the mBB and dBB methods directly, we
a
compared measurements of basal sulde levels in C57BL/6J
Spectroscopic measurements were performed at least in triplicate in
ꢄ
ꢀ/ꢀ
1
00 mM KCl and 50 mM PIPES buffer at pH 7.4 at 25.0 C. (wild type) and CSE
(CSE KO) mice. Based on previous work,
b
Quantum yields are referenced to 1 mM uorescein (F ¼ 0.95 in 0.1
mBB is sufficiently sensitive to differentiate and quantify
differential sulde levels in the wild type and homozygous CSE
knock out mice. Similarly, the mBB method allows for separa-
tion of the free, acid labile, and total sulde pools by either pre-
M NaOH).
yield. The absorption maxima (lmax), extinction coefficients (3),
emission maxima (lem), quantum yield (F), and brightness (3 ꢂ
F) were measured for both SdB and BTE and are shown in Table
37
treatment with acid or with a reductant. For this comparison,
both free and total plasma sulde (free + acid labile + bound
sulfur) was quantied using the optimized procedures for the
mBB assay from identical samples from the same mice. Based
1. As expected, the extinction coefficient for SdB is larger than
that of BTE because two bimane uorophores are present in the
molecule, thus increasing the absorption cross section.
Although the emission maxima of SdB and BTE are similar, the
quantum yield of BTE (62%) is signicantly higher than that of
SdB (8.3%). This enhancement is likely due to abolishment of
internal quenching mechanisms from the two bimane uo-
rophores in SdB. Furthermore, comparing the brightness of SdB
and BTE, which normalizes the quantum yield to the relative
molar absorptivity of each species, reveals that the BTE product
is over four times brighter than SdB. These direct comparisons
of the photophysical properties of SdB and BTE suggested that
detection limit of BTE should be signicantly lower than that of
SdB due to the greater brightness of the BTE product by
comparison to SdB.
on the results, both mBB and dBB clearly differentiate between
ꢀ/ꢀ
the C57BL/6J and CSE
mice (Fig. 3). In both cases, however,
the quantied sulde levels were signicantly different. The
mBB method produced sulde levels consistent with previous
measurements, however the dBB method provided measured
sulde levels that were signicantly higher, suggesting that dBB
may extract sulfur from other biological sources to which mBB
is unreactive. Additionally, the levels of free sulde measured by
dBB are higher than the total sulde levels, which suggests that
other volatile sulfur-containing species that react with dBB, but
not mBB, are volatilized in the procedure for free sulde
measurement, this providing another difference between the
mBB and dBB methods. Alternatively, the increased BTE
formation could also be due to reaction of dBB with proteins in
the plasma, such as albumin, which constitutes the majority of
thiols in the plasma, or by extrusion of sulfur from circulating
Based on the photophysical differences between SdB and
BTE, we next compared the H S detection limits of mBB and
2
dBB directly. For this comparison, the mBB and dBB reaction
products (SdB and BTE, respectively) were compared side-by-
side under identical conditions, and on the same instrument
used in the initial report of the mBB detection limit. Under
these identical conditions, BTE has a superior detection limit by
comparison to SdB (Fig. 2). Although SdB provides a 2.0 nM
detection limit, which is low enough for most practical biolog-
ical application of sulde detection, BTE provides a 0.6 pM
detection limit under identical conditions. This detection limit
46
sulfane–sulfur species, such as persuldes or polysuldes.
2
provides a signicantly larger window for H S detection and
Fig. 3 Comparison of free (a and b) and total (c and d) plasma sulfide
levels measured using dBB (a and c) and mBB (b and d) in C57BL/6J
ꢀ
/ꢀ
(
WT) and CSE
differences are observed in C57BL/6J and CSE
S detection limits of the mBB and dBB absolution sulfide levels quantified are different. ns ¼ non-significant;
knock out mice. For both mBB and dBB, significant
ꢀ/ꢀ
mice, however, the
Fig. 2 Comparison of the H
2
reaction products SdB and BTE, respectively, using fluorescence *p < 0.05, compared to control; ***p < 0.001. n ¼ 6 for C57BL/6J, 9 for
HPLC.
CSE KO.
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Comparison of sulfur extrusion by mBB and dBB
Because both mBB and dBB reported identical sulde levels
when treated with exogenous sulde sources, we interpreted
this result to suggest that dBB was sufficiently reactive to extract
sulfur from other sulfur sources, such as thiols. To test this
hypothesis, we treated dBB with N-acetyl cysteine (NAC) and
1
monitored the reaction by H NMR spectroscopy. Upon incu-
1
bation, new H NMR resonances corresponding to the BTE
product were observed in the H NMR spectrum and were
Fig. 5 Quantification of sulfur extrusion from GSH by (a) dBB to form
BTE and by (b) mBB to form SdB. BTE and SdB concentrations were
1
conrmed by the addition of an authentic sample of BTE quantified by HPLC. GSH concentrations were confirmed by reaction
with 4-fluoro-7-sulfobenzofurazan (SBD-F) followed by HPLC quan-
(
Fig. 4). These results suggest that dBB is sufficiently reactive to
tification. Treatment of dBB with 5 mM GSH resulted in detector
saturation (#).
extrude sulfur from biological thiols to form BTE, thus arti-
cially increasing the measured sulde levels, which is consis-
tent with the increased BTE formation observed from dBB
under biological conditions.
Mechanistic investigations into dBB sulfur extrusion
To quantify the amount of sulfur extracted from common
thiols by dBB, we next investigated and quantied the Based on these data, we sought to further investigate the
amount of BTE formed aer treatment with reduced gluta- mechanism by which sulfur is extruded by dBB. We viewed
thione (GSH) and measured the BTE product by HPLC. three possible mechanisms by which dBB could extract sulfur
1
Consistent with the H NMR studies, BTE formation was from thiols (Fig. 6). Each mechanism proceeds through an
observed by HPLC. To further determine the amount of sulfur initial nucleophilic attack of the thiol on one of the electrophilic
extruded from GSH, different concentrations of GSH were methylbromide groups to generate the thioether. Subsequent
added to dBB and the BTE product was quantied by HPLC intramolecular attack on the second electrophilic methyl-
(
Fig. 5). Treatment of mBB with increasing concentrations of bromide would generate the cyclic sulfonium intermediate.
GSH ranging from 5 mM to 5 mM only generated low nM From this point, we envisioned three potential mechanisms for
concentrations of SdB. By contrast, treatment of dBB with dealkylation to form the BTE product. If the sulfonium inter-
identical GSH concentrations results in generation of mediate maintains a sufficiently unhindered a-position, then
micromolar concentrations of BTE. Based on the data, aer a nucleophilic attack by a second equivalent of the thiol would
3
0 minute incubation, dBB extracts approximately 7.0% of generate the BTE product and one equivalent of the thioether
the sulfur from GSH to form BTE. By comparison, under derived from the incident thiol (Fig. 6c). Alternatively, if the
identical conditions the mBB method extruded less than sulfonium has accessible b-hydrogens, the elimination would
0
.01% sulfur from GSH. These extraction efficiencies not only extrude the BTE product with concomitant formation of a
explain the higher levels of biological sulde detected from terminal olen and regeneration of one equivalent of the inci-
dBB but also highlight that mBB does not extract appreciable dent thiol (Fig. 6d). The third possible mechanism could
sulde from endogenous thiol sources.
include radical fragmentation of the sulfonium intermediate to
form the BTE product (Fig. 6e).
To test between these different mechanistic pathways, we
chose multiple model thiols to investigate which pathways of
sulfur extrusion were operative and monitored the reactions by
1
H NMR spectroscopy. In addition to the biologically-relevant
cys, NAC, and GSH we also used other thiols to test specic
mechanistic considerations (Fig. 7). All of the thiols, except for
thiophenol (PhSH), produced the BTE product, which was
1
47
identied by H NMR spectroscopy and mass spectrometry.
Because tert-butyl thiol generates BTE, we know that nucleo-
philic attack cannot be the only mechanism of BTE formation
because nucleophilic attack on the tertiary carbon is not
possible. Similarly, benzyl thiol (BnSH) produced BTE, sug-
gesting that the elimination pathway cannot be the only oper-
ative pathway. Consistent with both nucleophilic and
elimination pathways leading to BTE formation, treatment of
dBB with PhSH, which cannot participate in either of these
reaction pathways, failed to produce BTE. If radical fragmen-
tation contributed appreciably to BTE formation, the BTE
1
Fig. 4 H NMR spectra of the reaction of dBB (50 mM) with N-acetyl should have been produced upon treatment with PhSH. To
3
cysteine (NAC, 20 mM) in CD CN. Growth of a new peak (*) at 3.8 ppm
corresponds to the BTE product.
further
exclude
the
radical
pathway,
we
used
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Fig. 6 Possible reaction routes of dBB reacting with thiols. (a) Addition of one thiol generates the sulfonium thioether. (b) Nucleophilic addition
of a thiol generates the dithiol bimane adduct. Possible mechanisms of BTE formation from thiols, including: (c) nucleophilic attack at the a-
position of the pendent thiol; (d) elimination from deprotonation of hydrogens in the b-position of the pendant thiols; and (c) radical
fragmentation.
Comparing the overall reactivity and selectivity reveals
that dBB is signicantly more sensitive for sulde than is
mBB under conditions without other thiols present. If thiols
are present, however, dBB is able to extrude sulfur from these
thiols with relatively high efficiency (Fig. 8). In such cases in
which thiols can be removed from the sample prior to anal-
ysis, dBB provides a highly-sensitive method of H S detection
2
and quantication. For biological samples containing other
sulydryl containing species, however, mBB is highly effi-
cient for H S quantication. Importantly, mBB very mini-
2
mally extracts sulfur from thiols, which is not signicant, and
can be corrected for by measuring total thiol concentrations
in a sample.
Fig. 7 (a) Reaction of dBB with thiols generates either the bis-thio-
ether or the BTE thioether product. (b) Model thiols used to investigate
the mechanism by which BTE is formed.
cyclopropylmethanethiol-containing 1 as a substrate to monitor
BTE formation. If the radical pathway were operative, this
substrate would generate a methylcyclopropyl radical, which
8
ꢀ1
would quickly react (k > 10 s ) to the corresponding open-
48,49
chain product.
Aer treatment of dBB with 1 under identical
conditions to those of the other thiol substrates, BTE formation
was observed but no cyclopropyl ring opening was observed by
1
H NMR spectroscopy, suggesting that persistent radicals are
not formed during the reaction. Similarly, treated dBB with GSH
50
in the presence of DMPO, a radical spin trap, did not produce
any spin-trapped product by EPR spectroscopy. Taken together,
these results suggest that both the nucleophilic and elimination
pathways are operative in the sulfur extrusion of dBB. Consis-
tent with these results, although BTE is stable at neutral pH, it
slowly decomposes in acidic conditions, which is consistent
with transient protonation of the thioether sulfur followed by
nucleophilic attack by thiol (or solvent) at one of the benzylic Fig. 8 General reaction scheme for (a) mBB and (b) dBB reactivity.
bimane carbons (Fig. S3†).
Extrusion of sulfur with mBB is inefficient whereas extraction of sulfur
with dBB is significantly more efficient.
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1
5 X. Cao, W. Lin, K. Zheng and L. He, Chem. Commun., 2012,
Conclusions
48, 10529–10531.
Complementing the mBB method, dBB provides a highly- 16 S. Chen, Z.-j. Chen, W. Ren and H.-w. Ai, J. Am. Chem. Soc.,
sensitive method for sulde quantication with a detection
2012, 134, 9589–9592.
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species, however, dBB extracts sulfur from other sources
4912.
thereby decreasing its delity for H
S quantication if other 18 V. S. Lin, A. R. Lippert and C. J. Chang, Proc. Natl. Acad. Sci.
thiols are present. Mechanistic investigations revealed that
U. S. A., 2013, 110, 7131–7135.
thiols with a- or b-hydrogens react to generate the BTE product. 19 A. R. Lippert, E. J. New and C. J. Chang, J. Am. Chem. Soc.,
Taken together, these results establish dBB as a highly-sensitive
2011, 133, 10078–10080.
method for H
S quantication, but also provide cautions for its 20 L. A. Montoya, T. F. Pearce, R. J. Hansen, L. N. Zakharov and
2
2
use in biological samples in which thiols are present.
M. D. Pluth, J. Org. Chem., 2013, 78, 6550–6557.
21 L. A. Montoya and M. D. Pluth, Chem. Commun., 2012, 48,
4
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This work was supported by NIH grants R00GM092970 (to MDP)
and HL113303 (to CGK). We thank Matt Hartle for assistance
with EPR measurements. The NMR facilities at the University of
Oregon are supported by NSF/ARRA CHE-0923589. The
Biomolecular Mass Spectrometry Core of the Environmental
Health Sciences Core Center at Oregon State University is sup-
ported, in part, by the NIEHS (P30ES000210) and the NIH.
672–9675.
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