Free Radical Biology & Medicine 47 (2009) 13461353

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Free Radical Biology & Medicine

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Methods in Free Radical Biology & Medicine

Making and working with hydrogen sulde

The chemistry and generation of hydrogen sulde in vitro and its measurement
in vivo: A review
Martin N. Hughes , Miguel N. Centelles, Kevin P. Moore
Centre for Hepatology, Department of Medicine, University College London Medical School, London

a r t i c l e

i n f o

Article history:
Received 15 July 2009
Revised 17 September 2009
Accepted 17 September 2009
Available online 19 September 2009
Keywords:
Hydrogen sulphide
Knockouts
Enzyme inhibitors
Methylaene blue
Hydrogen sulde
Sulde

a b s t r a c t
Hydrogen sulde is rapidly emerging as an important vasoactive mediator formed in health and disease. Its
biological action is centered on its reactivity with heme-proteins and its ability to activate KATP channels.
Hydrogen sulde is a signalling molecule of the inammatory and nervous systems, and in particular the
cardiovascular system where it regulates vascular tone, cardiac work, and exerts cardioprotection.
This has led to an explosion of papers in which the role of hydrogen sulde generated in vitro has been used
to stimulate biological responses, and where a variety of methods have been used to measure the
concentration of this compound in biological uids. Understanding the chemistry and the inherent problems
in the analytical techniques used to measure hydrogen sulde concentrations is critical to our expanding
knowledge on the biology of hydrogen sulde. In this brief review we will cover the chemistry of hydrogen
sulde, including sources of hydrogen sulde, its speciation at physiological pH, the susceptibility of sulde
to aerobic oxidation, and the methods used to measure hydrogen sulde concentrations in solution,
including biological uids. We also give a brief overview of knockout animals and inhibition of the enzymes
involved in the formation of hydrogen sulde in vivo.
2009 Elsevier Inc. All rights reserved.

Introduction

Pathways involved in the formation of hydrogen sulde in vivo

Hydrogen sulde, H2S, is continuing to emerge as an important

gaseous mediator in cellular physiology and pathology [13]. Its
toxicity has been known for many years, but it is only since the recent
discoveries of its actions in the vascular system, the liver and the brain
that its biological activity is being investigated. It is clear that
hydrogen sulde now sits with nitric oxide and carbon monoxide as
one of three gaseous mediators in biology. It is important, therefore, to
understand the chemistry and properties of this molecule, to be aware
of the problems associated with the choice of chemicals used to
generate hydrogen sulde in vitro and in vivo, and to appreciate the
limitations and errors that may be generated when measuring sulde
concentrations in biological uids. In this review the term sulde
will refer to the total sulde present in solution, irrespective of its
speciation. The species H2S, SH- and S2- will be named specically as
necessary.

In mammalian tissues, H2S is produced endogenously from

enzymic desulfydration of cysteine catalyzed by cystathionine-lyase (also termed -cystathionase CSE), cystathionine--synthase
(CBS), or 3-mercapto-sulfurtransferase [4,5] (Fig. 1), with CBS and CSE
dependent pathways being predominantly responsible for the
synthesis of H2S in vivo [4,6].
Thus, cysteine (cys) may be hydrolyzed by CBS to produce H2S,
with L-serine as the by-product, or hydrolyzed by CSE to produce H2S,
pyruvate and ammonia. In an alternative pathway, CSE may catalyze
the conversion of cystine (Cys-S-S-Cys) to thiocysteine, which is then
hydrolyzed by CSE to cysteine and H2S. These two key cytosolic
enzymes are also involved in the trans-sulfuration pathway of
methionine and homocysteine metabolism, with CBS catalyzing the
condensation of homocysteine and serine to form cystathionine in an
irreversible reaction. Cystathionine may then be hydrolyzed by CSE to
cysteine with ketobutyrate and ammonia as by-products (Fig. 1).
Recent studies have shown that carbon monoxide, formed through
the heme oxygenase pathways binds to the prosthetic heme of CBS,
stabilizing a 6-coordinated COFe(II)histidine complex to block its
activity [7].
In addition, several other pathways for H2S synthesis have been
described. Thus, cysteine may react with a ketoacid to form 3mercaptopyruvate, which undergoes desulfuration by 3-mercapto-

Corresponding author. Centre for Hepatology, Department of Medicine, University

College London Medical School, Royal Free Campus, Rowland Hill Street, London NW3
2PF, UK. Fax: +44 207 433 2871.
E-mail address: mhughes@medsch.ucl.ac.uk (M.N. Hughes).
0891-5849/$ see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2009.09.018

M.N. Hughes et al. / Free Radical Biology & Medicine 47 (2009) 13461353

1347

pyruvate sulfurtransferase (MPST) to form H2S and pyruvate [8,9].

Furthermore, CBS can catalyze the reaction on the thiol of cysteine to
form S-thiolate (Cys-S-R) with the concomitant release of H2S. Chen
et al. [10] reported also that CBS could catalyze the condensation of
cysteine and homocysteine to form cystathionine and H2S.
However, most studies suggest that H2S is synthesized in
mammalian tissues predominantly through the enzymatic action of
either CBS or CSE on cysteine. All the other pathways involving
additional enzymes and substrates are likely to play only a minor role
in the biosynthesis of H2S. However, CBS and CSE have different tissue
distributions; cystathionine -synthase is primarily responsible for
production of H2S in the central nervous system [11], while
cystathionine -lyase is primarily expressed in peripheral tissues,
including vascular and nonvascular smooth muscle [12,13].
The physiology of hydrogen sulde
Physiological concentrations of H2S are generally thought to be
quite high with many reports of concentrations of around 10100 mol L-1 or higher in blood or tissue samples [1416]. For a
long time we have believed that these values are too high, and expected much lower levels to be reported once the inherent problems
with current analytical methods to measure sulde were corrected.
More recently other investigators have estimated the concentration of
sulde in tissues or plasma to be in the nanomolar range [17,18].
H2S is known to exert effects in the cardiovascular, endocrine and
central nervous systems. Thus, H2S activates ATP-sensitive K+
channels, exhibits anti-inammatory effects and promotes healing
[19]. Human penile tissue also contains cystathionine--lyase and
cystathionine--synthetase and it appears that, like nitric oxide, H2S
is also involved in mediating penile erection in humans and other
mammalian animals [20].
An H2S-releasing group has been chemically linked to the nonsteroidal anti-inammatory drug diclofenac, with a resulting increase
in its anti-inammatory action [21]. The protective effects of garlic
against cardiovascular disease are thought to be secondary to the
formation of hydrogen sulde from garlic-derived organic polysuldes by the action of red blood cells [22]. Furthermore, a range of
sulfur compounds, shown in Fig. 2 (structures 1 to 5), and present in
algae, mushrooms and onions, are believed to undergo chemical and
enzymatic reactions during digestion that enhance the formation of
H2S in humans [23]. The physiological and pharmacological actions of
hydrogen sulde have recently been extensively reviewed [24].

Fig. 2. (a) Structures of some polysuldes in food that release H2S on digestion [21]. (1)
Allyl mercaptan, (2) Diallyl disulde, (3) Diallyl trisulde, (4) Diallyl tetrasulde, (5)
Lenthionine [21]. (b) Structure of an H2S releasing compound, GYY4137 [34].

Deciency of cystathionine--synthase and cystathionine gamma lyase

in humans
In humans, a deciency of CBS results in homocysteinuria, with
increased concentrations of homocysteine and methionine in
plasma and decreased levels of cysteine [25]. CBS deciency may
lead to mental retardation, optic lens dislocation, skeletal abnormalities, and a tendency to thromboembolic episodes that demonstrate the biochemical and medical signicance of H2S in health
[26].
Making and working with hydrogen sulde in the lab
The toxicity of H2S

Fig. 1. Hydrogen sulde can be generated by at least 3 metabolic pathways.

The toxicity of H2S is due to the binding of sulde to cytochrome c

oxidase in mitochondria. However, H2S also targets the olfactory
nerves, the eyes and the brain. Hydrogen sulde is more toxic than
hydrogen cyanide and must be treated with great care. The
preparation and use of solutions of H2S should always be carried out
under efcient fume hoods. It is important to note also that H2S is
heavier than air and so can accumulate in low, unventilated areas.
Investigators should be aware that one of the rst symptoms of H2S
toxicity is the loss of the ability to smell H2S, which begins at
concentrations of about 50 ppm after prolonged exposure. At
concentrations greater than 100 ppm the sense of smell is lost within
2 to 10 minutes, while exposure to 500 ppm H2S for 30 minutes may
be fatal. You cannot rely upon your sense of smell to tell how much
hydrogen sulde is present. If you are working with hydrogen sulde
and there is a strong smell, which disappears, be careful. In the
Appendix we provide information which may be useful for preparation of a safety assessment when working with hydrogen sulde (e.g.
COSSH). However, we strongly emphasize that it is important to seek
advice from your local safety ofcer when producing guidelines for
the laboratory (Appendix A).

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M.N. Hughes et al. / Free Radical Biology & Medicine 47 (2009) 13461353

Fig. 3. pH-dependent speciation of hydrogen sulde in aqueous solution.

Fig. 4. Effect of DTPA on the oxidation of SH- in aerobic solutions at 25C. The
concentration of the hydrogen sulde anion was measured by its absorbance at 230 nm
under aerobic conditions (exposure to air). The absorption was measured over time in
the absence (lower line) and presence of DTPA (10-4 mol/L). The results obtained in the
absence of the chelating agent show the induction period before the start of the
oxidation. The addition of DTPA prevents oxidation of the sulde ion.

The solution chemistry of H2S

Hydrogen sulde, H2S, is a weak acid, with pKa values of 6.98 at
25C and 6.76 at 37C. Data showing the pH-dependence of the
distribution of H2S at 25C and 37C are presented in Fig. 3. Thus, at
physiological pH and 37C, about 20% of sulde is present as H2S,
while at pH 7.4 and 25C about 40 % of the sulde will be present as
H2S. It should be emphasised that H2S and SH- may both contribute
directly to the biological action of hydrogen sulde, and that SH-, the
predominant sulde species under biological conditions, is a nucleophile, which readily binds to metal centres in biological molecules
(e.g. haemoglobin) or reacts with other compounds.
The second pKa value (pKa2) of H2S is now agreed to be 19 2 [27].
Therefore, the sulde anion S2- is present at extremely low concentrations at pH 7.4, with a mole fraction of 1.7 10-12 and is unlikely
to participate in the biological chemistry of H2S.
H2S is soluble in many solvents, including water, acetone, carbon
disulde, methanol, ethanol, ether, chloroform, and benzene. Some
data on the solubility of H2S in a range of non-aqueous solvents are
available [28,29]. Data on the solubility of H2S in water at various
temperatures are given in Table 1. H2S is a lipophilic molecule and
readily crosses cell membranes. It is commonly stated that its
solubility in lipophilic solvents is about ve times greater than its
solubility in water.
Aqueous solutions of hydrogen sulde are slightly acidic, with
pH 4.0. At room temperature in water, a saturated solution of H2S
has a concentration of 0.11 mol L-1, while at 0C the concentration is
0.21 mol L-1. If these solutions of H2S are accurately diluted into
deoxygenated carbonate buffer at pH 9.6, where the sulde is
entirely present as the hydrogen sulde anion HS-, the concentration
of the hydrogen sulde anion may be determined from the
absorption at 230 nm [30], using the molar absorptivity of 7200
mol-1 L cm-1. However, sulde in solution reacts with oxygen and so
the determination of sulde concentration by this UV method
depends on the use of thoroughly deoxygenated water to prepare
solutions.
Reactions of H2S and HSH2S/HS- has a shorter half-life in cells compared to aqueous
solutions because sulde may react with many intracellular targets

Table 1
Temperature-dependence of solubility of hydrogen sulde in water
Temp

0C

10C

20C

30C

37C

[H2S] mol L-1

0.209

0.152

0.116

0.0908

0.0815

such as peroxynitrite, peroxide and superoxide [3]. The hydrogensulde anion, HS-, reacts with metal centres to form iron-sulfur
clusters and with haemoglobin at the oxygen-binding site. It may
also add across carbon-carbon double bonds to form organic suldes.
Hydrogen sulde may be methylated in cells to form methanediol
(CH3SH), and this may be further methylated to form di-methanediol. The rates of these reactions are signicant and may result in a
substantial lowering of the anticipated concentration of sulde in the
cell.
Air oxidation of sulde in aqueous solutions
The air oxidation of sulde will occur when preparing and using
standard solutions of sulde during laboratory studies, particularly at
higher concentrations of sulde and pH. However, this may be
minimised. Solutions of sulde or H2S should be prepared in water
that has been thoroughly deoxygenated by bubbling with nitrogen or
helium for 40 minutes.
The air oxidation of H2S in aqueous solution is a slow reaction, so
at pH values below 6, where H2S is the dominant species, there is
little oxidation of H2S. As the pH of the solution increases above pH 6,
the concentration of the hydrogen sulde ion SH- increases: this
species readily undergoes oxidation in air [31]. The immediate
product of oxidation of HS- is sulphur, seen as a pale yellow or white
precipitate in solutions of H2S. This reaction involves an induction
period, suggesting that it proceeds through a chain mechanism with
a variety of intermediates. Such an oxidation reaction is probably
catalyzed by trace impurities of transition metals, such as Fe(III).
Thus, the stability of SH- with respect to air oxidation may be
increased if a chelating agent such as diethylenetriaminepentaacetic
acid (DTPA) is added to the solution. This is seen in Fig. 4, in which
we show that the autoxidation of HS- is prevented by the addition
of 10-4 mol L-1 DTPA.
Further oxidation of sulde leads to the formation of thiosulfate
S2O32-, which is the principal product at pH N 8.5, tetrathionate S4O62,
sulte SO32- and nally sulfate SO42-. The sulphur formed in the initial
oxidation step may also react with SH- to give polysuldes such as
S32-, S42- and S52-. Polysuldes may be oxidized by Fe3+ to give, for
example, the brilliant blue radical ion S3.- [32]. These species may be
present as contaminants in commercial samples of sodium sulde or
sodium hydrogen sulde.
The kinetics of oxidation of hydrogen sulde by hydrogen peroxide
have been studied [33,34]. The reaction with hydrogen peroxide
results in decreased levels of H2S in cells. The rst product of this
reaction is suggested to be HSOH, formed by nucleophilic attack of
SH- on H2O2, which may react further with either SH- to give

M.N. Hughes et al. / Free Radical Biology & Medicine 47 (2009) 13461353

polysuldes or with H2O2 to give polythionates. At 25C and pH 9,

with concentrations of HS- of 100 mol L-1 and H2O2 of 1200 mol L-1,
the half life of HS- is about 10 minutes. The nal product under these
conditions is probably sulphate.
Key Points

1. The stability of sulfide is greatest at acidic pH, where it is

present as H2S. However, under acidic conditions the gas is
more volatile.
2. Sulfide is stable in deoxygenated solutions.
3. Higher pH values results in deprotonation to give SH- (sulfide)
which is rapidly oxidized in air.
4. Sulfide is stabilised in the presence of DTPA which chelates
trace amounts of iron or copper, which otherwise catalyze the
oxidation of H2S in air.
Sources of H2S for biological experiments and reactions
Several methods are used for adding or generating H2S in aqueous
solution.
Sodium hydrogen sulde and sodium sulde
A convenient approach involves the addition of aqueous
solutions of NaSH.xH2O (sodium hydrogen sulde), or the
nanohydrate disodium salt Na2S.9H2O or their anhydrous forms.
These compounds are often referred to as H2S releasing drugs,
which is incorrect since they are related to H2S by an acid-base
equilibrium. At physiological pH hydrogen sulde is present
mainly as HS-.
There are major problems in the use of sodium suldes to generate
H2S (Table 1), primarily because their purity can vary enormously
from product to product. Sodium hydrogen sulde and sodium sulde
should be white. Yellow products should not be purchased. However,
white sulphide products may contain white impurities such as sulte
and thiosulfate formed by oxidation of the sulde. Contamination by
trace metal ions may also be important, as these catalyze oxidation
processes. The suldes should therefore be stored in a vacuum
dessicator to minimise oxidation. We have had reproducible results
using anhydrous sodium hydrogen sulde from Alfa Aesar, Heysham,
UK, (Cat. No.65122). This product was not hygroscopic and remained
white over several months when stored in a vacuum dessicator. The
UV spectrum of a solution of this compound in deoxygenated
carbonate buffer pH 9.6 gave a satisfactory molar absorptivity at
230 nm, conrming its purity. A solution of a yellow sample of NaSH.
xH2O from another supplier showed an absorption band with max
around 420 nm, suggesting the presence of the polysulde S32-. On
exposure to air for some hours, a sample of this yellow compound
became green and then blue, due to the formation of the radical ion
S3.-, and the presence of the Fe3+ impurity necessary to catalyze this
reaction.
Sodium sulde nonahydrate is used to a much lesser extent than
other suldes despite being a cleaner compound. Solid Na2S.9H2O
exists as white hygroscopic blocks. It may contain white impurities
such as sodium salts of thiosulfate or higher oxidation state sulfur
oxyanions [31]. Several authors comment on the need to wash off
surface oxidation products from the nanohydrate before use. We
have observed blue-purple particles on the surface of Na2S.9H2O
due to the radical ion S3.- formed by the Fe3+ catalyzed oxidation of
polysulde. The purity of suldes may be measured by determining the sulde content either by titration with bromate, as described in standard analytical chemistry texts, or by UV spectroscopy in the case of sodium hydrogen sulde, at pH 9, which has
an absorption maximum at 230 nm with a molar absorptivity of
7200 L mol-1 cm-1.

1349

Key Points

1. The water content of sodium hydrogen sulfide is often

unknown and variable.
2. All sources of sodium hydrogen sulfides or sodium sulfide
have metal ion contamination which accelerates oxidation of
sulfides.
3. Sodium hydrogen sulfide should be white in colour. A yellow
colour is due to oxidation to polysulfides. White products are
likely to have greater purity, but may contain thiosulfates and
sulfites.
4. Anhydrous sodium sulfide is less susceptible to aerobic
oxidation.
5. Solutions of sodium hydrogen sulfide should be clear. Yellow
solutions contain oxidation products.

Using hydrogen sulde gas

The purest source of hydrogen sulde is the gas. The preparation
of aqueous solutions using H2S from a cylinder (Sigma Lecture
bottle) without allowing oxidation of SH- in solution is fairly
straightforward. This must be carried out in an efcient fume
cupboard. H2S from a cylinder is passed through a series of
Dreschler bottles, connected with plastic tubing, and with glass
sinters attached to the entry tubes in order to generate ne bubbles
of gas (Fig. 5). Nitrogen or preferably argon should rst be passed
through the system for about 40 minutes to deoxygenate the
solutions before the H2S is admitted.
The rst Dreschler bottle should contain distilled water to dissolve
the H2S to saturation. Once saturated with H2S at room temperature,
this solution will be acidic ( pH 4) with the concentration of H2S at
0.10 mol/L. Alternatively, solutions containing only the hydrosulde
anion SH- can be generated by passing H2S into a slightly alkaline
buffer (pH 9). The H2S that passes through the rst Dreschler bottle is
then passed through three further Dreschler bottles, each of which
contains about 200 ml of an H2S trapping solution. This solution (in
practice, usually a slurry) is prepared by dissolving 10 g zinc acetate,
2.94 g sodium citrate and 4 g sodium hydroxide in 1000 ml of water
[35]. The sulde is precipitated as zinc sulde in the trapping solution.
The ow rate of H2S through the system should be low enough to
ensure that no bubbles of H2S escape from the last trapping bottle. The
concentration of the H2S solution may be measured by its absorbance
at 230 nm, after serial dilution into deoxygenated carbonate buffer,
and using the molar absorptivity of 7200 dm3 mol-1 cm-1 at 230 nm
[36].
Using H2S releasing molecules
Only a small number of H2S releasing compounds are available.
The existence of compounds that release H2S at various rates would
be of great value. Thioacetamide, CH3CSNH2, releases H2S on decomposition in water. However, it is carcinogenic and highly toxic
to the liver. Lawesson's reagent, 2,4-bis(4-methoxyphenyl)1,3,2,4dithiaphosphetane-2,4-disulde is also an H2S releaser. It contains a
four-membered ring of alternate P and S atoms. The ring opens to give
two molecules of a very reactive dithiophosphine, R-PS2, that
decomposes to give H2S. However, this reagent has an exceptionally
foul odour and is toxic. Nevertheless, it is still used as an H2S generator
to a limited extent.
H2S releasing compounds have been synthesized which possess
vasodilator and antihypertensive activity. An example is morpholin4-ium 4-methoxyphenyl- (morpholino)phosphinodithioate (coded
GYY4137) (Fig. 2, structure 6) which acts as a slow releaser of
H2S [37]. Dithiolthiones have proved to be successful H2S releasing
compounds. The 5-(4-hydroxyphenyl)-3H-1,2-dithiol-3-thione group
has been attached to several drugs, notably non-steroidal antiinammatory drugs, in the expectation that the anti-inammatory

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M.N. Hughes et al. / Free Radical Biology & Medicine 47 (2009) 13461353

Fig. 5. Preparing saturated hydrogen sulde solution: This should only be carried out in an efcient fume cupboard: Extreme caution must be used. The rst Dreschler bottle should
contain distilled water or solvent in which to dissolve the H2S to saturation. Once saturated with H2S at room temperature, this solution will be acidic ( pH 4), and in our experience
the concentration of H2S is 0.11 mol L-1 H2S. Alternatively, solutions containing the hydrogen sulde anion SH- can be generated by passing H2S into a slightly alkaline buffer (pH 9).
After passing through the rst bottle of water, any remaining hydrogen sulde needs to be removed completely so that no free gas escapes into the atmosphere. This is achieved by
passing the hydrogen sulde gas sequentially through three Dreschler bottles in series, and each containing 200 ml of an H2S trapping solution. This solution (usually a slurry) is
made up by dissolving 10 g zinc acetate, 2.94 g sodium citrate and 4 g sodium hydroxide in 1000 mls of water [33].

effects of H2S release would alleviate toxic effects due to the drug, such
as gastrointestinal ulceration and bleeding and also improve its
efciency [38]. Specic examples of attachment of a H2S releasing
group to drugs include the nonsteroidal anti-inammatory drug
diclofenac [39,40] and aspirin [41].
Measurement of sulde in biological samples
Colorimetric Methods
The use of colorimetric assays is based on the assumption or belief
that plasma or tissue concentrations of sulphide are high. If recent
estimates of the nanomolar concentrations of hydrogen sulde turn
out to be correct [17,18], then none of the colorimetric assays will be
sensitive enough.
The methylene blue method involves the reaction of sulde with
N, N-dimethyl-p-phenylenediamine sulfate in the presence of the
oxidising agent Fe3+ in hydrochloric acid to form methylene blue (Fig.
6) [42]. The reaction involves a 2:1 stoicheiometry of reagent to
sulde.
The formation of methylene blue is widely used for the
determination of sulde in biological samples such as plasma,
although there are major problems with this assay. Firstly, although
methylene blue has a high molar absorptivity of 71 090 mol-1 L cm-1
with an absorption maximum of 667 nm [43], unfortunately the
absorption spectra of aqueous solutions of methylene blue do not
obey Beer's law: i.e. there is no linear dependence of absorbance on
the concentration of methylene blue. This is due to the formation of
dimers and trimers of methylene blue (Fig. 7) [44]. We discovered this
during our own experimental validation of the methylene blue assay
and then went back through the literature to see if this phenomenon
was well known. In fact, it has been known for many decades that
methylene blue only obeys Beer's Law at very low concentrations, i.e.
below 1 mol L-1, which is considerably lower than concentrations
generated typically during analysis of sulde-containing biological

uids. The monomeric, dimeric and trimeric forms of methylene blue

have absorption maxima at 667, 610 and 595 nm, respectively [44].
Dissociation constants for the dimer and trimer are 1.5 10-4 mol L-1
and 3.2 10-11 mol L-1 respectively [44]. The self-association of
methylene blue is favoured at high concentrations of anions (as
methylene blue has a positive charge).
It should be noted that N, N-dimethyl-p-phenylenediamine
sulfate is extremely toxic. Gloves should always be used when
working with this compound. The reagent is also unstable and
should be stored in a refrigerator. However, it is liable to decompose over time to form a solid block of material. Solutions of the
reagent are readily oxidized to a purple product. The reagent bottle
should be covered in foil and stored in a refrigerator or in a vacuum
dessicator.
Therefore, if you decide to use this method, despite the inherent
problems, the procedure should be validated by recording spectra of
the methylene blue solutions obtained for both standards and
samples over the 550 to 700 nm range, measuring the extent of
formation of monomers and dimers and then conrming that the
distribution of the dimer-monomer equilibrium is similar in both
standards and samples.
A closely related colorimetric method for sulde involves the
formation of the dye ethylene blue, by using the reagent N,N-diethylp-phenylenediamine [45]. We have found this method to be an
improvement over the methylene blue method. The reagent is much
less toxic and more stable with respect to oxidation. However,
solutions of the reagent turn purple after a few days. The ethylene
blue dye has a higher molar absorptivity (87,700 mol-1 L cm-1) than
does methylene blue. Ethylene blue also has a lower tendency to
aggregate [45].
Electrochemical methods
Electrochemical methods have the advantage of allowing sulde
concentrations to be continuously monitored with time.

Fig. 6. Analysis of sulde by formation of methylene or ethylene blue. The reaction between sulde and the reagent involves a 1:2 stoicheiometry. The N, N-dimethyl-pphenylenediamine sulphate reagent is toxic and should be used with care. It is also unstable. This compound is a free-running crystalline compound but decomposes to give a solid
mass. Aqueous solutions of the reagent decompose to give a purple solution, and should be prepared freshly as required. All analytical solutions should be prepared in thoroughly
deoxygenated distilled water.

M.N. Hughes et al. / Free Radical Biology & Medicine 47 (2009) 13461353

1351

display pronounced hypertension and diminished endotheliumdependent vasorelaxation. Serum H2S levels in CSE/ mice and
CSE/+ mice were reduced by about 50 and 20% respectively,
compared to the wild type [48].
Alternatively, mice heterozygous for disruption in the CBS gene
may be purchased from Jackson Labs (Bar Harbor, MI), and mated to
generate homozygous knockouts. The CBS (-/-) animals exhibited
strong biological phenotypes, including facial alopecia, osteoporosis,
endoplasmic reticulum stress in the liver and kidney, they suffered
from severe growth retardation and a majority of them died within
5 weeks after birth [49].
Measurement of CBS/CSE mRNA

Fig. 7. Concentration dependence of the speciation of methylene blue. Once formed,

methylene blue undergoes dimerisation and trimerisation in a concentration
dependent manner. At a product concentration of 100 mol L-1 only 55% of the
methylene blue is present as the monomeric form. Thus, when working with high
sulde concentrations (N10 mol L-1), the absorbance due to methylene blue increases
on dilution. This paradox is explained by the facts that dilution leads to the formation of
the monomer and loss of the dimer.

Sulde electrodes have usually been developed for use in

environmental contexts and function optimally at pH values higher
than pH 7.4. Seary and Peterson (2004) [46] have recently assessed
the use of a commercial sulde specic electrode. This was originally
used at pH 13 on the assumption that the electrode responded to S2(due to the erroneous belief that the value of pKa2 for H2S was about
13). However, it has been shown that this electrode functions well
at pH values 4, 7, and 13. At pH 7 the electrode responded to
concentrations of hydrogen sulde anion as low as 10-15 mol L-1,
although the electrode equilibrates slowly at low concentrations.
Searcy and Peterson [46] have noted that the biological reactions of
sulde could be studied at constant concentrations of sulde in a
chemostat using a sulde electrode. The use of this electrode (and
other sulfude electrodes) suffers from precipitation of metal suldes
from the media on the electrodes and the electrode needs conditioning before use.
Polarography is an important method for determining H2S
concentration. It is well known for its use in measuring the
concentrations of nitric oxide. Polarography is a type of voltammetry
where the working electrode is a mercury cathode made up of a
succession of small mercury drops falling from a ne capillary tube.
The anode is a pool of mercury. The electrode is described in standard
text books. Recently [47] the method has been applied to the study of
the rates of production and consumption of sulde in mammalian
tissues, with detection limits near to 10 nmol L-1. The polarographic
method analyses specically for H2S. The total sulde concentration
may then be calculated from the Henderson Hasselbach equation,
knowing the pKa value of H2S at the temperature of the experiment
and the pH of the solution. Fig. 3 gives the percentage of sulde
present as H2S at different pH values. However, Whiteld et al. (2008)
[18] found little evidence for free sulde in vertebrate blood.
Furthermore in experiments where sulde was added to blood
there was a rapid decrease in its concentration. This implies that
sulde does not circulate in plasma at measurable concentrations.
Some reactions that may contribute to the loss of sulde have been
noted earlier.

Since CBS and CSE have different tissue distributions, the CBS and
CSE enzymes may be localized and quantied by the use of molecular
probes and RT-PCR, northern blotting or in situ hybridisation, see
reference 48 for information on primers [48].
Inhibitors for the CBS and CSE enzymes
The formation of H2S in vivo and in vitro can be inhibited by
compounds that are selective towards either cystathionine -lyase
(CSE) or cystathionine--synthase (CBS).
CSE catalyses the conversion of cysteine to H2S, pyruvate and
ammonia. This reaction is irreversibly inhibited by D,L-propargylglycine and competitively inhibited by -cyano-L-alanine in a dosedependent manner. The doses used in a rat model for L-propargylglycine and -cyano-L-alanine range between 10 to 50 mg kg-1
intravenous injection [50,51].
Potent inhibitors for the cystathionine--synthase (CBS) are also
available, namely aminooxyacetate (AOA) and hydroxylamine (HA)
[52]. Aminooxyacetate at 1 mM concentration abolished H2S
production in liver tissues. However, the specicity of these inhibitors
is presently unknown.
Qu et al. [53] have studied the effect of these four inhibitors in a
stroke model. They showed that all four inhibitors inhibited H2S
production in vitro in a dose-dependent manner. Values of IC50 were
0.0126 mmol L-1, with 98 % inhibition at 0.50 mmol L-1, for
aminooxyacetate and CBS; 0.50 mmol L-1 for hydroxylamine with
CBS; 2.5 mmol L-1 and 70 % inhibition at 10 mmol L-1 for cyanoalanine with CSE; and 7.1 mmol L-1 and 55% inhibition at
10 mmol L-1 for L propargylglycine with CSE. The ability of these
inhibitors to effect neuroprotection in vivo paralleled their ability to
inhibit the synthesis of H2S invitro, suggesting that inhibition of H2S
synthesis may have potential in the protection against strokes.
Conclusions
This short review describes some of the basic experimental
approaches involved in studying the biological activity of hydrogen
sulde, and highlights the importance of the care needed to avoid
oxidation artifacts when generating hydrogen sulphide in vitro, and
the problems inherent in current colorimetric assays of hydrogen
sulphide concentration. As with all new elds of research it takes a
while before we understand the problems and pitfalls inherent in
assay development and sample handling.
Appendix A

Enzyme studies of hydrogen sulde in vivo and in vitro

Safe use of H2S in the Laboratory

Genetic knockouts

Hydrogen sulde is very toxic. When working with H2S, it is

essential that good safety practices are maintained, in particular that
fume cupboards operate efciently and that researchers do not work
alone in the laboratory. H2S is heavier than air and accumulates in

In order to study H2S production in vivo, experiments may be

carried out in transgenic mice models. Mutant mice lacking CSE

1352

M.N. Hughes et al. / Free Radical Biology & Medicine 47 (2009) 13461353

Table 2
Toxic effects of increasing concentrations of H2S
H2S concentration in air
(ppm)

Physiological effects

0.010
5
25
100

Odour detectable
Odour readily detectable
Odour strongly detectable
Loss of sense of smell after about 3-5 minutes; irritation
of the eyes.
Effects on the respiratory system after extended
exposure (N 1 hour).
Loss of consciousness; death may occur after
30 minutes.
Loss of consciousness, respiration failure, death.

200-300
300-700
Above 700

low-lying unventilated areas. If a build-up of H2S is suspected the

laboratory should be evacuated immediately.
The toxic effects of H2S are most likely to result from inhalation,
leading initially to a loss of smell (see Table 2). It is essential that you
do not depend on your sense of smell to warn you that there is a build
up in H2S concentration in the laboratory. If the initial warning over
the smell of H2S is ignored the consequences may be serious. In cases
of concern over the possible inhalation of H2S, the subject must be
removed immediately into the fresh air, and medical attention sought
if necessary. H2S has wide-ranging toxic effects, including those on the
olfactory nerves, lungs, eyes, brain and respiration.
In experiments where H2S gas is being passed through a solution,
the leaving ow of gas should be passed into about 250 ml of a
trapping solution containing zinc acetate (10 g), sodium citrate
(2.94 g) and sodium hydroxide (4 g) per litre to trap the H2S by
precipitation as zinc sulde. The gas should not pass through the H2S
trap.
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