Antioxidant chemistry: Reactivity and oxidation of DL-cysteine by some common oxidants

Article abstract of DOI:10.1039/a708863i

The reactivity of DL-cysteine, a physiologically important aminothiol, was studied by reacting it with several well known oxidants. No activity was observed on the amino and carboxyl groups. The only reactivity of physiological significance was at the sulfur centre. Reactions of cysteine with hydrogen peroxide show that the thiol group is capable of mopping up free radicals by forming thyl radicals, as expected in its role as an antioxidant. A four-electron oxidation of cysteine gave reasonably stable cysteine sulfinic acid. Oxidants in the form of peracids do oxidize cysteine only as far as the sulfinic acid. Stronger oxidizing agents can oxidize cysteine as far as the cysteine sulfonic acid. No further oxidation can be detected as the C-S bond is not cleaved. The inertness of the amino group in cysteine makes it incapable of reversibly mopping up the dangerous oxyhalogens HOCl and HOBr which are produced by myeloperoxidase-catalysed oxidation of halides by hydrogen peroxide, as is the case with taurine. A detailed mechanism, together with a computer simulation study of the oxidation of cysteine by acidified bromate, is proposed.

Full text of DOI:10.1039/a708863i

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Antioxidant chemistry
Reactivity and oxidation of DL-cysteine by some common oxidants¤
James Darkwa,a Claudius Mundomab and Reuben H. Simoyib
a Department of Chemistry, University of the North, Private Bag X1106, Sovenga 0727, South
Africa
b Department of Chemistry, West Virginia University, Morgantown, WV 26506-6045, USA
The reactivity of DL-cysteine, a physiologically important aminothiol, was studied by reacting it with several well known oxidants.
No activity was observed on the amino and carboxyl groups. The only reactivity of physiological signiÐcance was at the sulfur
centre. Reactions of cysteine with hydrogen peroxide show that the thiol group is capable of mopping up free radicals by forming
thyl radicals, as expected in its role as an antioxidant. A four-electron oxidation of cysteine gave reasonably stable cysteine
sulÐnic acid. Oxidants in the form of peracids do oxidize cysteine only as far as the sulÐnic acid. Stronger oxidizing agents can
oxidize cysteine as far as the cysteine sulfonic acid. No further oxidation can be detected as the CwS bond is not cleaved. The
inertness of the amino group in cysteine makes it incapable of reversibly mopping up the dangerous oxyhalogens HOCl and
HOBr which are produced by myeloperoxidase-catalysed oxidation of halides by hydrogen peroxide, as is the case with taurine.
A detailed mechanism, together with a computer simulation study of the oxidation of cysteine by acidiÐed bromate, is proposed.
There has not been, to date, an exhaustive study of the meta-
bolic functions of small-molecular-weight amino acids. The
sulfur-based amino acids, especially, have not been adequately
characterized. In some of our recent work, we studied the
reactivity of the most common amino acid in the human
body, taurine.2 Taurine has been implicated in several physio-
logical roles, including antioxidant activity,3 and yet no pos-
sible mechanism through which taurine could act as an
antioxidant had been established.
that can inactivate free radicals,10,11 especially the reactive
oxygen species, ROS, and thus can protect and preserve
cells.12 Thus it should act physiologically as an antioxidant
and can extend cell life span and protect against toxic sub-
stances.12 Cysteine is supposed to protect glutathione from
powerful oxidants because it is much more easily oxidizable
than glutathione.13 It is also supposed to control protein
folding in the endoplasmic reticulum by maintaining the redox
state of the extracellular Ñuids.14
We recently began a series of studies on oxyhalogenÈsulfur
chemistry in which we tried to elucidate systematically the
oxidation of a sulfur centre in small organic molecules.4 Our
studies established an oxidation algorithm which involves suc-
cessive two-electron oxidations of the sulfur centre up to the
sulfonic acid stage before cleavage of the CwS bond to form
sulfate.5 For the physiologically active sulfur-based amino
acids which we have studied so far: taurine,2 hypotaurine,6
methionine7 and glutathione,7 this algorithm, however, is not
followed. Oxidation of taurine occurs only on the amino
group, with no activity on the sulfonic acid moiety. Hypo-
taurine, when oxidized by oxyhalogens, is oxidized to taurine
and then to bromo- and chlorotaurines and no further.6
Methionine is oxidized to give a mixture of the sulfoxide and
the sulfone, while glutathione is oxidized only to the SwS
dimeric species. No cleavage of the CwS bond is observed in
any of the cases. The reversible nature of the oxidations (even
by very strong oxidants like HOCl and HOBr) observed with
these aminothiols and aminosulfonic acids2 tends to lend
them well to physiological uses, as there is the possibility of
substrate regeneration for further use.
Some clinically negative aspects related to cysteine mention
that it contributes to kidney stones and is contraindicated for
diabetics.15 Also, cysteineÏs homologue, homocysteine, has
been implicated as playing a critical role in destroying arteries
and, subsequently, contributing to heart attacks.16 Homo-
cysteine, however, is extremely inert and reacts exceedingly
slowly (reaction times of the order of days) even with the most
powerful oxidants (e.g. aqueous bromine). Removing
a
wCH w group from homocysteine gives cysteine, a much
2
faster reacting thiol.
The sulfhydryl group in cysteine seems to play a physiologi-
cally signiÐcant role.17 Here, we report on the reaction
dynamics, stoichiometries and mechanisms of the oxidation of
cysteine by peroxyacetic acid, hydrogen peroxide, acidiÐed
bromate and aqueous bromine. It is hoped that this study will
help in evaluating the oxidation products of cysteine and lead
to a plausible mechanism for its action as an antioxidant. We
also hope to determine whether there is any activity on the
amino group of the cysteine molecule.
Experimental
Cysteine is an aminothiol with three reactive centres: the
carboxylic acid, the amino group and the sulfur centre. Cyste-
ine is particularly interesting in that it is a non-essential
amino acid which can be synthesized by the human body
through the metabolism of methionine,8 a process which pro-
duces the very important amino acids homocysteine, hypo-
taurine and taurine.9
Materials
All stock solutions and their subsequent dilutions were pre-
pared from doubly distilled deionized water. The following
reagents were used without further puriÐcation: sodium
bromate, sodium bromide, perchloric acid, 72%, (Fisher);
nitrate metal salts, (Aldrich); DL-cysteine, DL-cystine, DL-
cysteine sulÐnic acid, stabilized hydrogen peroxide (30 wt.%
solution in water), (Acros), and peroxyacetic acid (32 wt.%)
(Sigma). Bromine solutions were stored in sealed brown
winchester bottles as a precautionary measure. All cysteine
Several claims have been made for the physiological rele-
vance of cysteine. Cysteine is said to contain sulfur in a form
¤
Part 32 in the series: Nonlinear dynamics in chemistry derived
from chemistry. Part 31: ref. 1.
J. Chem. Soc., Faraday T rans., 1998, 94(14), 1971È1978
1971

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solutions were prepared fresh to ensure reproducibility.1 All
reagents were stored as recommended by the vendor.
Stoichiometry
The stoichiometric studies were performed on a Perkin-Elmer
Lambda 2S UVÈVIS spectrophotometer. The stoichiometry of
the reaction of cysteine with bromate was determined by
varying the ratios of bromate to DL-cysteine in an acidic
medium. The mixtures were incubated for at least a 24 h
period before stoichiometric determinations were performed.
The amount of Br produced or consumed was quantiÐed
2
spectrophotometrically by measuring its absorbance at 390
nm and the product was identiÐed by 1H NMR spectroscopy.
1H NMR studies
1
H NMR spectra were run at 270 MHz on a JEOL-270 MHz
spectrometer at 25 ¡C. Commercial grade deuterated water
(
D O) and DClO (Aldrich) were used as supplied. The
2
4
solvent peak, D O d \ 4.67), was used as the internal stan-
dard.
2
Fig. 1 Determination of the stoichiometry of the bromateÈcysteine
reaction. The initial amount of cysteine was varied at constant
bromate (0.080 mmol). Cell path length is 10 mm, volume 4 cm3.
Wavelength used 390 nm. The reactions were run in a medium with
Kinetic determinations
an acid strength, [H`] , of 0.065 M. The reaction mixtures of varying
ratios of DL-cysteine to bromate were incubated for a period of at
least 24 h, after which aliquots from each mixture were analysed by
taking absorbance readings at 390 nm.
Reactions were slow enough to be followed on a conventional
Perkin-Elmer UVÈVIS Lambda 2S spectrophotometer inter-
faced to a 486 DX DEC computer for data acquisition,
storage and manipulation. Solutions were Ðrst vigorously
mixed with a magnetic stirrer prior to introduction to the
spectrophotometer. A constant time lag of 25 s was main-
tained between mixing of reagents and commencement of data
acquisition.
0
[
BrO ~] P [cysteine] , the amount of Br obtained starts to
3
0
0
2
fall with increase in cysteine, to the point where the cysteine
concentrations used are sufficient to completely consume the
BrO ~ ions, leaving no more BrO ~ ions to produce Br .
This is the stoichiometric point for the bromateÈcysteine reac-
tion. The data in Fig. 1 were used to evaluate the stoichio-
metry (1).
3
3
2
Stopped-Ñow measurements
The kinetics of the cysteineÈbromate, metal ion e†ect and the
bromineÈcysteine reactions were followed on a Hi-Tech Scien-
tiÐc SF61-DX2 double mixing stopped-Ñow spectrophoto-
In the highly acidic environments used in this series of
experiments (pH \ 2) it is expected that both the amino and
thiol centres will be highly protonated:
meter interfaced to
a
pentium 133 MHz computer.
Time-dependent absorbance changes corresponding to the
formation of bromine were measured at 390 nm (e \ 142 dm3
mol~1 cm~1)18 for a duration of at least 1000 s. All concen-
trations reported are post mixing and reactions were run at
H N`CH(CO ~)CH SH ] H` H
3
2
2
H N`CH(CO ~)CH S`H (2)
3
2
2
2
2
5.0 ^ 0.5 ¡C with a Neslab RTE-101 thermostat. All reac-
The oxidation of cysteine was performed using four oxi-
dants: hydrogen peroxide, peroxyacetic acid, sodium bromate
and aqueous bromine. When reactions were performed in an
NMR tube, the organic products could be identiÐed by 1H
NMR spectroscopy. The 1H NMR spectrum of DL-cysteine
has two main distinguishing features. First, there is an unre-
solved doublet of a doublet at a chemical shift of 3.85 ppm,
arising from the proton of the wCHw group. The other peak
comprises two sets of doublets due to the inequivalent
tions were run with at least a two-fold excess of bromate con-
centrations over cysteine in highly acidic environments
(
pH \ 1.00).
Results
Stoichiometry and product identiÐcation
Product identiÐcation by 1H NMR shows that the Ðnal
sulfur-containing product of the oxidation of cysteine by
wCH w protons20 coupling with each other and with the
2
wCHw proton. By monitoring each of the oxidation reac-
acidic
bromate
is
cysteine
sulfonic
acid
tions by 1H NMR it was possible to establish that the oxida-
[
H N`CH(CO ~)CH SO H] and the stoichiometry of the
3
2
2
3
tion products of the H O reaction are di†erent from those of
reaction was experimentally deduced to be:
2
2
the peroxyacetic acid reaction, which in turn are di†erent from
those of the bromate and bromine oxidation reactions (Fig.
BrO ~ ] H N`CH(COO~)CH SH ]
3
3
2
2
A). It is clear from the 1H NMR spectra that H O oxida-
H N`CH(COO~)CH SO H ] Br~ (1)
2 2
3
2
3
tion of DL-cysteine leads to a mixture of unidentiÐable pro-
ducts. The peroxyacetic acid oxidation cleanly forms cysteine
sulÐnic acid. This could be established by comparing the 1H
NMR of the oxidation product with commercial cysteine sul-
Ðnic acid (Aldrich). On the other hand, BrO ~ and Br oxida-
Quantitative tests for the presence of sulfate19 were negative,
indicating that the CwS bond was not cleaved under these
conditions. The centre of reactivity is the sulfur atom, with the
other functionalities merely modifying its reactivity.
3
2
It was recognized that the reaction that forms Br is an
tion of DL-cysteine show the 1H NMR spectrum of cysteine
sulfonic acid (Fig. 2B). By reacting an authentic sample of
2
extraneous pure oxyhalogen reaction that occurs in excess
BrO ~. Br would only be produced when BrO ~ was in stoi-
DL-cysteine sulÐnic acid with Br and running the 1H NMR
3
2
3
2
chiometric excess (see Fig. 1). When [BrO ~] A [cysteine]
spectrum of the product, it was shown that the product is the
3
0
0
the amount of Br formed was proportional to the cysteine
sulfonic acid (Fig. 2B). The oxidized DL-cysteine products, i.e.
the sulÐnic and the sulfonic acids, show a downÐeld shift for
2
concentration up to some maximum value. However, when
1972
J. Chem. Soc., Faraday T rans., 1998, V ol. 94

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Fig. 2 A 1H NMR traces of oxidation products of cysteine with di†erent oxidants. (a) Spectrum of pure CySH, (b) CySH ] H O , (c)
2
2
CySH ] peroxyacetic acid, (d) spectrum of pure DL-cysteine sulÐnic acid. The shift observed in spectrum (c) [from the standard spectrum (d)] is
due to the acidic environment. B 1H NMR traces of the products of cysteine oxidation by: (a) pure CySH, (Aldrich), (b) CySH ] acidiÐed
bromate, (c) cysteine ] Br (aq). The shift observed again in spectra (b) and (d) is due to the acidic environment of spectrum (b). Bromate
2
oxidations occur only at highly acid conditions.
both the wCHw and the wCH w peaks compared to those
mined by the initial conditions, there is a sudden and rapid
2
of cysteine. Unlike the wCHw peaks of cysteine, those of sul-
production of aqueous Br . No bromine production was
2
Ðnic and sulfonic acids are well resolved doublets of a doublet.
The clearly distinguishing features of the 1H NMR spectra
of the sulÐnic acid and sulfonic acid derivatives are the gaps
observed with r \ 1.
Acid dependence. As expected from most oxyhalogen oxida-
tions, acid strongly catalysed the reaction. Fig. 3A shows a
series of experiments at varying acid concentrations. Increase
in the acid concentration reduced the induction period and
also increased the rate of formation of bromine at the end of
the induction period. The acid did not, however, a†ect the
Ðnal amount of bromine formed. A plot of induction period
vs. the square of the inverse of the acid concentrations gave a
straight line (Fig. 3B), indicating that the precursor reaction to
the formation of bromine is catalysed by acid to the second
power.22
between the high-intensity doublets for the CH group of each
2
acid. The sulÐnic acid gap is much larger; *d \ 0.07 ppm
compared with *d \ 0.02 ppm for the sulfonic acid.21 Apart
from this, all other spectral features are essentially the same
for both acids. Based on the in situ NMR studies above, it was
established that the strong oxidants, BrO ~ and Br , cleanly
3
2
oxidise DL-cysteine all the way to the sulfonic acid. The reac-
tion dynamics of these reactions are described below.
Reaction dynamics
Bromate dependence. Bromate also decreased the length of
the induction period (Fig. 4A). When 1 \ r O 1.2 the Ðnal
amount of bromine formed was proportional to the initial
bromate concentration. At r [ 1.2 the ratio of cysteine to the
amount of bromine formed was 3 : 2 exactly. The stoichiom-
etry of the reaction, taking into account the formation of
bromine was:
In the presence of excess BrO ~ ([BrO ~] /[cysteine] ratio
3
3
0
0
r [ 1), the oxidation of cysteine gives a long quiescent period
with no absorbance activity at 390 nm and very little activity
in the redox potential of the reaction mixture (calomel
reference). After an induction period, whose length was deter-
J. Chem. Soc., Faraday T rans., 1998, V ol. 94
1973

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Fig. 3 A, Absorbance traces at j \ 390 nm in two-fold excess of
Fig. 4 A, Absorbance traces at j \ 390 nm in excess [BrO ~] over
[
[
0
BrO ~] over cysteine at varying initial acid, HClO , concentration.
3
0
[
CySH] at varying [BrO ~] and constant acid strength. Final
3
0
4
CySH] \ 0.0026 M, [BrO ~] \ 0.0050 M; [H`] \ (a) 0.30, (b)
0
3
0
amount of bromine formed is the same for all traces. [H`] \ 0.70 M;
0
3
0
0
.40, (c) 0.50, (d) 0.70, (e) 0.90 and (f) 1.00 M. B, Acid dependence of the
0
[
CySH] \ 0.0026 M; [BrO ~] \ (a) 0.003, (b) 0.0040, (c) 0.0060, (d)
induction period for data shown in A.
0
3
0
0
.0080 and (e) 0.012 M. B, Inverse of induction time vs. [BrO ~] for
3
0
conditions in A.
6
BrO ~ ] 5Cys-SH ] 6H` ]
3
constant and Ðxed at 0.0026 M, thus conÐrming stoichiometry
(1) of 1 : 1.
5
Cys-SO H ] 3Br (aq) ] 3H O (3)
3
2
2
where Cys-SH is cysteine and Cys-SO H is cysteine sulfonic
3
acid. This is the stoichiometry obtained at the formation of
Cysteine dependence. The cysteine dependence of the reac-
tion is more complex than that for acid or bromate. The ratio,
r, was the most important parameter in this set of experi-
ments. With very large values of r ([15), variations in the
cysteine concentration did not a†ect the induction period. The
cysteine concentration, however, did a†ect the rate of forma-
the maximum amount of Br in Fig. 1.
2
All Br~ formed in stoichiometric reaction (1) reacts with the
excess bromate to give bromine:23
BrO ~ ] 5Br~ ] 6H` ] 3Br (aq) ] 3H O
(4)
3
2
2
The stoichiometry of reaction (3) is a composite of
5 ] reaction(1)] ] reaction(4). The induction period is
inversely proportional to the initial bromate concentrations
tion of Br . With small r (1.2 \ r \ 5.0) increasing cysteine
2
[
concentration increased the induction period (see Fig. 5A).
This would suggest that the complete consumption of cysteine
(
Fig. 4B). The plot shown, with the inverse of the induction
could be the precursor reaction to the formation of Br .
Lower cysteine concentrations need very little time for com-
2
period vs. [BrO ~] is valuable in conÐrming the deduced
3
0
stoichiometric reaction (1).24 The [BrO ~] axis intercept
plete consumption (oxidation) by bromate. The amount of Br2
formed was proportional to the initial cysteine concentration
so long as r [ 1.2 (Fig. 5B). 1 mole of cysteine, according to
stoichiometry (1), gives 1 mole of Br~. This Br~ will determine
3
0
shows that the reaction produces no Br (t \ O) when
ind
BrO ~] \ 0.0026 M. This is the limiting bromate concentra-
2
[
3
0
tion before formation of Br . Formation of Br denotes an
2
2
excess of BrO ~ over that required for stoichiometry (1). The
cysteine concentration used for the series of experiments was
the Ðnal amount of Br formed, according to the stoichiom-
3
2
etry of reaction (3). Fig. 5C shows the induction period data
1974
J. Chem. Soc., Faraday T rans., 1998, V ol. 94

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E†ect of bromide. Bromide appeared to have no e†ect on
the reaction. Most bromate oxidations show bromide cataly-
sis, since the bromateÈbromide reaction is responsible for the
production of the reactive species which perform the bulk of
the oxidation: HBrO , HOBr, and Br .
2
2
BrO ~ ] Br~ ] 2H` ] HBrO ] HOBr
(5)
3
2
Reaction (5) is recognized as the initiation reaction for all
bromate oxidations, and it is usually the rate-determining
step.25 With the BrO ~Ècysteine reaction being insensitive to
3
initial bromide concentrations, then there must be another
reaction that can produce Br~ and subsequently HBrO ,
2
HOBr and Br , more rapidly than reaction (5) for the reac-
2
tionÏs initiation. There is no unambiguous way of testing for
the efficiency of the direct bromateÈcysteine reaction, but the
following experiment was performed. Dry crystals of NaBrO3
and DL-cysteine were mixed together into an intimate
powdery mix. Without addition of acid, water was carefully
added drop-wise (in a fume hood) to this mixture. As soon as
the Ðrst drop of water was added, the mixture exploded with
rapid formation of bromine (observed as a yellowish-brown
gas) due to the violent bromateÈcysteine reaction. Thus the
reaction is capable of initiating itself to the extent that the
addition of bromide delivers no catalytic e†ect. Fig. 6 shows
data for a series of experiments with varying initial bromide
concentrations. [Br~] was changed by a factor of 20 with no
0
noticeable e†ect on the reaction. Even for [Br~] concentra-
0
tions in excess of the initial cysteine concentrations, there was
no e†ective variation in the induction period. The only
(
expected) di†erence was the increase in the Ðnal Br concen-
2
trations with increased bromide.
Direct Br –cysteine reaction. The direct oxidation of cyste-
2
ine by aqueous bromine was extremely fast (see Fig. 7) and,
for approximately equimolar Br Ècysteine solutions, the reac-
2
tion was essentially over within 0.1 s. The stoichiometry of the
reaction was determined by both spectrophotometric and
titrimetric techniques26 to be:
3
Br ] Cys-SH ] 3H O ] Cys-SO H ] 6Br~ ] 6H` (6)
2
2
3
The reaction occurred in two stages. The Ðrst was so rapid (to
the point of di†usion control) that it could not be resolved,
even on the advanced Hi-Tech ScientiÐc DX2 stopped-Ñow
Fig. 5 A, E†ect of varying [CySH] in excess [BrO ~] conditions.
0
3
0
[
0
H`] \ 0.70 M; [BrO ~] \ 0.005 M; [CySH] \ (a) 0.001, (b),
0 3 0 0
.0015, (c) 0.0025, (d) 0.0030, and (e) 0.0035 M. B, Maximum absorb-
ance at j \ 390 nm for the data shown in A. The absorbances were
taken after 1000 s, the values did not change signiÐcantly after 48 h.
C, Induction time vs. [CySH] for the conditions in A.
0
for small values of r (1.2 \ r \ 5.0). These data show a discon-
tinuity as r goes below unity because the induction period
increases to O.
Fig. 6 E†ect of Br~ on the bromateÈCySH reaction. [CySH] \
0
0.0025 M; [H`] \ 0.70 M; [BrO ~] \ 0.005 M; [Br~] \ (a)
0
3
0
0
control, no Br~ added, (b) 0.0005, (c) 0.0010, (d) 0.005 and (e) 0.0100 M
J. Chem. Soc., Faraday T rans., 1998, V ol. 94
1975

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E†ect of metal ions. A number of metal ions were used to
examine possible metal-ion catalysis. The only notable set of
results was obtained with CuII ions. The results obtained were
unexpected. CuII ions seemed to have an inhibitory e†ect on
the overall oxidation of cysteine (see Fig. 8). Higher concentra-
tions CuII ions considerably lengthened the induction period
of the reaction. The inhibitory e†ect was much more pro-
nounced when the cysteine solutions were Ðrst incubated with
the CuII ions for between 1È6 h before subjecting them to
reaction with acidiÐed bromate. Unincubated solutions did
not show any CuII ion e†ects.
Mechanism
The bromide e†ect suggests that the Ðrst step of the reaction is
production of the reactive species by direct reaction of
bromate with DL-cysteine:
BrO ~ ] Cys-SH ] H` ] Cys-SOH ] HBrO
(9)
3
2
The sulfenyl acid formed in reaction (9), Cys-SOH, is very
unstable and has never been isolated.27 It can be oxidized
further [reaction (10)] or dimerize to form cystine [reaction
Fig. 7 Direct reaction of bromine with cysteine. [Br ] \ 0.00392 M,
2
0
and [CySH] \ 0.0005 M.
0
(11)]:28
HBrO ] Cys-SOH ] Cys-SO H ] HOBr
(10)
(11)
2
2
spectrophotometer used for these studies. The second (and
slower) stage of the reaction occurs after 2 moles of bromine
have been consumed for each mole of cysteine. Thus the very
Ðrst part of the reaction involves the oxidation of cysteine to
cysteine sulÐnic acid:
Cys-SOH ] Cys-SH ] Cys-SwS-Cys ] H O
2
In the presence of excess oxidant, however, further oxidation
of the sulfenyl acid is the predominant pathway.29 The cystine
can also be further oxidized, provided it is not produced in the
reaction to the level where it precipitates, as it is sparingly
soluble in water.
2
Br ] Cys-SH ] 2H O ] Cys-SO H ] 4Br~ ] 4H` (7)
2
2
2
Fig. 7 shows that by the time the reaction is captured by the
stopped-Ñow spectrophotometer (mixing time, essentially), it is
essentially complete. The initial absorbance expected for Fig. 7
was 0.265.
Cys-SwS-Cys ] 2H O ] 2Cys-SOH ] 2H` ] 2e~ (12)
2
Both products of reactions (10) (cysteine sulÐnic acid) and (11)
(
cystine) are extremely stable and have been isolated as stable
The second stage of the reaction is the further oxidation of
the more stable sulÐnic acid:
products.30 Further reaction of HOBr releases Br~ which
reacts with BrO ~ as in reaction (4) to release more reactive
3
species:
Br ] Cys-SO H ] H O ] Cys-SO H ] 2Br~ ] 2H` (8)
2
2
2
3
HOBr ] Cys-SH ] Cys-SOH ] Br~ ] H`
(13)
From the kinetics data we estimate an upper-limit di†usion-
controlled rate constant for the Ðrst stage of the reaction to be
The Br~ formed in reaction (13) can also form Br :31
2
4.5 ^ 1.3 ] 106 dm3 mol~1 s~1.
HOBr ] Br~ ] H` ] Br ] H O
(14)
2
2
The Br , as soon as it is formed, will immediately react with
2
DL-cysteine or any other cysteine species in solution not yet
oxidatively saturated (at the sulfur centre):
Br ] Cys-SH ] H O ] Cys-SOH ] 2Br~ ] 2H`
2
2
(15)
(16)
(17)
Br ] Cys-SOH ] H O ] Cys-SO H ] 2Br~ ] 2H`
2
2
2
Br ] Cys-SO H ] H O ] Cys-SO H ] 2Br~ ] 2H`
2
2
2
3
Fig. 7 data show that reactions (15) and (16) are extremely fast
and nearly di†usion-controlled. Reaction (17), though not
di†usion-controlled, is still one of the fastest reactions in the
reaction mixture. In excess bromate, we expect the cysteine
species to mop up all bromine species in solution as soon as
they are formed. The only time Br production is observed
2
(
end of the induction period) is after all the cysteine species
have been oxidized to cysteine sulfonic acid. Stoichiometry (1)
Fig. 8 E†ect of Cu2` on the CySHÈbromate reaction. [CySH]
should go to completion before Br production can occur. In
0
0.0026 M) was premixed with acid, [H`] (0.70 M) and varying
2
(
excess bromate, the rate of formation of Br should be pro-
0
amounts of Cu2` for traces (b)È(e). These mixtures were incubated for
2
portional to the initial cysteine concentration, as the compos-
a period of 6 h before they were reacted with [BrO ~] (0.0050 M). (a)
3
0
ite reaction for formation of Br is given by reaction (4). The
control, no Cu2` added, [Cu2`] \ (b) 0.0025, (c) 0.0050, (d) 0.0075
2
0
and (e) 0.025 M. Trace (f) is direct reaction of cystine (0.0012 M) with
data in Fig. 5A conÐrm this assertion. The formation of Br is
2
same [BrO ~] and [H`] conditions as in (a)È(e).
a pure oxyhalogen reaction with no interference from the
3
0
0
1976
J. Chem. Soc., Faraday T rans., 1998, V ol. 94

View Article Online
cysteine species. This could be proved by running control
experiments with the cysteine substituted by an equal concen-
Computer simulation
The proposed mechanism was distilled to yield the set of reac-
tions shown in Table 1. The Ðrst three reactions, (M1)È(M3)
are pure oxyhalogen reactions whose kinetics parameters were
obtained from the literature.40 In this mechanism, the bulk of
the oxidation was assumed to be carried out by HOBr and
Br . Oxidation of cysteine by HOBr and Br was assumed to
tration of Br~ and observing the rate of formation of Br
which was identical to the post-induction period rate.
2
Other oxidants
While the oxidation of cysteine is extremely facile, the oxida-
tion of cysteine sulÐnic acid is not as rapid. Peroxyacetic acid
can easily oxidize cysteine to its sulÐnic acid but cannot take
it all the way to the sulfonic acid. Hydrogen peroxide is a
powerful oxidizing agent and should oxidize cysteine to the
sulfonic acid. The oxidation by H O , however, is predomi-
2
2
be fast and not rate-determining. The rate-determining steps
were taken as the rates of formation of HBrO and Br as well
2
2
as the rate of oxidation of cysteine sulÐnic acid. The simula-
tions were very sensitive to the kinetics parameters for reac-
tion (M4) as it is the one which initiates the reaction. Owing
to the non-linear production of HOBr, [as in reaction (M2)]
reaction (M4) is not the rate-determining step. Reaction (M5)
was not very important in this reaction scheme. Owing to the
rapid reaction (M2), reaction (M5) could be omitted from the
mechanism without loss of structure in our simulations. Reac-
tions (M6) and (M7) were rendered fast and non-rate-deter-
mining. Reaction (M8) was slower than reactions (M6) and
2
2
nantly via free radical mechanisms.32 Physiologically, cysteine
is known as a free radical scavenger because of the thiol
group. H O can produce a variety of free radical species,
2
2
including the acidic form of the superoxide radical HO ~.33
2
and the hydroxyl radical, HO~.34 Both the superoxide and
hydroxyl radicals are one-electron oxidants, which can form
thiyl free radicals with cysteine, Cys-S~.35 The predominant
pathway for free-radical mediated oxidation of thiols is via the
dimer:35
(M7) (see data in Fig. 7) and controlled the rate of formation
of Br in the later stages of the reaction. Thus, although the
2
kinetics parameters for reactions (M5)È(M8) were estimated,
RSH [ e~ ] RS~ ] H`
RS~ ] RS~ ] RS [ SR
(18)
(19)
they had no e†ect on the simulations so long as they were fast
and not rate-determining.
Reactions (M9)È(M11) were treated in the same manner as
their HOBr counterparts [reactions (M6)È(M8)]. The simula-
tions were thus controlled only by the kinetics parameters of
reactions (M1)È(M4) with (M4) being the only reaction with
adjustable kinetics parameters as the other values were set to
literature data. The ordinary di†erential equations generated
from Table 1 were numerically integrated by semi-implicit
RungeÈKutta techniques.41 The simulations gave reasonably
good Ðts to the experimental data (see Fig. 9). The simulations
were also able to predict quantitatively both the acid depen-
dence (Fig. 3A and B) and bromate-dependence (Fig. 4A and
B).
As noted before, the dimer, cystine, is extremely insoluble (and
only sparingly soluble in highly acid conditions), and is
rapidly precipitated. Other possible products include sul-
foxides and thiosulÐnates.36 There is lack of product control
with free radical mechanisms. This explains the unidentiÐable
solid products obtained with hydrogen peroxide, as well as the
complex 1H NMR spectrum. Rapid and quantitative forma-
tion of cystine yields a precipitate.
E†ect of CuII ions
It has been reported that reactions of sulfur compounds, espe-
cially thiols, are strongly catalysed by Cu` and Cu2` ions.37
The decomposition of S-nitrosothiols is one such reaction that
is catalysed by Cu` ions.38 Cu2` ions strongly inhibited the
reaction (see Fig. 8). The retardation is due to the formation of
cystine. The CuII ions can oxidize the thiol group via a one-
electron free radical mechanism:39
Conclusion
The most important result is that cysteine is extremely
capable of acting as an antioxidant with respect to the reactive
oxygen species: superoxide and peroxoradicals and hydroxyl
radicals. The mechanism of this interaction involves the for-
mation of the cystine dimer via the thyl radicals. The dimer
can be reduced back to the cystine through an appropriate
2e~ reduction, such that cysteine can continue to moderate
radical toxicity. For physiological purposes, the regeneration
of the antioxidant is essential. Cysteine can also mop up
harmful oxidants such as HOCl and HOBr. The efficiency of
cysteine as a radical and oxyhalogen scavenger is related to
the rate at which it reacts with radicals, oxyhalogens and
halogens. Thus, cysteine should be preferentially oxidized over
2
Cu2` ] 2RSH ] 2Cu` ] RSSR ] 2H`
(20)
The cystine dimeric species, RSSR, is very stable and only
slightly soluble in water. The conversion of the cysteine to the
more stable, slower reacting cystine, signiÐcantly retards the
reaction. The cystine that precipitates is lost to further reac-
tion, but the dissolved cystine can be oxidized further to give
the unstable sulfenyl acid as in reaction (12). In the presence of
excess oxidant, the sulfenyl acid is rapidly oxidized to the
cysteine sulÐnic acid [e.g. reaction (16)].
Table 1 Mechanism for the oxidation of DL-cysteine by bromate in an acidic medium
forward and reverse
rate constant(s)
reaction
reaction
(M1)
(M2)
(M3)
(M4)
(M5)
(M6)
(M7)
(M8)
(M9)
(M10)
(M11)
BrO ~ ] 2H` ] Br~ H HOBr ] HBrO
2.1; 1.0 ] 104
3
2
HBrO ] Br~ ] H` H 2HOBr
2.0 ] 106; 2.0 ] 10~5
8.9 ] 108; 110
2
HOBr ] Br~ ] H` H H O ] Br
2
2
BrO ~ ] CySH ] H` ] CySOH ] HBrO
210
3
2
HBrO ] CySH ] CySOH ] HOBr
4.0 ] 103
1.0 ] 105
5.0 ] 105
3.2 ] 103
4.5 ] 106
1.2 ] 104
1.0 ] 103
2
HOBr ] CySH ] CySOH ] Br~ ] H`
HOBr ] CySOH ] CySO H ] Br~ ] H`
2
HOBr ] CySO H ] CySO H ] Br~ ] H`
2
3
Br ] CySH ] H O ] CySOH ] 2Br~ ] 2H`
2
2
Br ] CySOH ] H O ] CySO H ] 2Br~ ] 2H`
2
2
2
Br ] CySO H ] H O ] CySO H ] 2Br~ ] 2H`
2
2
2
3
Rate constants separated by a semi-colon denote forward and reverse rate constants. Rate constant units were deduced from the reactionÏs
molecularity, except for those that involve water, where the water was not considered.
J. Chem. Soc., Faraday T rans., 1998, V ol. 94
1977

View Article Online
1
1
1
1
1
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1
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T he Sadtler Standard Spectra, Nuclear Magnetic Resonance
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Fig. 9 Computer simulations using the model shown in Table 1. The
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0
[
BrO ~] \ 0.005 M, [H`] \ 0.70 M. This simple model was able to
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1
We would like to thank the University of the North for giving
leave of absence to J.D. This work was supported by the
National Science Foundation grant Number CHE-9632592
awarded to R.H.S. We also acknowledge several useful dis-
cussions with Prof. Gannett.
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