Small molecular, macromolecular, and cellular chloramines react with thiocyanate to give the human defense factor hypothiocyanite

Article abstract of DOI:10.1021/bi902089w

Thiocyanate reacts noncatalytically with myeloperoxidase-derived HOCl to produce hypothiocyanite (OSCN^-), thereby potentially limiting the propensity of HOCl to inflict host tissue damage that can lead to inflammatory diseases. However, the efficiency with which SCN^- captures HOCl in vivo depends on the concentration of SCN^- relative to other chemical targets. In blood plasma, where the concentration of SCN^- is relatively low, proteins may be the principal initial targets of HOCl, and chloramines are a significant product. Chloramines eventually decompose to irreversibly damage proteins. In the present study, we demonstrate that SCN ^- reacts efficiently with chloramines in small molecules, in proteins, and in Escherichia coli cells to give OSCN^- and the parent amine. Remarkably, OSCN^- reacts faster than SCN^- with chloramines. These reactions of SCN^- and OSCN^- with chloramines may repair some of the damage that is inflicted on protein amines by HOCl. Our observations are further evidence for the importance of secondary reactions during the redox cascades that are associated with oxidative stress by hypohalous acids.

Full text of DOI:10.1021/bi902089w

2068 Biochemistry 2010, 49, 2068–2074
DOI: 10.1021/bi902089w
Small Molecular, Macromolecular, and Cellular Chloramines React with Thiocyanate To
Give the Human Defense Factor Hypothiocyanite†
Bheki A. Xulu and Michael T. Ashby*
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019
Received December 6, 2009; Revised Manuscript Received January 19, 2010
ABSTRACT: Thiocyanate reacts noncatalytically with myeloperoxidase-derived HOCl to produce hypothio-
cyanite (OSCN-), thereby potentially limiting the propensity of HOCl to inflict host tissue damage that can
lead to inflammatory diseases. However, the efficiency with which SCN- captures HOCl in vivo depends on
the concentration of SCN- relative to other chemical targets. In blood plasma, where the concentration of
SCN- is relatively low, proteins may be the principal initial targets of HOCl, and chloramines are a significant
product. Chloramines eventually decompose to irreversibly damage proteins. In the present study, we
demonstrate that SCN- reacts efficiently with chloramines in small molecules, in proteins, and in Escherichia
coli cells to give OSCN- and the parent amine. Remarkably, OSCN- reacts faster than SCN- with
chloramines. These reactions of SCN- and OSCN- with chloramines may repair some of the damage that
is inflicted on protein amines by HOCl. Our observations are further evidence for the importance of secondary
reactions during the redox cascades that are associated with oxidative stress by hypohalous acids.
Organismic oxidative stress involves complex redox cascades
that inflict widespread damage to cells and tissue constituents (1).
Hypohalites (including OCl-, OBr-, and OSCN-), in particular,
are implicated in human health and disease (2-6). Hypohalites
are produced by the oxidation of halides (or a pseudohalide, as in
the case of SCN-) with hydrogen peroxide in reactions that are
catalyzed by the human peroxidases, including lactoperoxidase
(LPO), salivary peroxidase, myeloperoxidase, and eosinophil
peroxidase (7), primarily for the purpose of combating host
infection:
physiologic fluids depends upon the source of the fluid and any
smoking habits of the individual. We have suggested that eq 2
dominates in the oral cavity (where [SCN-] is millimolar) (14),
but a recent study of conditions similar to those of human plasma
(where [SCN-] is micromolar) suggests that HOCl would pre-
ferentially react with proteins (15). The protein chloramines, that
are a significant initial product of HOCl-mediated protein
oxidation, eventually undergo further reactions, including hydro-
lysis to carbonyls, one-electron reductions to N-centered radicals,
and chlorine transfer reactions (including some that are directed
toward protein residues by the protein tertiary structure) (16). In
the present study, we demonstrate that chloramines also react
with SCN- to give OSCN- (R = small molecule, protein, or
cellular component; Figure 1):
X- þ H2O2 peroxidase OX- þ H2O ðX ¼ Cl, Br, SCNÞ ð1Þ
f
The relative oxidation potentials of the halides are Cl- > Br->
SCN-. Thus, SCN- may serve as a scavenging agent in vivo for
the more powerful oxidants hypochlorous acid (X = Cl) (8) and
hypobromous acid (X = Br) (9) in reactions that also yield
OSCN- (10):
k1
f
RNHCl þ SCN- þ H3Oþ
s
RNH þ þ OSCN- þ Cl- ð3Þ
3
Thus, SCN- is capable of repairing some of the damage to
proteins that is caused by HOCl but at a cost of producing
potentially injurious OSCN- (17, 18).
HOX þ SCN- f OSCN- þ X- þ Hþ
ð2Þ
These and related noncatalytic reactions, such as the reaction of
HOX with the antioxidant glutathione (11-13), are believed to
restrict the lifetimes of HOX and thereby limit their propensities
to inflict host tissue damage that can lead to inflammatory
diseases (2-6).
The second-order rate constants for the reactions of HOX with
SCN- are very large (107 and 109 M-1 s-1 for X = Cl and Br,
respectively) (8, 9), but the efficiency with which SCN- captures
HOX (X = Cl and Br) in vivo depends upon its concentration
relative to other chemical targets. The [SCN-] in various human
EXPERIMENTAL PROCEDURES
Materials. All chemicals were ACS certified grade or better
and were used as received from Sigma-Aldrich. Water was
doubly distilled in glass. Unless otherwise stated, all solutions
were prepared using 0.1 M phosphate buffer at pH 7.4 (I = 1.0).
The buffer was prepared using NaH2PO4 H2O and Na2HPO4.
3
The ionic strength was adjusted with NaCl. Stock solutions of
NaOCl were prepared by sparging Cl2 into a 0.3 M solution of
NaOH. The sparging was stopped when the [OCl-] achieved
approximately 100 mM (pH 12), as determined spectrophotome-
trically (ε(OCl-)292nm = 350 M-1 cm-1).
Instrumental Measurements and Data Analysis. UV-vis
spectra were collected using an Ocean Optics UBC2000 CCD
spectrometer equipped with a 1 m WPI fiber optic cell and an
†This work was funded by the National Science Foundation (CHE-
0911328) and the National Institutes of Health (1 R21 DE016889-
01A2).
*To whom correspondence should be addressed. Tel: 405-325-2924.
Fax: 405-325-6111. E-mail: MAshby@ou.edu.
r
pubs.acs.org/Biochemistry
Published on Web 01/19/2010
2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 9, 2010 2069
resulting chloramines (Ub*Cl) were quantified with 5-thio-2-
nitrobenzoic acid (TNB, λmax = 412 nm, ε412nm = 14150 M-1
cm-1). Ub*Cl was incubated with TNB for 3 min before the
UV-vis measurements were made. Based on the TNB analysis,
33-41% (n = 3) of the original HOCl oxidizing equivalents were
recovered from Ub*Cl.
Reaction of Ub*Cl with SCN- and Quantification of
OSCN-. Equal volumes (5 mL) of 6.8 μM Ub*Cl with 0.50 mM
SCN- were mixed. After 20 min, the Ub*Cl/SCN- mixture was
reacted with TNB (59 μM) in a stopped-flow spectrophotometer
with 1:1 mixing in single-mixing mode. The rate constant
observed ((3.73 ( 0.01) ꢀ 105 M-1 s-1) is consistent with the
value that was independently measured for the reaction of TNB
with authentic samples of OSCN-. The absorption change
(ΔAbs) was constant with the recovery of 80% of the redox
equivalents that were measured for Ub*Cl using TNB.
F
IGURE 1: (A) Reaction of taurine chloramine (TauCl) with excess
SCN- gives a quantitative yield of taurine (Tau) and OSCN-. (B)
Reaction of ubiquitin (Ub) with HOCl and subsequent reaction with
SCN- gives OSCN-. (C) Chlorination of Escherichia coli (MG1655)
cells with HOCl, pelleting by centrifugation, suspension in phosphate
buffer (to give bacterial cells with “chlorine cover”), reaction of the
cells with SCN-, and filter sterilization through a 0.2 μM polyamide
filter yields OSCN-.
Chlorination of E. coli and Quantification of Oxidizing
Equivalents. Cultures of E. coli (MG1655) were grown from
frozen stocks to their terminal density in 15 h at 37 °C in
Luria-Bertani medium using a shaking water bath. A portion
of the culture (40 mL) was centrifuged (10 min at 5000g and 5 °C)
and washed twice with 0.1 M phosphate buffer (2 ꢀ 4 mL), and
the resulting cell pellet was resuspended in phosphate buffer
(4 mL) to give a cell density of ca. 109 cells/mL (OD600 = 0.76).
The cell suspension was treated with OCl- for a period of time
(Table 1). After incubation, the chlorinated cells were centrifuged
(10 min at 5000g and 5 °C), the supernatant was removed, and the
cells were resuspended in 0.1 M phosphate buffer (4 mL). This
washing procedure was repeated three times. Following the final
resuspension (107 cells/mL), the oxidizing equivalents (i.e.,
“chlorine cover”) were determined by treating the chlorinated
cells with TNB in 0.10 M phosphate buffer for 12 min (Table1).
Reaction of Chlorinated MG1655 with SCN- and Quan-
tification of OSCN-. Equal volumes (5 mL) of chlorinated E.
coli (107 cells/mL, with an experimentally determined number of
two-electron oxidizing equivalents, vide supra) and excess SCN-
were mixed. After 12 min, the cell suspension was filtered through
a 0.2 μm polyamide filter. The supernatant was reacted with an
excess of TNB in a stopped-flow spectrophotometer with 1:1
mixing in single-mixing mode. Quantification of OSCN- was
computed from ΔAbs at 412 nm within the time frame of reaction
of OSCN-, as explained in the Results and Discussion, Table 1,
and Figure 5.
ATS D 1000 CE UV light source. pH measurements were made
with an Orion Ion Analyzer EA920 using a Ag/AgCl combina-
tion pH electrode. Kinetic measurements were made using a Bio-
Logic SFM-400/Q mixer and a MOS-450 spectrophotometer
equipped with a Xe arc lamp and a PMT detector. Single-mixing
mode was used for all of the stopped-flow experiments. The
pseudo-second-order rate constants were obtained by nonlinear
least-squares fits of the data with KaleidaGraph 3.6 (Synergy
Software) or by Euler’s method using Mathematica 7.0 code (for
Figure 3), available in the Supporting Information. All stopped-
flow kinetic traces represent the average of at least nine mixing
cycles.
Preparation of TauCl. A 2.5 mM stock solution of TauCl
solution, free of dichloramine (TauCl2) and excess taurine (Tau),
was prepared by adding 5.0 mM OCl- in 0.10 M NaOH dropwise
to 5.0 mM Tau in 0.10 M NaOH while vortexing (19). The
formation of TauCl was confirmed by observation of the
characteristic absorbance spectrum (ε252nm = 429 M-1 cm-1).
Solutions of TauCl in 0.10 M phosphate buffer were prepared
from the stock solution by adjusting the pH to 7.4 using a 10 mM
solution of HCl.
Reaction of TauCl with SCN- and Quantification of
OSCN-. Solutions of OSCN- for Figure 2 were prepared by
addition of 5 mL of TauCl (100 μM) to 5 mL of SCN- (100, 200,
and 500 μM) while vortexing over a period of ∼1 min. The
samples were transferred to 10 mL plastic syringes, the solutions
were injected into the 1 m fiber optic cell, and the UV-vis spectra
were recorded.
RESULTS AND DISCUSSION
Preparation and Quantification of Ubiquitin Chlora-
mines (Ub*Cl). Quantitative oxidation of ubiquitin (Ub)
Met-1 to a sulfone (Ub*) was accomplished with performic acid
using a literature procedure (20). The performic acid was
prepared by mixing 0.50 mL of 30% hydrogen peroxide with
9.5 mL of 99% formic acid (HCOOH) and allowing the mixture
to stand for 2 h at room temperature. Before use, this freshly
prepared performic acid (10 mL) was precooled to 0 °C (ca. 1 mL
of methanol was added to the reagent to prevent freezing). Ub
(3.0 mg) was dissolved in the performic acid (3.0 mL), the mixture
was allowed to stand for 4 h at 20 °C, and cold HBr (2.0 mL of
48%) was added to destroy the excess performic acid. The
volatiles were removed using a rotary evaporator at 37 °C, and
the residue was redissolved in 0.10 M phosphate buffer at pH 7.4.
Oxidized ubiquitin (25 μM Ub*, as determined by the absorption
at 280 nm) was treated with OCl- (250 μM) for various amounts
of time (0-2 h) in 0.1 M phosphate buffer at pH 7.4. The
Reaction of Small Molecular Chloramines with Thio-
cyanate. The mechanisms of nucleophilic reactions of taurine
chloramine (TauCl) have been investigated recently by Calvo
et al. (21). Thiocyanate was included in that study, but the initial
SCN--derived product of the reaction was not identified (it was
speculated to be ClSCN or (SCN)2). In this study, we show that
the mechanism is more complicated than previously thought. The
second-order rate constant for the reaction of protonated TauCl
(TauClHþ) with SCN- was determined by Calvo et al. to be close
to the diffusion limit (1.9 ꢀ 109 M-1 s-1) (21). However, the pKa
of TauClHþ is approximately 0 (22), so the reaction of TauCl
with SCN- is relatively slow at physiologic pH (128.6 ( 0.1
M-1 s-1 at pH 7.4). Nonetheless, the rate of reaction of SCN-
with chloramines is comparable to the best biological nucleo-
philes (e.g., thiolates and thioethers) (21, 23).
On the basis of our earlier studies of the reactions of electro-
philic halogenating agents with SCN- (8, 9), we suspected that

2070 Biochemistry, Vol. 49, No. 9, 2010
Xulu and Ashby
Table 1: Experimental Conditions for Reacting E. coli Cells (109 Cells/mL) with Hypochlorous Acid, Oxidation Equivalents Recovered by the TNB Analysis,
and Amount of Hypothiocyanite Produced upon Reaction of the Chlorinated Cells (107 Cells/mL) with Thiocyanate
[HOCl]0 (μM)
time of exposure (h)
oxidizing equiv recovered (μM, %)a
OSCN- produced (μM, %)a
200
350
0.5
0.5
0.5
5.0
66 (33)b
120 (34)b
194 (39)b
423 (42)c
66 (100)d,e
ND
500
ND
300 (71)d,f
1000
aCorrected for a 100-fold dilution of the cells before their reaction with TNB or SCN-. b[TNB]0 = 74 μM. c[TNB]0 = 61 μM. dPercentage based upon the
two-electron oxidizing equivalents recovered by the TNB assay. e[SCN-]0 = 180 μM. f[SCN-]0 = 2.5 mM.
F
IGURE 2: UV-vis spectra obtained for the reactions of TauCl (50
μM) with SCN- (50-250 μM) in phosphate buffer (100 mM, pH 7.4)
at 20 °C. The spectra were recorded with a 1 m fiber optic cell. The
chemical yield of OSCN- is indicated versus [SCN-]0.
F
IGURE 3: Chemical yield of OSCN- for [SCN] = 10 mM and
[TauCl]0 = 0.25, 0.50, 1.0, and 2.0 mM in phosphate buffer (100
mM, pH 7.4) at 20 °C.
[TauCl]0 is varied from 250 μM to 2 mM (Figure 3). A similar
decrease in yield of OSCN- is observed if [TauCl]0 is kept
constant and the [SCN-] is decreased (data not shown). These
observations indicate that OSCN- is also capable of reacting
with TauCl:
the product of the reaction of TauCl with SCN- was in fact
OSCN-. Employing a unique UV-visible spectroscopic signa-
ture for OSCN- (λmax = 376 nm, ε = 26.5 M-1 cm-1) (10), we
found that the reaction of TauCl with SCN- does indeed produce
OSCN- (Figure 2). Because of the small molar extinction
coefficient, in order to employ physiologically relevant concen-
trations of the reactants, it is necessary to use a spectrophoto-
metric cell with a long (1 m) path length. The data of Figure 2 are
for [TauCl]0 = 50 μM and 50 e [SCN]0 e 250 μM, with chemical
yields of OSCN- of 43-100%. The concentration of oxidant
(TauCl) that was used is within the range that might be expected
for an immune response (although the oxidant flux, not an
absolute concentration, is more relevant in vivo). The range of
[SCN-] that was used is comparable to the normal reference
values in human physiological fluids (33.5 ( 25.4 and 111.2 (
92.1 μM in plasma and 542 ( 406 and 1655 ( 841 μM in saliva,
for smokers and nonsmokers, respectively) (24). Results similar
to those of Figure 2 were also obtained for [TauCl]0 = 80 μM and
0.5 e [SCN]0 e 5 mM, with chemical yields of OSCN- of
27-100% (Supporting Information Figure S1).
k2
f
RNHCl þ OSCN- þ H2O
s
RNH þ þ O2SCN- þ Cl- ð4Þ
3
The simulation of Figure 3 (see the Supporting Information)
yields an estimate of k2 ≈ 5 ꢀ 103 M-1 s-1 for eq 4 at neutral pH,
about 50 times larger than the rate constant for SCN- (eq 3, k1 =
128.6 ( 0.1 M-1 s-1 at pH 7.4). To further test this model, we
briefly investigated the reaction of TauCl with OSCN-. Un-
fortunately, using existing methods, OSCN- is always prepared
in the presence of excess SCN-, a competing reductant for
TauCl. Of the methods available to prepare OSCN-, we have
found the LPO-catalyzed oxidation of SCN- by H2O2 to be the
most effective means of preparing high concentrations of OSCN-
that are relatively free of excess SCN- and overoxidation
products (10). The LPO-catalyzed oxidation of 5.0 mM SCN-
by 5.0 mM H2O2 produces 3.5 mM OSCN-. Unreacted SCN-
and H2O2 remain after catalysis, and the latter can be destroyed
by catalase if desired; however, for the present study, because we
know that the uncatalyzed reaction of H2O2 with SCN- is very
slow (26, 27), and because the effects of catalase on the TauCl (28)
and on other components of the reaction (vide infra) are
unknown, we elected not to add catalase to our stock solution.
Figure 4 illustrates the reaction of equimolar concentrations of
TauCl and OSCN- (mixed second-order conditions). The reac-
tion was monitored at two wavelengths, 252 nm (filled circles,
λmax for TauCl, ε252nm = 429 M-1 cm-1) and 376 nm (open
circles, λmax for OSCN-, ε376nm = 26.5 M-1 cm-1). The observed
absorption change at 252 nm (ΔAbs(obs) = 0.38 AU) was
We have previously observed (10, 25) that excess SCN- is
required to cleanly produce OSCN- using other electrophilic
halogenating agents (e.g., HOCl (8) and HOBr (9)), because
OSCN- reacts with the oxidants at competitive rates to produce
“overoxidized” products, vide infra. Within the chloramine
concentration range we investigated ([TauCl]0 = 50-80 μM),
a 5-10-fold molar excess of SCN- is required to cleanly produce
OSCN- (Figure 2 and Supporting Information Figure S1). In
addition, the amount of excess SCN- that is required to stoichio-
metrically produce OSCN- depends inversely upon the
[chloramine]0. For example, when [SCN-] = 10 mM, the che-
mical yield of OSCN- drops from 53% to 13%, when the

Article
Biochemistry, Vol. 49, No. 9, 2010 2071
chemical yields, we suspect that the rate laws of alternative
mechanisms may also explain the available data. Equation 5
describes the stoichiometry of a limiting oxidation of SCN- by an
excess of an electrophilic halogenating agent (e.g., where Xþ is the
Clþ of HOCl or TauCl):
2 -
4Xþ þ SCN- þ 5H2O f 4X- þ SO4 þ OCN- þ 10Hþ
ð5Þ
Xþ is incapable of further oxidizing SO4 or OCN-. Accord-
2-
ingly, we have at best only described the first two steps of the
redox cascade that is defined by eq 5. At least two additional two-
electron oxidation reactions and at least one hydrolysis reaction
are necessary to relate the sequence of eqs 3 and 4 to eq 5. The
observations of Figure 4 suggest the formation of transient species
that continue to react (e.g., -d(Abs252nm)/dt ¼ -d(Abs376nm)/dt,
despite the fact that [TauCl]0 and [OSCN-] were carefully
measured independently before the reaction was initiated). One
possible explanation for the data of Figure 4 is that the observed
absorption changes that are attributed to TauCl (following the
consumption of OSCN-) are actually due to a transient species
that is produced by the initial oxidation (e.g., O2SCN-) that
undergoes subsequent hydrolysis and/or disproportionation re-
actions. Alternatively, it is possible that one or more of the
intermediates of the redox cascade of eq 5 are efficient scavengers
of chloramines and/or OSCN-. For the reaction conditions of
Figure 4, such scavenging reactions could produce a nonstoichio-
metric ratio of the original reactants (leaving behind excess TauCl
or OSCN-). For example, SO32-, which has previously been
observed during the decomposition of OSCN- (32) and which is
known to react efficiently with chloramines, is a possible inter-
mediate in the redox cascade of eq 5 (33-36). Furthermore, we
have recently shown that cyanide, another product observed
during the decomposition of OSCN-, reacts with OSCN- to
give reactive dicyanosulfide, NCSCN (which is itself an unstable
species) (37). Accordingly, a more detailed mechanistic study of
the reaction of eq 4 must await a better understanding of the
overall redox cascade that is defined by the net reaction of eq 5.
Reaction of Protein Chloramines with Thiocyanate. The
low molar absorptivity of the distinguishing UV-vis spectro-
scopic signature of OSCN- (Figure 2) precludes using electronic
spectroscopy to identify the products of the reactions of protein-
derived and cell-derived chloramines with SCN-. To overcome
this challenge, we took advantage of the selectivity of OSCN- for
thiols to develop a “kinetic signature” for the presence of
F
IGURE 4: Concentrations determined at 252 nm (solid circles, λmax
forTauCl) and 376nm(opencircles, λmax for OSCN-) versustimefor
the reaction of [TauCl]0 = 0.74 mM and [OSCN-]0 = 0.74 mM in
phosphate buffer (100 mM, pH 7.4) at 20 °C. The OSCN- was
prepared by the LPO-catalyzed oxidation of 5 mM SCN- by 5 mM
H2O2 to give a 3.5 mM stock solution of OSCN- (determined
spectrophotometrically). The solid lines are imperfect nonlinear
least-squares fit of the experimental data (for clarity, 1% and 10%
shown for TauCl and OSCN-, respectively) using a mixed second-
order rate equation (k = (1.182 ( 0.002) ꢀ 103 and (2.84 ( 0.03) ꢀ
103 M-1 s-1 at 276 and 376 nm, respectively). The dashed line is the
expected absorption change for a second-order absorption decay for
k = 128.6 ( 0.1 M-1 s-1 (the independently measured rate constant
for the reaction of TauCl and SCN- under the same reaction
conditions).
comparable to the change expected for the consumption of
0.74 mM TauCl (ΔAbs(calc) = 0.32 AU). The observed absorp-
tion change at 376 nm (ΔAbs(obs) = 0.008 AU) was about half
that expected for the consumption of 0.74 mM OSCN-
(ΔAbs(calc) = 0.019 AU). Since the [OSCN-]0 was confirmed
spectrophotometrically before the reaction was initiated, we
attribute the shortfall in absorbance to the concomitant absorp-
tion increase of one or more of the products. For the purpose of
fitting the data to a mixed second-order rate equation (Figure 4,
solid lines), the absorption changes were assumed to be due to the
consumption of 0.74 mM of TauCl and OSCN-, respectively.
For comparison, the expected kinetic trace for the reaction of
0.74 mM of TauCl with 0.74 mM SCN- is also illustrated
(Figure 4, dashed line). Given the order-of-magnitude difference
in absorptivities of TauCl and OSCN-, the data at 252 nm are
more precise than the data at 376 nm. Nonetheless, three
observations are apparent: (1) the observed reactions are much
faster than the rate expected for a reaction of TauCl with
SCN-, (2) the rate that is associated with -d[OSCN-]/dt (2.8 ꢀ
103 M-1 s-1) is faster than -d[TauCl]/dt (1.2 ꢀ 103 M-1 s-1), and
(3) the mixed second-order model incompletely describes the
observed kinetics. As evidenced by the second observation, TauCl
appears to continue to react after the OSCN- has been consumed.
The computed second-order rate constants are comparable to that
estimated from Figure 3 (within factors of 2 and 5, respectively),
but for reasons discussed next, we are not confident that the
available kinetic data define the rate constant or the mechanism.
The overoxidized product(s) of OSCN-, depicted as cyano-
sulfite (O2SCN-) in eq 4, remain(s) uncharacterized for the
present reaction conditions (although O2SCN- has been pre-
viously characterized in nonaqueous medium) (29-31). While the
relatively simple sequential reaction sequence of eqs 3 and 4
adequately explains the trends we observe for the kinetics and the
OSCN-. Employing 5-thio-2-nitrobenzoic acid (TNB, λmax
=
412 nm, ε = 14150 M-1 cm-1) as a colorimetric reagent and by
monitoring the kinetics of its oxidation to the disulfide, we are
able to readily identify and quantify the oxidant (Figure 5). This
assay was vetted using OSCN- produced by an enzyme system
(LPO þ SCN- þ H2O2) (10), and by using the reaction of TauCl
with SCN-, with independent spectroscopic confirmation of the
intermediary OSCN- (cf. Figure 2 and Supporting Information
Figures S4 and S5) prior to the subsequent quantification using
the TNB kinetic method.
Ubiquitin (Ub), a highly conserved 8.5 kDa eukaryotic
regulatory protein, was selected for our investigation of protein
chloramines. The expected order of reaction of Ub residues
(number in parentheses) with HOCl is Met (1) . Lys (7) >
Arg (4) > His (1) > Tyr (1) (38). For our studies, the N-terminal
Met of Ub was oxidized up to a sulfone (hereafter Ub*) via a
procedure that is known not to affect the structure of Ub (39).

2072 Biochemistry, Vol. 49, No. 9, 2010
Xulu and Ashby
modification of the cells that leaves them with redox-active
functionality) (44). Chlorine cover is believed to be the first step
in the bactericidal action of active chlorine biocides (e.g.,
chlorine, hypochlorite, chloramines). By analogy to the reaction
of HOCl with proteins, the modification of cells with HOCl
presumably involves the formation of chloramines. To distin-
guish between cell-associated functionalization and medium-
derived chemical species, we employed the following protocol:
reaction of 109 cells/mL with HOCl for a period of time (Table 1),
pelleting by centrifugation and washing with phosphate buffer,
reaction of the cells with excess SCN- for 12 min (Table 1), and
filter sterilization through a 0.2 μm polyamide filter. Using this
procedure, but without the addition of SCN-, we were able to
recover 33-42% of the two-electron oxidizing equivalents (based
upon the original amount of HOCl) upon reaction of the cells
with a TNB assay (Table 1 and Supporting Information Figure
S8). The assay presumably involves the direct reaction of TNB
with cellular chloramines. When SCN- was included in the assay,
we recovered 71-100% as OSCN- from the supernatant (based
upon the two-electron oxidizing equivalents present, 33-42% of
the original amount of HOCl) (Table 1 and Supporting Informa-
tion Figure S9). The latter assay presumably involves the reaction
of SCN- with cellular chloramines to give OSCN-, which is in
turn quantified by the TNB.
F
IGURE 5: Kinetic trace for the pseudo-first-order reaction of
OSCN- (4 μM, produced by the LPO-catalyzed oxidation of SCN-
by H2O2) with TNB (56 μM) in phosphate buffer at pH 7.4 and I =
1.0 (for clarity, 20% of the data are illustrated). A nonlinear least-
squares simulation (shown) yields k = (5.00 ( 0.01) ꢀ 105 M-1 s-1
.
For comparison, simulated traces for the reactions of HOCl (dashed
line, (1.9 ( 0.1) ꢀ 109 M-1 s-1) and TauCl (dot-dashed line, (1.38 (
0.08) ꢀ 104 M-1 s-1) with TNB are also illustrated (dashed line).
Note that the reaction of HOCl and TNB occurs during stopped-flow
mixing, but a subsequent reaction occurs that involves an unidenti-
fied intermediate. The rate constants for OSCN- derived from the
reactions of TauCl, Ub*Cl, and chlorinated E. coli with SCN-
were (4.44 ( 0.01) ꢀ 105, (3.73 ( 0.01) ꢀ 105, and (9.1 ( 0.1) ꢀ
105 M-1 s-1, respectively.
Biological Significance of the Reduction of Chloramines
by SCN- and OSCN-. Employing experimental rate constants
measured for model compounds and concentration estimates,
Pattison et al. have recently employed a mathematical model to
predict the targets of HOCl in human blood plasma and to
predict the fates of some of the oxidized reactive intermedi-
ates (15). Their model provides a suitable starting point for
discussing the possible biological significance of the reactions of
chloramines with SCN- and OSCN-. Proteins, free amino acids,
lipids, antioxidants, DNA bases, and other potential targets
(including SCN-) were treated as separate classes of biomole-
cules in the model. It was predicted that HOCl reacts principally
with proteins, free amino acids, antioxidants, and SCN-, with
SCN- being the major individual target (2-8%, depending upon
whether the plasma contained 50 or 175 μM SCN-), but with
proteins as a class consuming most of the HOCl (∼94%). The
computational model predicted that about 5% of the HOCl
would produce chloramines, with >80% of the chloramines
associated with plasma proteins and with the remainder asso-
ciated with small molecules. The model employed by Pattison
et al. was an incomplete one, and in particular it employed
experimental rate constants for small molecules, not proteins,
despite the fact that proteins were a major target of HOCl. Some
effort was made to compensate for the anticipated reactivity
differences in proteins (e.g., by arbitrarily decreasing the rate
constant for protein Met by a factor of 10 relative to free Met),
but no effort was made to distinguish between reactions that
involve two small molecules, between reactions that involve a
small molecule and a protein, or between reactions that involve
two proteins. It is reasonable to expect that transhalogenation
(and other two-electron redox processes) between two proteins
are kinetically disfavored, relative to the reaction pathways that
involve two small molecules and small molecules with proteins.
The redox cascade that begins with HOCl produces both
chemically inert derivatives (e.g., chlorotyrosine and methionine
sulfoxide) and biological reactive intermediates. Chloramines are
a major fraction of the reactive secondary derivatives. The
present study suggests SCN- may be particularly effective at
While we have obtained similar results for the native Ub (data not
shown), employing Ub* circumvents the highly competitive
reaction of chloramines with Met-1 (which we have recently
shown produces a reactive dehydromethionine derivative) (40).
Thus, Ub* was reacted with HOCl at pH 7.4 for various amounts
of time, and the number of oxidizing equivalents that were
incorporated into the protein (Ub*Cl) were subsequently quan-
tified with TNB. We note that Ub*Cl reacts with TNB with
apparent biphasic kinetics (Supporting Information Figure S6).
The pseudo-second-order rate constants of ca. 103 and 104
M-1 s-1 are comparable to the value of (1.38 ( 0.08) ꢀ 104
M-1 s-1 that is observed for the reaction of TauCl with TNB
under the same reaction conditions (Supporting Information
Figure S3), but these rate constants indicate that chloramines of
different reactivities are produced in the protein. If insufficient
time is allowed for the reaction of Ub* and HOCl, unreacted
HOCl remains (which was quantified by its kinetic signature; cf.
Figure 5). Once the HOCl is consumed, further delay results in
decomposition of the protein chloramines. Once optimized,
between 33% and 41% of the oxidizing equivalents in Ub*Cl
(based upon the amount of HOCl) are recovered by its reactions
with TNB. The percent recovery we observe is comparable to that
reported by Hawkins and Davies for the reaction of plasma
proteins with HOCl (41). It is also similar to the maximum of
45% chloramine Pattison et al. recovered from the reaction of
lysozyme with HOCl (16). When Ub*Cl is reacted with excess
SCN- and subsequently reacted with TNB, we observe a rate
constant ((3.73 ( 0.01) ꢀ 105 M-1 s-1) that is comparable to that
independently measured for the reaction of TNB with authentic
OSCN- (Figure 5) (42, 43). In addition, the ΔAbs is what is
expected for 80% yield of OSCN- from the Ub*Cl (based upon
the amount of chloramines present).
Reaction of Cellular Chloramines with Thiocyanate. To
investigate whether the reaction of eq 3 is observed in cells,
we treated E. coli (MG1655) cells suspended in phosphate
buffer with HOCl to produce a “chlorine cover” (an ill-defined

Article
Biochemistry, Vol. 49, No. 9, 2010 2073
scavenging chloramines in protein. The rate constant for the
reaction of SCN- with chloramines (129 M-1 s-1 at pH 7.4) is
comparable to the rate constants for the best proteinaceous
nucleophiles Cys (200-900 M-1 s-1) and Met (40-300
3. Mainnemare, A., Megarbane, B., Soueidan, A., Daniel, A., and
Chapple, I. L. C. (2004) Hypochlorous Acid and Taurine-N-
Monochloramine in Periodontal Diseases. J. Dent. Res. 83, 823–831.
4. Venglarik, C. J., Giron-Calle, J., Wigley, A. F., Malle, E., Watanabe,
N., and Forman, H. J. (2003) Hypochlorous Acid Alters Bronchial
Epithelial Cell Membrane Properties and Prevention by Extracellular
Glutathione. J. Appl. Physiol. 95, 2444–2452.
M
-1 s-1) (45). While intraprotein redox reactions are undoubt-
edly favorable when the protein-derived reaction partners are in
close proximity, it is likely that protein chloramines that are
reactively isolated from other protein-derived reaction partners
will instead find kinetically competent reactions with small
molecules. Since small molecular derivatives of Cys and Met
are relatively scarce in plasma (10-20 μM) (15) and they are
subject to depletion in the presence of HOCl, we suspect that
SCN- may be the major small molecule reductant for protein
chloramines in vivo. However, SCN- is also expected to be a
major target of HOCl (15). Without thiol reaction partners,
OSCN- may accumulate under conditions of oxidative stress.
Further contributing to an expected surplus of OSCN- is the fact
that SCN- is a major substrate of the myeloperoxidase system
for most physiological fluids (ca. eqimolar amounts of HOCl
and OSCN- are produced for [Cl-] = 100 mM and [SCN-] =
100 μM) (46, 47). An unexpected finding of the present study is
the observation that OSCN- is more reactive toward chlora-
mines than SCN-. Thus, one fate of OSCN- that is produced
under conditions of HOCl-induced oxidative stress may be as a
reductant of chloramines.
5. Kruidenier, L., Kuiper, I., Lamers, C. B. H. W., and Verspaget, H. W.
(2003) Intestinal Oxidative Damage in Inflammatory Bowel Disease:
Semi-Quantification, Localization, and Association with Mucosal
Antioxidants. J. Pathol. 201, 28–36.
6. Krasowska, A., and Konat, G. W. (2004) Vulnerability of Brain
Tissue to Inflammatory Oxidant, Hypochlorous Acid. Brain Res. 997,
176–184.
7. Davies, M. J., Hawkins, C. L., Pattison, D. I., and Rees, M. D. (2008)
Mammalian Heme Peroxidases: From Molecular Mechanisms to
Health Implications. Antioxid. Redox Signaling 10, 1199–1234.
8. Ashby, M. T., Carlson, A. C., and Scott, M. J. (2004) Redox Buffering
of Hypochlorous Acid by Thiocyanate in Physiologic Fluids. J. Am.
Chem. Soc. 126, 15976–15977.
9. Nagy, P., Beal, J. L., and Ashby, M. T. (2006) Thiocyanate Is an
Efficient Endogenous Scavenger of the Phagocytic Killing Agent
Hypobromous Acid. Chem. Res. Toxicol. 19, 587–593.
10. Nagy, P., Alguindigue, S. S., and Ashby, M. T. (2006) Lactoperox-
idase-Catalyzed Oxidation of Thiocyanate by Hydrogen Peroxide: A
Reinvestigation of Hypothiocyanite by Nuclear Magnetic Resonance
and Optical Spectroscopy. Biochemistry 45, 12610–12616.
11. Nagy, P., and Ashby, M. T. (2007) Reactive Sulfur Species: Kinetics
and Mechanisms of the Oxidation of Cysteine by Hypohalous Acid to
Give Cysteine Sulfenic Acid. J. Am. Chem. Soc. 129, 14082–14091.
12. Nagy, P., and Ashby, M. T. (2007) Kinetics and Mechanism of the
Oxidation of the Glutathione Dimer by Hypochlorous Acid and
Catalytic Reduction of the Chloroamine Product by Glutathione
Reductase. Chem. Res. Toxicol. 20, 79–87.
CONCLUSION
13. Harwood, D. T., Nimmo, S. L., Kettle, A. J., Winterbourn, C. C., and
Ashby, M. T. (2008) Molecular Structure and Dynamic Properties of
a Sulfonamide Derivative of Glutathione That Is Produced under
Conditions of Oxidative Stress by Hypochlorous Acid. Chem. Res.
Toxicol. 21, 1011–1016.
14. Ashby, M. T. (2008) Inorganic Chemistry of Defensive Peroxidases in
the Human Oral Cavity. J. Dent. Res. 87, 900–914.
15. Pattison, D. I., Hawkins, C. L., and Davies, M. J. (2009) What Are the
Plasma Targets of the Oxidant Hypochlorous Acid? A Kinetic
Modeling Approach. Chem. Res. Toxicol. 22, 807–817.
16. Pattison, D. I., Hawkins, C. L., and Davies, M. J. (2007) Hypochlor-
ous Acid-Mediated Protein Oxidation: How Important Are Chlor-
amine Transfer Reactions and Protein Tertiary Structure?
Biochemistry 46, 9853–9864.
17. Wang, Z., Nicholls, S. J., Rodriguez, E. R., Kummu, O., Hoerkkoe,
S., Barnard, J., Reynolds, W. F., Topol, E. J., DiDonato, J. A., and
Hazen, S. L. (2007) Protein Carbamylation Links Inflammation,
Smoking, Uremia and Atherogenesis. Nat. Med. 13, 1176–1184.
18. Lloyd, M. M., van Reyk, D. M., Davies, M. J., and Hawkins, C. L.
We have previously suggested that when available in sufficient
quantities SCN- might be effective at scavenging HOX (X = Cl
and Br) in vivo to produce OSCN- (8, 9). The present study
evidences the importance of reactions of SCN- with secondary
oxidants that are produced in HOCl-induced redox cascades.
Thus, when direct reaction of HOX with SCN- is inefficient, Clþ
can be subsequently scavenged by reaction of the resulting
chloramines with SCN-. One surprising result of the present
study is the observation that OSCN- reacts with chloramines
faster than SCN- does. When the chloramines are protein-
derived, their reactions with SCN- and OSCN- may repair
some of the damage that is inflicted by HOCl. We note that it has
been previously shown that the biological function of proteins
can be lost following side-chain oxidation by HOCl, and chlor-
amines mediate further oxidation reactions and protein unfold-
ing (48). The health/disease consequences of the additional
production of OSCN- from chloramines are unclear. While
HOCl and HOBr are more chemically reactive than HOSCN,
there is mounting evidence that the production of excess OSCN-
may also have deleterious human health consequences (17, 18).
(2008) Hypothiocyanous Acid Is
a More Potent Inducer of
Apoptosis and Protein Thiol Depletion in Murine Macrophage Cells
Than Hypochlorous Acid or Hypobromous Acid. Biochem. J. 414,
271–280.
19. Thomas, E. L., Grisham, M. B., and Jefferson, M. M. (1986)
Cytotoxicity of Chloramines. Methods Enzymol. 132, 569.
20. Simpson, R. J. (2003) Proteins and Proteomics: A Laboratory Man-
ual, p 370, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
21. Calvo, P., Crugeiras, J., Rios, A., and Rios, M. A. (2007) Nucleophilic
Substitution Reactions of N-Chloramines: Evidence for a Change in
Mechanism with Increasing Nucleophile Reactivity. J. Org. Chem. 72,
3171–3178.
22. Antelo, J. M., Arce, F., Calvo, P., Crugeiras, J., and Rios, A. (2000)
General Acid-Base Catalysis in the Reversible Disproportionation
Reaction of N-Chlorotaurine. J. Chem. Soc., Perkin 2, 2109–2114.
23. Peskin, A. V., and Winterbourn, C. C. (2001) Kinetics of the Reac-
tions of Hypochlorous Acid and Amino Acid Chloramines with
Thiols, Methionine, and Ascorbate. Free Radical Biol. Med. 30,
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24. Tsuge, K., Kataoka, M., and Seto, Y. (2000) Cyanide and Thiocya-
nate Levels in Blood and Saliva of Healthy Adult Volunteers.
J. Health Sci. 46, 343–350.
25. Nagy, P., Lemma, K., and Ashby, M. T. (2007) Kinetics and
Mechanism of the Comproportionation of Hypothiocyanous Acid
and Thiocyanate to Give Thiocyanogen in Acidic Aqueous Solution.
Inorg. Chem. 46, 285–292.
SUPPORTING INFORMATION AVAILABLE
Mathematica code (used to fit the data of Figure 3), Figure S1
(analogous to Figure 2, albeit for different reaction conditions),
and Figures S2-S9 (representative time-resolved spectral data
used to vet the “kinetic signature” of OSCN- and quantify the
[OSCN-] in various samples). This material is available free of
charge via the Internet at http://pubs.acs.org.
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