Formation and reactions of S-nitroso proteins

Article abstract of DOI:10.1039/b008112o

Nitrosation of Bovine Serum Albumin (BSA) by nitrous acid occurs in two stages: a rapid step, which is consistent with an S-nitrosation reaction of a free cysteine unit, followed by a slower reaction which is consistent with an N-nitrosation reaction of a tryptophan residue. The fast reaction is catalysed by chloride and bromide ions (as is the reaction of free cysteine), whereas the slower reaction is not halide ion catalysed (like the reaction of tryptophan). Kinetic results were obtained for both reactions. The derived rate constants for the first stage for the reaction of ClNO and BrNO are reasonably close to the reported values for the reactions of cysteine. The second stage is a reversible process and we can estimate from measured infinity values and also from the variation in the measured rate constant for reactions at different [HNO2], values for the equilibrium constant of 3500 +/- 200 and 2600 +/- 200 dm³ mol^-1 which compare reasonably with the reported value for the reaction of tryptophan of 850 dm³ mol^-1. The pK_a values of the cysteine residue in both BSA and Human Serum Albumin (HSA) were determined from rate measurements of the reactions with dipyridin-4-yl disulfide (4-aldrithiol), and yielded values of 8.32 and 8.18 respectively, which are close to the accepted value of 8.4 for cysteine itself, and which are substantially higher than the much quoted literature values. The S-nitroso derivatives of both BSA and HSA generated in solution (at ca. 1 x 10^-4 mol dm^-3) showed very little sign of decomposition at pH 7.4 when measured spectrophotometrically, even in the presence of added Cu²⁺. There was, however, clear evidence of rapid decomposition at much lower reactant concentration (ca. 1 x 10^-6 mol dm^-3) yielding nitric oxide in increasing amounts and rates, as Cu²⁺ is added. These results are discussed in terms of the complexation of the Cu²⁺ catalyst at different albumin derivative concentrations. Transnitrosation between the S-nitroso derivatives of both BSA and HSA and excess cysteine occurred very readily. These experiments were carried out in the presence of added Cu²⁺, and the decomposition of the resulting S-nitrosocysteine (S-NOCys) was monitored. The results support the suggestion that S-nitroso proteins can, in theory, act as a reservoir for NO which can readily be made available, either by the Cu²⁺-catalysed reaction, or more readily by a direct NO⁺ transfer to a low molecular weight thiol, such as cysteine, which is much more prone to yield NO rapidly and quantitatively by the Cu²⁺-catalysed pathway. The S-nitroso derivative of BSA also underwent a relatively slow decomposition reaction initiated by cysteine, even in the presenc...

Full text of DOI:10.1039/b008112o

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Formation and reactions of S-nitroso proteins
Darren R. Noble and D. Lyn H. Williams*
Department of Chemistry, Durham University, South Road, Durham, UK DH1 3LE.
E-mail: D.L.H.Williams@durham.ac.uk
2
Received (in Cambridge, UK) 9th October 2000, Accepted 22nd November 2000
First published as an Advance Article on the web 13th December 2000
Nitrosation of Bovine Serum Albumin (BSA) by nitrous acid occurs in two stages: a rapid step, which is consistent
with an S-nitrosation reaction of a free cysteine unit, followed by a slower reaction which is consistent with an N-
nitrosation reaction of a tryptophan residue. The fast reaction is catalysed by chloride and bromide ions (as is the
reaction of free cysteine), whereas the slower reaction is not halide ion catalysed (like the reaction of tryptophan).
Kinetic results were obtained for both reactions. The derived rate constants for the first stage for the reaction of
ClNO and BrNO are reasonably close to the reported values for the reactions of cysteine. The second stage is a
reversible process and we can estimate from measured infinity values and also from the variation in the measured rate
constant for reactions at different [HNO2], values for the equilibrium constant of 3500 200 and 2600 200 dm3
molϪ1 which compare reasonably with the reported value for the reaction of tryptophan of 850 dm3 molϪ1. The
pKa values of the cysteine residue in both BSA and Human Serum Albumin (HSA) were determined from rate
measurements of the reactions with dipyridin-4-yl disulfide (4-aldrithiol), and yielded values of 8.32 and 8.18
respectively, which are close to the accepted value of 8.4 for cysteine itself, and which are substantially higher than
the much quoted literature values. The S-nitroso derivatives of both BSA and HSA generated in solution (at
~1 × 10Ϫ4 mol dmϪ3) showed very little sign of decomposition at pH 7.4 when measured spectrophotometrically,
even in the presence of added Cu2ϩ. There was, however, clear evidence of rapid decomposition at much lower
reactant concentration (~1 × 10Ϫ6 mol dmϪ3) yielding nitric oxide in increasing amounts and rates, as Cu2ϩ is
added. These results are discussed in terms of the complexation of the Cu2ϩ catalyst at different albumin derivative
concentrations. Transnitrosation between the S-nitroso derivatives of both BSA and HSA and excess cysteine
occurred very readily. These experiments were carried out in the presence of added Cu2ϩ, and the decomposition of
the resulting S-nitrosocysteine (S-NOCys) was monitored. The results support the suggestion that S-nitroso proteins
can, in theory, act as a reservoir for NO which can readily be made available, either by the Cu2ϩ-catalysed reaction, or
more readily by a direct NOϩ transfer to a low molecular weight thiol, such as cysteine, which is much more prone to
yield NO rapidly and quantitatively by the Cu2ϩ-catalysed pathway. The S-nitroso derivative of BSA also underwent
a relatively slow decomposition reaction initiated by cysteine, even in the presence of EDTA. This is discussed in terms
of a reaction yielding nitrite anion which is not derived from NO.
The chemistry of S-nitrosothiols RSNO continues to be of
much interest within the context of the formation, storage and
transport of nitric oxide within the body. Nitric oxide itself,
synthesised in vivo from -arginine is known to be responsible
for the control of a range of physiological functions, yet its
half-life under these conditions is believed to be very short,1 too
short, it is thought, for it to be transported in the body as free
NO. There is a growing belief that transport and storage of
NO could occur if it is bonded to thiols as RSNO species2
and that NO can be obtained from them when required. There
is an alternative possibility, which has yet to be convincingly
demonstrated, that RSNO species (which show similar bio-
logical properties to NO) could directly transfer the NO group
to an active site without the necessity for free NO formation.
Whatever the answer to these questions it is important to
establish the chemistry of S-nitrosothiols under conditions
resembling as closely as possible those in vivo.3
The spontaneous thermal decomposition of S-nitrosothiols
(often confused in the biological literature with the much
faster Cu2ϩ-catalysed reaction) is negligibly slow at room tem-
perature, and the photochemical breakdown requires incident
radiation at the appropriate wavelength. In aqueous solution
RSNO compounds react at physiological pH to give nitric
oxide and the disulfide RSSR4 in a process catalysed by even
trace impurity levels of Cu2ϩ. Here the true reagent5 is Cuϩ,
generated by reduction of Cu2ϩ by low levels of thiolate ion
6
(also catalytic) always present in S-nitrosothiol solutions,
or by any other added reducing agent, e.g. ascorbic acid7
[eqns. (1)–(3)]. Reaction can be effectively completely halted by
RSNO ϩ H2O
RSH ϩ HNO2
(1)
(2)
(3)
2Cu2ϩ ϩ 2RSϪ → 2Cuϩ ϩ RSSR
RSNO ϩ Cuϩ → RSϪ ϩ Cu2ϩ ϩ NO
removal of Cu2ϩ with EDTA, or alternatively by taking steps to
reduce the thiolate concentration to trivial levels, by generating
RSNO in solution with a large excess of nitrous acid over
thiol.
Another important reaction of S-nitrosothiols in the context
of possible NO formation is that wherein the NO group is
transferred directly in the NOϩ sense to a nucleophilic centre
without generating a free NO entity. We have described a
range of such reactions to nitrogen,8 sulfur9 and oxygen7,10
nucleophilic centres. This transnitrosation to a different thiolate
group11,12 is of particular interest, since by this means a new
RЈSNO is generated [eqn. (4)] which might be much more
RSNO ϩ RЈSϪ
RSϪ ϩ RЈSNO
(4)
DOI: 10.1039/b008112o
J. Chem. Soc., Perkin Trans. 2, 2001, 13–17
This journal is © The Royal Society of Chemistry 2001
13

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susceptible to decomposition to give NO by the Cu2ϩ-catalysed
route than is RSNO itself. The equilibrium reaction can be
driven to the right with a large excess of the thiol RЈSH. It
has been shown from measurements at different pH values
that reaction occurs via the thiolate anion, and the mechanism
shows all the characteristics of a nucleophilic reaction by thio-
late anion at the nitroso nitrogen atom.
Another decomposition reaction of S-nitrosothiols occurs
at relatively high thiol concentration. This has been studied
as reactions of RSNO with RSH, i.e. with the same R group, to
avoid complications due to transnitrosation. The disulfide
is formed together with ammonia, nitrous oxide and nitrite
ion (which is not derived from nitric oxide by oxidation and
subsequent hydrolysis). This reaction is quite independent of
Cu2ϩ and takes place in the presence of metal ion chelators. The
pH–rate profile shows that the reactive entity is the thiolate
ion. Reaction pathways have been proposed that account for
the range of products, but these have not yet been conclusively
demonstrated.13,14
Fig. 1 Values of kobs as a function of pH in the reaction of BSA with
dipyridin-4-yl disulfide.
In the main, all of these reactions of RSNO compounds have
been studied and mechanisms established for simple low
molecular weight substrate S-nitroso species, such as those of
many cysteine derivatives and glutathione. In recent times a
number of S-nitroso compounds including S-nitroso proteins
have been detected in vivo15,16 and an S-nitroso protein deriva-
tive of serum albumin has been isolated and characterised.17,18
Since NO itself has a very short half-life in vivo, it has been
proposed that NO circulates in mammalian plasma mainly as
the S-nitroso derivative of serum albumin.15,19
It is clearly important to establish if S-nitroso proteins can
undergo the reactions already established for simple low
molecular weight RSNO structures. A number of observations
have already been made, e.g. that in solution at pH 7.4, S-
nitroso proteins are apparently much more resistant to break-
down to give NO than is S-nitrosocysteine itself17,20 and
a number of transnitrosation reactions have been reported21
from S-nitrosocysteine (S-NOCys) and other nitrosothiols to
the –SH group in proteins.
We chose to work, as others have done, with the proteins
bovine serum albumin (BSA) and human serum albumin
(HSA), which are reasonably plentiful in circulating plasma, are
readily available and which possess a single free –SH group as
Cys-34.22
Fig. 2 Absorbance–time plots for the nitrosation of BSA (8.8 × 10Ϫ5
mol dmϪ3 free thiol) with excess nitrous acid (1.83 × 10Ϫ4 mol dmϪ3) in
HCl (0.4 mol dmϪ3).
4-yl disulfide) as a function of the pH of the solution. This
reaction generates 4-mercaptopyridine which absorbs at 324
nm, and measurements were made at this wavelength. Our
results for BSA are shown in Fig. 1, and show the expected
sigmoid shape, if reaction occurs via the thiolate anion. Mere
inspection of the graph indicates that the pKa value of the thiol
is in the region of 8.5. A full analysis of the data using the
Scientist package27 gave a value of 8.32 0.02. A similar graph
was obtained for the reaction of HSA with 4-aldrithiol, and
analysis yielded a pKa value of 8.18 0.02. Both of these values
are quite close to the value for cysteine itself, so according to
our measurements and analysis, there is no need to invoke
unusually low pKa values for these albumins, since they behave
in a very similar fashion to free cysteine.
Results and discussion
Thiol analysis and pKa determination of BSA and HSA
Most preparations of serum albumins contain a mixture of
the one free thiol group (Cys-34) and mixed disulfides derived
from it. To establish how much free thiol is present we used the
analytical procedure using Ellman’s reagent,23 which develops
a yellow species with a maximum absorbance at 412 nm. Our
commercial samples were found to contain 57% (BSA) and
26% (HSA) free thiol respectively. These figures changed a little
with each new batch of protein, and were redetermined each
time.
The pKa values of the free thiol groups in serum albumins
have been the subject of some curiosity and uncertainty.
A review article24 on the structure of serum albumins quotes
an unusually low value of ~5 for the –SH ionisation, whilst
the source of the determination25 actually derives a value of
about 7, which is much closer to the literature value26 of 8.4 for
cysteine itself. The ‘much lower’ pKa value for the protein-
bound thiol group is much quoted in the biological literature,
although it has never been satisfactorily explained. We decided
to redetermine these values, given the uncertainty and un-
usually low values quoted. We adopted the same procedure as
previously25 of determining the rate constants of the reaction
between the albumin and a disulfide (4-aldrithiol or dipyridin-
Nitrosation of BSA and HSA
Nitrosation of both albumins was examined spectrophoto-
metrically in aqueous acidic (~0.4 mol dmϪ3 HCl) solution
using sodium nitrite. Within seconds the characteristic peaks at
345 and 545 nm were generated, indicative of an S-nitrosation
process. However when excess nitrous acid (over the concen-
tration of free thiol group) was used, an additional, much
slower reaction occurred resulting in a further increasing
absorbance in the 345 nm region, but not in the 545 nm region
of the spectrum (see Fig. 2). This does not occur when the
free thiol group is present in excess over nitrous acid. It has
already been suggested28 that the second nitrosation occurs at
the indole nitrogen atom of a tryptophan residue within the
albumin. This is borne out by these results since it has been
reported29 that N-nitrosotryptophan itself has an absorbance
peak at ~340 nm with an absorption coefficient of ~12000 dm3
molϪ1 cmϪ1, which is an order of magnitude greater than the
absorption coefficient of S-nitrosothiols at this wavelength.
Tryptophan derivatives have been much used as model com-
pounds in studies of the nitrosation of peptides and proteins.30
14
J. Chem. Soc., Perkin Trans. 2, 2001, 13–17

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Fig. 3 Catalysis by bromide ion (upper line) and chloride ion (lower
line) in the S-nitrosation of HSA in HCl (0.1 mol dmϪ3).
Fig. 4 Variation of A∞ at 340 nm with initial [HNO2] in the reaction of
BSA with excess HNO2 in HCl (0.4 mol dmϪ3).
Kinetic measurements were made on the S-nitrosation reac-
tion by measuring the increasing absorbance at 340 nm in a
stopped-flow spectrophotometer, working as far as possible
with equal concentrations of thiol function and nitrous acid.
This complicated the kinetic analysis somewhat, but good fits
to second-order behaviour were obtained using the Scientist
package. Values of kobs [eqn. (5)] were measured as a function of
Rate = kobs[Thiol] [HNO2]
(5)
added chloride and bromide ion for reactions of both BSA and
HSA. The results for HSA are shown graphically in Fig. 3,
where it is clear that there is halide ion catalysis, with BrϪ >ClϪ.
This is typical of S-31 and many other (but not all) nitrosation
reactions. Further, from the slopes of the lines in Fig. 3 we can
determine the values of the bimolecular rate constants for
reaction of ClNO and BrNO with the thiol function. These are
determined as 1.0 × 106 and 6.5 × 104 dm3 molϪ1 sϪ1 for BSA
and 1.1 × 106 and 13.2 × 104 dm3 molϪ1 sϪ1 for HSA. These
values are remarkably similar to the corresponding figures
obtained for cysteine32 of 1.2 × 106 and 5.8 × 104 dm3 molϪ1 sϪ1
respectively. There is a similar correspondence between the
reactivity of the uncatalysed process (as measured by the value
of the third-order rate constant k3 [eqn. (6)]), between S-
Fig. 5 Plots of kobs against [HNO2] in the reaction of BSA (7.7 ×
10Ϫ5 mol dmϪ3 free thiol) with HNO2 in HCl (0.4 mol dmϪ3).
[thiol], and the absorbance measurements fitted to the first-
order rate equation after allowance was made for the initial
rapid S-nitrosation step. As is clear from the plot of the rate
constants against [HNO2] (Fig. 5), there is a significant inter-
cept to the straight line plot, characteristic of reversible reac-
tions [eqn. (8)]. The ratio of the slope (forward rate constant k1)
Rate = k3[RSH][HNO2][Hϩ]
(6)
kobs = kϪ1 ϩ k1[HNO2]
(8)
nitrosation in the proteins and free cysteine, although these
values are less precisely obtained from the intercepts of the
plots in Fig. 3. These results argue strongly that we are in the
present work looking at S-nitrosation of a cysteine residue
within the albumins and that its reactivity is not significantly
modified by its bonding within the protein.
to the intercept (reverse rate constant kϪ1) gives the equilibrium
constant for the second nitrosation as 2800 200 dm3 molϪ1
,
in reasonable agreement with the earlier value. These results
are consistent with the suggestion that the second nitrosation
occurs at the indole nitrogen atom of a tryptophan residue. The
equilibrium constant for N-acetyltryptophan itself37 has been
determined as 850 dm3 molϪ1—reasonably close to our values.
We have one further piece of evidence in support of the second
nitrosation being at the tryptophan residue. The rate of the
second nitrosation step in BSA carried out in 0.1 mol dmϪ3 HCl
is totally unaffected by the presence of added sodium bromide
in the range 0–0.1 mol dmϪ3, in sharp contrast to the catalysis
noted for the faster first S-nitrosation. This is exactly what is
reported for the nitrosation of free N-acetyltryptophan29,37
at pH values <1, when it is believed that proton loss from a
nitrosated intermediate is the rate-limiting step.
In summary, we have measured the pKa value of the single
cysteine residues in both BSA and HSA and find values close
to that of cysteine itself, contradicting the widely held belief
in the biological literature that it has an unusually low value
in the protein environment. The rapid S-nitrosation of the
cysteine residue in BSA and HSA has been observed spectro-
photometrically, and we have been able to determine rate
constants for the process, which shows the normal charac-
teristics of S-nitrosation processes of thiols, including halide
ion catalysis. When nitrous acid is in excess, a second reaction
The second, slower reaction, when nitrous acid is in excess
over the thiol, gave final absorbance values (A∞) at 340 nm
which were dependent on the initial nitrous acid concentration
in such a way as to suggest that the process is a reversible
one, i.e. A∞ values increased with [HNO2] at constant [thiol].
Reversibility in nitrosation is well-known. It is very pronounced
in the nitrosation of alcohols33 and has been identified in
other cases, including the reactions of amides,34 guanidines,35
some enols,36 and N-acetyltryptophan.29 A quantitative analysis
of the variation of A∞ with [HNO2], assuming a reversible
process yields Fig. 4, where the points are the experimental
ones, and the curve is the calculated one from the reversibility
model, using the Scientist package for the fitting. This yielded a
value of 3500 200 dm3 molϪ1 for the equilibrium constant
[eqn. (7)]. Kinetic measurements also gave clear evidence of
RЈRЉNH ϩ HNO2
RЈRЉNNO ϩ H2O
(7)
reversibility for the second nitrosation step. Measurements were
carried out with a varying large excess of nitrous acid over the
J. Chem. Soc., Perkin Trans. 2, 2001, 13–17
15

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Fig. 6 Yield (%) of NO in the reaction of S-NO BSA (0.92 × 10Ϫ6 mol
dmϪ3) at pH 7.4 in the presence of (A) EDTA (1 × 10Ϫ4 mol dmϪ3),
(B) no added EDTA or Cu2ϩ, (C) added Cu2ϩ (1 × 10Ϫ5 mol dmϪ3),
(D) added Cu2ϩ (3 × 10Ϫ5 mol dmϪ3) and (E) added Cu2ϩ (5 × 10Ϫ5 mol
dmϪ3).
Fig. 7 Absorbance (at 340 nm)–time plots for the decomposition of
S-NO BSA (1.5 × 10Ϫ4 mol dmϪ3) in the presence of Cu2ϩ (5 × 10Ϫ5 mol
dmϪ3) and cysteine (Cys) in the range 2.5–15.5 × 10Ϫ3 mol dmϪ3
.
impurity in the buffer solution. When this is removed with
EDTA, no nitric oxide is formed, confirming that in these
occurs, which shows significant reversibility and no halide ion
catalysis, and which is fully consistent with an earlier suggestion
that this is an N-nitrosation process taking place at the indole
nitrogen atom of a tryptophan residue in the albumin. It is
known38 that BSA contains a single tryptophan unit—
tryptophan 214.
reactions we are observing NO formation via the Cu2ϩ
-
catalysed reaction.
It is now clear that although the nitrosoalbumin is very stable
to decomposition in solution at millimolar concentrations,
this is not the case at micromolar and lower concentrations,
which is the more likely scenario in vivo. It is known40 that
serum albumins will bind Cu2ϩ at a Gly-Gly-His peptide
sequence, so it is entirely plausible that at millimolar concen-
trations of S-NO BSA there is effectively no free Cu2ϩ to bring
about reaction. This effect will be much reduced as the S-NO
BSA concentration is reduced to micromolar levels.
Reactions of S-nitroso proteins
(a) Decomposition. The S-nitroso derivative of BSA (S-NO
BSA) generated in situ (at 1.5 × 10Ϫ4 mol dmϪ3) in acid solution
and adjusted to pH 7.4 showed very little sign of decomposition
over several hours, as measured spectrophotometrically, noting
the decreasing absorbance at 340 nm. Typically about 15%
decomposition occurred in three hours and about 25% in 14
hours. The absorbance–time traces were unaffected by added
Cu2ϩ up to 1 × 10Ϫ4 mol dmϪ3 or by the presence of EDTA at
1 × 10Ϫ4 mol dmϪ3. This shows that under these conditions
S-NO BSA is relatively stable and that any decomposition is
not via the Cu2ϩ-catalysed pathway, but rather by the thermal
decomposition route. This bears out earlier reports17,20 on the
stability of these solutions. We have shown recently39 that
whilst S-nitrosoglutathione (GSNO) is similarly very stable
in solution at millimolar concentrations, it does break down
rapidly and reasonably quantitatively to give nitric oxide at
the micromolar concentration level. The explanation for this
apparently very unusual result is that the disulfide product of
the reaction (GSSG) binds Cu2ϩ very strongly, via its glutamate
residues, effectively removing the catalyst. This is very much
a concentration-dependent effect, and at micromolar concen-
trations enough Cu2ϩ catalyst is available. We thus examined
the decomposition of S-NO BSA at much lower reactant con-
centrations using the NO probe to monitor the reaction. We
found, as we did for GSNO, that at around and below micro-
molar concentrations, nitric oxide is generated, typically over
2–3 minutes. The yield of NO increased progressively from
~10% at an initial concentration of 1.4 × 10Ϫ6 mol dmϪ3 to
~60% at 3.5 × 10Ϫ8 mol dmϪ3. These results refer to reaction
again at pH 7.4 and in the presence of added Cu2ϩ (1 × 10Ϫ5
mol dmϪ3) .We were unable to get results at lower [nitrosothiol],
because the electrode system was very noisy at these low con-
centrations. In addition, we were not able to get reliable rate
constants for NO release. We showed that this process is a
Cu2ϩ-catalysed reaction by carrying out some experiments,
again with the NO probe, on the decomposition of S-NO BSA
(~1 × 10Ϫ6 mol dmϪ3) as a function of added Cu2ϩ in the range
1–5 × 10Ϫ5 mol dmϪ3. The results are shown in Fig. 6, where it is
clear that the yield of NO increases with [Cu2ϩ] to a maximum
of 60% in this case. Even without added Cu2ϩ some 10% NO is
generated, presumably from the residual copper present as an
There is very little free Cu2ϩ in the body, but significant
amounts are bound to proteins and peptides. We have
previously shown41 that Cuϩ can be accessed from Cu2ϩ bound
in this way, so long as there is a reducing agent, such as a thiol
or ascorbic acid present in sufficient quantity. This at least
allows the possibility that NO could in principle be obtained
from S-nitroso proteins in vivo.
(b) Transnitrosation. Reactions between a nitrosothiol and
a different thiol have been described, both qualitatively and
quantitatively. Although in a few instances rate measurements
have been made,12 it is a difficult procedure experimentally since
the absorption coefficients of RSNO and RЈSNO are generally
very similar and absorbance changes are consequently very
small. We decided to use an alternative procedure of carrying
out the experiments in the presence of added Cu2ϩ and to use a
thiol RЈSH such that the new generated nitrosothiol RЈSNO is
one known to decompose rapidly to RЈSSRЈ and NO under
these conditions, and we chose cysteine (Cys) to this end. These
reactions were followed spectrophotometrically, with initial
[S-NO BSA] at 1.5 × 10Ϫ4 mol dmϪ3. The absorbance at 340 nm
decreased rapidly in the presence of an excess of Cys in a
first-order process (Fig. 7). These reactions were orders of
magnitude faster than the corresponding decompositions of S-
NO BSA (at the same concentration and [Cu2ϩ]) in the absence
of added Cys—i.e. the slow thermal reaction and slow Cu2ϩ
-
catalysed decomposition. The observed first-order rate con-
stants showed a first-order dependence upon [Cys]. Similar
results were obtained for the HSA derivative, S-NO HSA. An
almost identical series of absorbance–time traces were obtained
by generating S-nitrosocysteine (S-NOCys) in situ from
Cys and nitrous acid, and observing its decay at 340 nm as a
function of the same added excess of Cys. This demonstrates
that on the time scale of our measurements with S-NO BSA
and S-NO HSA, transnitrosation to form S-NOCys takes place
very rapidly and we are only able to measure the subsequent
Cu2ϩ-promoted decomposition of the latter [eqn. (9)]. These
results confirm an earlier suggestion20 that transnitrosation
from an S-nitrosoalbumin is a facile process.
16
J. Chem. Soc., Perkin Trans. 2, 2001, 13–17

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S-NOAlbumin ϩ Cys → S-NOCys → NO (9)
References
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Other workers21 have noted that, as would be expected from
studies on simple low molecular weight systems, S-nitroso-
albumins can be formed by transnitrosation from any RSNO
to e.g. BSA. This is clearly a quite general reaction [eqn. (4)]
for all R and RЈ groups and the final product will depend on the
relative concentrations of the reactants: the equilibrium can be
driven in either direction by the use of an excess of either RSH
or RЈSH.
So it is clear that these S-nitrosoalbumins behave very much
like the simple low molecular weight S-nitrosothiols in their
ability to transfer the NO group directly to a thiolate ion,
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6 P. H. Beloso and D. L. H. Williams, Chem. Commun., 1997, 89.
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We have looked briefly at one other possible S-nitroso pro-
tein decomposition reaction (which occurs also in simple
RSNO species) by treatment with thiols at relatively high thiol
concentration. It was found that the absorbance at 340 nm
decayed in a first-order process when S-NO BSA (1.5 × 10Ϫ4
mol dmϪ3) was allowed to react in the presence of cysteine
(1 × 10Ϫ2 mol dmϪ3) and of EDTA and varying concentrations
of BSA (0.2–4.3 × 10Ϫ4 mol dmϪ3). Under these conditions it is
expected that effectively all of the S-NO BSA will be converted
to S-NOCys, so we are looking at the Cu2ϩ-independent
decomposition of S-NOCys brought about by BSA. The
observed first-order rate constants increased linearly with
[BSA]. A similar set of traces emerged when reaction occurred
between S-NOCys (generated in situ) and BSA under the same
conditions. We detected no nitric oxide or ammonia in the
reaction products, but did find nitrite ion, although it was not
formed quantitatively. These results are consistent with a rapid
transnitrosation from S-NO BSA to form S-NOCys, which
undergoes some reaction with BSA. The same behaviour was
found for the decomposition of S-NO HSA in the presence
of excess cysteine and HSA. As a check, measurements
made using S-NO BSA and BSA in the presence of EDTA but
no cysteine showed very little decomposition. We have not
established the nature of these reactions further, but it is likely
that they follow the complex course suggested by others13,14,42
for the reaction of simple RSNO species with thiols at reason-
ably high [thiol], in which Cu2ϩ is not involved. It has been
suggested that nucleophilic attack by RSϪ occurs at the sulfur
atom of RSNO leading to a variety of products, including
nitrite ion and possibly involving intermediate formation of
HNO.
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Experimental
All materials were commercially available samples at the highest
purity grade available. S-Nitrosothiols were generated in
solution by conventional S-nitrosation of the corresponding
thiol in acid solution, and subsequent pH adjustment. Kinetic
measurements were carried out spectrophotometrically at
25 ЊC at pH 7.4, following the disappearance of the band at
340 nm due to RSNO in a conventional spectrophotometer
interfaced with a PC, or in some cases for the faster reactions,
using a stopped-flow spectrophotometer. Standard software
packages were used to obtain the rate constants. Nitric oxide
detection was carried out with a commercial WPI NO Mark 2
electrode calibrated using the reaction of ascorbic acid with
nitrous acid. Nitrite ion was determined using the Griess
analytical procedure, free thiol by the Ellman reaction and the
attempted ammonia analysis using the standard diagnostic kit
(Sigma).
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34 See ref. 31, p. 102.
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Acknowledgement
We thank the EPSRC for a Research Grant which enabled this
work to be carried out.
41 A. P. Dicks and D. L. H. Williams, Chem. Biol., 1996, 3, 655.
42 P. S.-Y. Wong, J. Hyun, J. M. Fukuto, F. N. Shirota, E. G. DeMaster,
D. W. Shoeman and H. T. Nagasawa, Biochemistry, 1998, 37, 5362.
J. Chem. Soc., Perkin Trans. 2, 2001, 13–17
17