Regiospecific Nitrosation of N-terminal-blocked Tryptophan
Derivatives by N2O3 at Physiological pH*
Michael
Kirsch
§,
Anke
Fuchs
, and
Herbert
de Groot
From the Institut für Physiologische Chemie,
Universitätsklinikum, Hufelandstrasse 55, D-45122 Essen,
Germany
Received for publication, January 9, 2003, and in revised form, January 22, 2003
 |
ABSTRACT |
N2O3 formed from
nitric oxide in the presence of oxygen attacks thiols in proteins to
yield S-nitrosothiols, which are believed to play a central
role in NO signaling. In the present study we examined the
N-nitrosation of N-terminal-blocked (N-blocked) tryptophan derivatives in the presence of N2O3 generating
systems, such as preformed nitric oxide and nitric oxide donor
compounds in the presence of oxygen at pH 7.4. Under these conditions
N-nitrosation of N-acetyltryptophan and
lysine-tryptophan-lysine, respectively, was proven unequivocally by
UV-visible spectroscopy as well as 15N NMR spectrometry.
Competition experiments performed with the known
N2O3 scavenger morpholine demonstrated that the
selected tryptophan derivatives were nitrosated by
N2O3 with similar rate constants. It is further
shown that the addition of ascorbate (vitamin C) induced the release of
nitric oxide from N-acetyl-N-nitrosotryptophan as monitored polarographically with a NO electrode. Theoretical considerations strongly suggested that the reactivity of protein-bound tryptophan would be high enough to compete effectively with
protein-bound cysteine for N2O3. Our
data demonstrate conclusively that N2O3 nitrosates the secondary amine function
(Nindole) at the indole ring of N-blocked
tryptophan with high reactivity at physiological pH values.
 |
INTRODUCTION |
Nitric oxide (·NO) is involved in a great variety of
physiological and pathophysiological processes, including the
regulation of vascular tonus, immune response, and neurotransmission
(1). In mammals the basal level of ·NO (in the aqueous phase) is
in the nanomolar range (2). However, the local ·NO
concentrations can be increased substantially in areas with high NO
synthase activity and/or in hydrophobic regions (3, 4), thereby
facilitating the formation of dinitrogen trioxide (N2O3) (Equations 1 (5) and 2 (6)).
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(Eq. 1)
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(Eq. 2)
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Because N2O3 is highly effective in
nitrosating sulfhydryl groups (7) (Equations 3 (8) and 4 (2)),
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(Eq. 3)
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(Eq. 4)
|
the putative formation of S-nitrosothiols is now
generally believed to be of high physiological importance in
vivo (Ref. 9 and references therein). For instance,
S-nitrosothiols can operate as nitric oxide-donating
compounds in vitro (Equation 5 (10)), and they are believed
to induce ·NO-like biological activities in vivo,
probably by releasing ·NO (11, 12).
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(Eq. 5)
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Further, S-nitrosation of thiols is postulated to
induce several functional protein modifications in vivo
(13-18). In addition to thiols, secondary amines also react rapidly
with N2O3 (7). Consequently, the question arose
whether (protein-bound) tryptophan could be nitrosated at the nitrogen
atom of the indole ring at physiological pH 7.4. It has been
demonstrated recently that melatonin (N-acetyl-5-methoxytryptamine) is N-nitrosated by
NaNO2/HCl as well as by ·NO/O2 at pH
7.4. However, the reported rate constant for the latter reaction is
rather low (k(melatonin + ·NO) = 0.5 M
1s
1) (19). Keeping the low
physiological concentrations of melatonin in mind (e.g. the
maximal concentration in human serum is ~75 pg/ml (20)), this
reaction therefore should not proceed to a significant extent in
vivo. A similar reaction has not been established thus far for
tryptophan at pH 7.4. However, there are indications that the secondary
amine of tryptophan (Nindole) can be nitrosated when the primary amine function (Nalanine) is
N-blocked.1 Consequently,
albumin (3, 21-23) or, more simply, N-acetyltryptophan (24)
were N-nitrosated by N2O3 generated
in situ by HCl/NaNO2 (Scheme
1). Nitrosamines in general are well
known carcinogens (25). Similarly, Venitt et al.
(26) observed mutagenic effects of
N-acetyl-N-nitrosotryptophan in bacteria. On the
other side, as protein-bound N-nitrosotryptophan was able to
both stimulate vasorelaxation and inhibit platelet aggregation (21),
there might be a putative physiological significance of
N-nitrosotryptophan derivatives. This idea, however, has not
been developed further, probably because thiols are now believed to be
the main physiological targets for N2O3 in
proteins.
In contrast to this opinion, we demonstrate unequivocally here the
N2O3-mediated nitrosation of N-blocked
tryptophan (N-acetyltryptophan and the tripeptide
lysine-tryptophan-lysine) by N2O3 at
physiological pH 7.4. In addition, competition experiments performed
with the common N2O3 scavenger morpholine
demonstrate that N2O3 reacts quickly with
N-terminal-blocked tryptophan. Finally, as liberation of ·NO is
evident after reaction of
N-acetyl-N-nitrosotryptophan with ascorbate,
N-nitrosotryptophan is expected to exhibit similar ·NO-donating capabilities as
S-nitrosocysteine.
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MATERIALS AND METHODS |
Chemicals--
L-Tryptophan,
N-acetyltryptophan, melatonin, lysine-tryptophan-lysine,
L-cysteine, morpholine, piperazine and
15N-labeled sodium nitrite were obtained from Sigma.
MAMA NONOate and spermine NONOate were purchased from Situs
(Düsseldorf, Germany).
Experimental Conditions--
Because nitrosation reactions are
sensitive to the presence of metal ions, solutions were exposed to
chelating resin (Chelex 100) as described previously (27).
Nitrosation by Preformed ·NO Gas--
Nitric oxide
(14·NO) or 15N- labeled nitric oxide
(15·NO) was prepared daily by the addition of
glacial acetic acid to an aqueous solution of
K4[Fe(CN)6] (0.7 M) containing
either NaNO2 or Na15NO2 (0.72 M) under oxygen-free conditions. The overall stoichiometry is presented by the equation,
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(Eq. 6)
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The reliability of this procedure was confirmed by detecting the
release of nitric oxide polarographically with a graphite nitric
oxide-sensing electrode (World Precision Instruments, Berlin, Germany).
N-blocked derivatives of tryptophan were nitrosated in
oxygen-saturated phosphate/triglycine buffer (250/25
mM, pH 8.0) with the above described preformed
·NO gas by bubbling it gently into the solution through a needle until the pH value had dropped to 7.4.
Nitrosation by NO Donor Compounds--
MAMA NONOate and spermine
NONOate were prepared as 100-fold stock solutions in 10 mM
NaOH at 4 °C and used immediately. A stock solution of
nitrosocysteine was prepared by S-nitrosation of 100 mM cysteine with equimolar amounts of NaNO2 in
acidic solution (pH 2) at 0 °C (24). Nitrosation reactions were
performed in 1 ml of potassium
phosphate/NaHCO3/CO2 buffer (50 mM/25 mM/5%, pH 7.4, 37 °C) in 35-mm dishes.
Nitrosation by NaNO2 under Acidic
Conditions--
Substances were exposed to equimolar concentrations of
sodium nitrite in H2O equilibrated with glacial acetic acid
to pH 3.5.
15N NMR Identification of Nitrosated
Products--
Immediately after nitrosation, sample aliquots were
supplemented with 10% D2O and analyzed by 15N
NMR spectrometry. The adducts were identified by 50.67 MHz
15N NMR spectrometry on a Bruker AVANCE DRX 500 instrument.
Chemical shifts (
) are given in ppm relative to neat nitromethane
(
= 0) as the external standard.
Reactivity of N-Acetyltryptophan toward
N2O3--
Morpholine (0-100 mM)
or piperazine (0-20 mM) and L-tryptophan (2 mM) or its derivatives were incubated with 0.5 mM MAMA NONOate in potassium phosphate buffer (50 mM, pH 7.5, 37 °C) for 30 min. The reaction products
were analyzed spectrophotometrically at 335 nm (or 346 nm for
nitrosomelatonin) with a SPECORD S 100 spectrophotometer from Analytik
Jena (Jena, Germany). A similar experiment was carried out with
piperazine as the competitor. Control experiments demonstrated that
transnitrosation reactions did not proceed at this pH between nitrosomorpholine + N-acetyltryptophan,
nitrosopiperazine + N-acetyltryptophan, morpholine + N-acetyl-N-nitrosotryptophan, or piperazine + N-acetyl-N-nitrosotryptophan, in line with
observations reported by Meyer et al. (28).
Decay Kinetic Experiments--
The decay kinetics of
N-acetyl-N-nitrosotryptophan (100 µM) at various temperatures (15-45 °C) were
determined in air-tight quartz cuvettes by rapid scan monitoring the
UV-visible absorption at
max = 335 nm
(
335 = 6100 M
1cm
1) (24). Between
recordings the samples were protected from light. The temperature was
maintained at ± 0.1 °C.
 |
RESULTS |
Formation of N-Nitrosotryptophan in Phosphate Buffer, pH
7.4--
Evidence exists that under acidic conditions similar to those
in the stomach the NaNO2-dependent nitrosation
of human serum albumin, bovine serum albumin, or the dipeptide
glycine-tryptophan yields N-nitrosotryptophan (21, 23). The
main nitrosating species in this reaction is postulated to be
dinitrogen trioxide (N2O3) formed from
HNO2 dehydration (7, 29). Provided that this is indeed the
case, N2O3 formed from ·NO in the
presence of oxygen at pH 7.4 should also nitrosate
N-terminal-blocked tryptophan derivatives, like
N-acetyltryptophan and peptide-associated tryptophan (lysine-tryptophan-lysine).
To verify the nitrosation of N-blocked tryptophan derivatives by
N2O3 at pH 7.4, we selected various
N2O3-generating systems. We employed preformed
nitric oxide as well as in situ generation of ·NO
from S-nitrosocysteine and spermine NONOate, respectively. In these systems, N2O3 is formed from oxidation
of nitric oxide (·NO autoxidation) according to Equations 1 and
2 (see the Introduction). Because the formation of
N-acetyl-N-nitrosotryptophan from the reaction of
N-acetyltryptophan with NaNO2 at pH 3.5 has been
described in detail (24, 30), we used this reaction as a reference
system. In analogy to the data of Bonnett and Holleyhead (24) we
recorded the UV-visible spectrum of
N-acetyl-N-nitrosotryptophan with an absorption
maximum at 335 nm (Fig. 1A,
trace b). Interestingly, an almost identical UV-visible
spectrum was observed when N-acetyltryptophan was allowed to
react with preformed nitric oxide in the presence of oxygen at
physiological pH 7.4 (Fig. 1A, trace e). This
finding indicated very strongly that
N2O3 can indeed nitrosate the secondary amine
of the indole system in tryptophan. Likewise, the UV-visible spectrum
of N-acetyl-N-nitrosotryptophan was also detected
during reaction of N-acetyltryptophan with the above
mentioned nitric oxide-donating compounds in the presence of air (Fig.
1A, traces c and d).

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Fig. 1.
UV-visible absorption spectra of
N-nitroso-tryptophan residues generated from the
reactions of both N-acetyltryptophan and
lysine-tryptophan-lysine with various NO sources. A, spectra
of N-acetyltryptophan (trace a) and authentic
N-acetyl-N-nitrosotryptophan (trace b)
in air-saturated potassium phosphate buffer/NaHCO3, at pH
7.4, and spectra observed after reaction of
N-acetyltryptophan (1 mM) with spermine NONOate
(2 mM) (trace c), S-nitrosocysteine
(3.4 mM) (trace d, dilution 1:4) in the same
buffer solution, or preformed ·NO gas in oxygenated 250 mM potassium phosphate buffer, pH 8.0, 25 mM
triglycine (trace e, dilution 1:10). B, spectrum
of lysine-tryptophan-lysine (5 mM) (trace a) in
air-saturated potassium phosphate/NaHCO3/CO2
buffer (50 mM/25 mM/5%, pH 7.4, 37 °C);
spectrum observed after reaction of lysine-tryptophan-lysine with
NaNO2 (5 mM each) at pH 3.5 (trace
b); and spectra observed after reaction of
lysine-tryptophan-lysine (5 mM) with
S-nitrosocysteine (3.4 mM) (trace c,
dilution 1:2), spermine NONOate (2 mm) (trace d) in
air-saturated potassium phosphate/NaHCO3/CO2
buffer (50 mM/25 mM/5%, pH 7.4, 37 °C), or
preformed ·NO gas in oxygenated potassium phosphate/triglycine
buffer (250/25 mM, pH 8.0, 37 °C) (trace e,
dilution 1:10). In the latter buffer, the pH decreased to 7.4 during
the experiment.
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Because in vivo N-terminal-blocked tryptophan is present
primarily in proteins, the N2O3-mediated
nitrosation of the nitrogen atom of the indole ring was also
investigated for peptide-bound tryptophan by employing
lysine-tryptophan-lysine. In full agreement with the experiments
performed with N-acetyltryptophan (see above), all applied
N2O3-generating systems were able to nitrosate
peptide-bound tryptophan effectively at the selected pH values (Fig.
1B). In contrast, the nitrosation of
L-tryptophan by the
N2O3-generating systems was much less effective
(data not shown), which is in line with data from the literature (28,
30). Conclusively, only N-terminal-blocked tryptophan but not
L-tryptophan is a relevant target for
N2O3 at physiological pH.
Detection of N-Nitrosotryptophan by 15N NMR
Spectrometry--
Because UV-visible absorption spectra cannot provide
unambiguous evidence that the secondary amine function of the indole ring was truly nitrosated, we used 15N NMR spectrometry as
a more reliable analytical tool. In 1986, Dorie et al. (31)
recorded the 15N NMR spectrum of 15N-labeled
N-acetyl-[15N]nitrosotryptophan from
the reaction of N-acetyltryptophan with Na15NO2 at pH 4. As commonly observed for
N-nitroso compounds (32) the 15N NMR spectrum
exhibited two resonances, at 184.6 and 169.6 ppm relative to neat
nitromethane, of the Z- and E-conformer of
N-acetyl-N-nitrosotryptophan, respectively. Fig.
2A shows the 15N
NMR spectrum of preformed (authentic)
N-acetyl-15N-nitrosotryptophan at pH 7.4, exhibiting two resonance lines at 180.8 and 166.3 ppm, respectively.
The small shift difference of ~3 ppm compared with the data of Dorie
et al. (31) may be explained by differences in the recording
conditions, e.g. different pH values as well as improvements
of the 15N NMR spectrometric techniques during the past
years. Nevertheless, nitrosation of the nitrogen atom at the indole
ring is proven by this characteristic 15N NMR spectrum.
When N-acetyltryptophan was reacted with authentic 15NO in the presence of oxygen, only the two new
15N NMR resonances at 180.1 and 165.6 ppm were detected,
consistent with the formation of
N-acetyl-N-nitrosotryptophan (Fig.
2B). To the best of our knowledge, this is the first direct
proof that a tryptophan derivative can be nitrosated by
N2O3 at the nitrogen atom of the indole system
at physiological pH.

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Fig. 2.
15N NMR spectra of
15N-labeled N-nitrosotryptophan
derivatives. A, 15N-labeled
N-acetyl-N-nitrosotryptophan prepared from
reaction of N-acetyltryptophan with NaNO2 (20 mM each) in acidified aqueous solution at pH 3.5 (after
completion of the reaction, the reaction mixture was adjusted to pH 7.4 by the addition of 1 N NaOH). 15N-Labeled
N-acetyl-N-nitrosotryptophan (B) and
15N-labeled
lysine-15N-nitrosotryptophan-lysine (C)
were produced from the reaction of either 100 mM
N-acetyltryptophan or 200 mM
lysine-tryptophan-lysine with preformed 15·NO in
oxygenated potassium phosphate/triglycine buffer (250 mM/25
mM, pH 8.0, 37 °C). Under the conditions in B
and C, the pH decreased to 7.4 during the experiment.
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Analogous to N-acetyltryptophan the tripeptide
lysine-tryptophan-lysine was nitrosated at pH 7.4 by preformed
15NO as proven by the 15N NMR resonance lines
at 181.1 and 166.7 ppm, respectively (Fig. 2C).
Reactivity of N-blocked Tryptophan Derivatives Toward
N2O3--
For a discussion of the putative
significance of tryptophan nitrosation in vivo it is
necessary to determine the rate constants of the reaction of
N-acetyltryptophan or its derivatives with N2O3
at pH 7.5. As mentioned above, Blanchard et al. (19)
reported that melatonin reacts with ·NO in the presence of
oxygen at pH 7.4 with a second-order rate constant of only 0.5 M
1 s
1. However, these
experiments were performed in the presence of 200 mM Hepes
buffer, which is known to react with reactive nitrogen/oxygen species
to yield both H2O2 (33, 34) and a
·NO-donating compound (35). Therefore we evaluated the rate
constants of N-nitrosation of N-blocked tryptophan
derivatives with N2O3 by a competition method
employing morpholine as the N2O3 scavenger (2,
36, 37). The reaction of 2 mM N-acetyltryptophan
with 0.5 mM MAMA NONOate yielded about 360 µM
N-acetyl-N-nitrosotryptophan. Morpholine
competitively inhibited the MAMA NONOate-induced nitrosation of
N-acetyltryptophan in a nonlinear manner with an
IC50 value of 13.4 mM (Fig.
3). The protonated form of morpholine,
that is morpholinium, with a pKa value of 8.23 at 37 °C (38), is not expected to react with
N2O3 (39). Thus, the fraction of unprotonated morpholine is about 15.7% at pH 7.5. Taking this fraction into account, the true IC50 value is given by 13.4 mM × 0.157 = 2.1 mM. Conclusively, as 2 mM N-acetyltryptophan was used (see above), N2O3 reacts at 37 °C with virtually
identical rate constant with both N-acetyltryptophan and
morpholine. To verify the presumption that only the unprotonated
fraction of the secondary amine effectively reacts with
N2O3, a control experiment was performed with
piperazine as a competitive N2O3 scavenger. At
pH 7.5 about 99% of piperazine (pKa = 5.55 (7))
exists in the unprotonated form; thus, as expected, piperazine was more
effective than morpholine in inhibiting the MAMA NONOate-induced
nitrosation of N-acetyltryptophan (Fig. 3). From inspection
of Fig. 3 it can be deduced that MAMA NONOate-induced nitrosation of
N-acetyltryptophan was half-maximally inhibited at a
piperazine concentration of 2.7 mM. The observation that the corrected IC50 value of morpholine is lower than the
experimental IC50 value of piperazine (Table
I) is in agreement with the report that
N2O3 reacts faster with morpholine than with
piperazine (7). Not unexpectedly, the reactivity of other
N-terminal-blocked tryptophan derivatives toward
N2O3 was found to be similar (Table I). To compare the rate constant of these nitrosation reactions with other
nitrosation reactions reported in the literature, one experiment with
N-acetyltryptophan was performed at 25 °C and a rate
constant of 4.4 × 107
M
1s
1 was obtained. Thus,
N-blocked tryptophan reacts rather fast with N2O3 (see the Discussion).

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Fig. 3.
Influence of morpholine and piperazine on
MAMA NONOate-induced nitrosation of
N-acetyltryptophan. MAMA NONOate (0.5 mM) was incubated for 30 min with 2 mM
N-acetyltryptophan in potassium phosphate buffer (50 mM, pH 7.5, 37 °C) in the presence of various
concentrations of morpholine or piperazine, respectively.
N-Acetyl-N-nitrosotryptophan formation was
monitored at 335 nm. Each value represents the mean ± S.D. of
three determinations.
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Decomposition of N-Acetyl-N-nitrosotryptophan--
In contrast to
common, stable nitrosamines, protein-bound
N-nitrosotryptophan has been reported to undergo slow decay
at pH 2 (23). To quantify this capability at pH 7.4, we analyzed the decomposition of N-acetyl-N-nitrosotryptophan by
monitoring its absorption at 335 nm. At 37 °C
N-acetyl-N-nitrosotryptophan decayed in a
first-order manner (Fig. 4A).
Similarly, first-order decay kinetics were also observed at other
temperatures (15-45 °C, data not shown). From the excellent
Arrhenius plot of the rate data (Fig. 4B) an activation
barrier of Ea = 13.2 ± 0.1 kcal
mol
1 and an A-factor of 1.7 ± 0.3 × 105 s
1 were extracted. This
A-factor appears to be extremely low for a simple
first-order (homolysis) decomposition. From the Arrhenius parameters
the half-life of N-acetyl-N-nitrosotryptophan at
physiological conditions (T = 37 °C, pH 7.4) can be
calculated to 140 min. Thus, the N-nitrosamines of N-blocked
tryptophan derivatives are rather long-lived intermediates.

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Fig. 4.
Kinetic data of the first-order rate decay of
N-acetyl-N-nitrosotryptophan.
A, decay kinetic of 100 µM
N-acetyl-N-nitrosotryptophan in potassium
phosphate buffer (50 mM, pH 7.4, 37 °C). B,
Arrhenius plot of the first-order rate constants of decay of 100 µM N-acetyl-N-nitrosotryptophan in
the same buffer. Each value represents the mean ± S.D. of three
determinations.
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Because the half-life of peptide-bound N-nitrosotryptophan
was found to be similar to that of
N-acetyl-N-nitrosotryptophan (data not shown),
one may ask whether it would make any sense for physiological functions
to nitrosate tryptophan in vivo. Recently, Harohalli
et al. (23) reported that
N-nitrosotryptophan very slowly releases ·NO at
pH 2, and Blanchard-Fillion et al. (40) observed that nitrosomelatonin spontaneously decays at pH 7.4, thereby
releasing nitric oxide at a yield of 71%. In our hands,
however, authentic nitrosomelatonin released only negligible amounts of
nitric oxide on decomposition in Hepes-free buffer solution (data not
shown). Noticeably neither Harohalli et al. (23) nor
Blanchard-Fillion et al. (40) directly detected ·NO,
e.g. with a NO electrode.
As nitrosothiols release nitric oxide in the presence of vitamin C
(10), we compared the potential of ascorbate to induce ·NO
release from S-nitrosoglutathione versus
N-acetyl-N-nitrosotryptophan (Fig.
5). In the absence of copper ions, the
release of ·NO from
N-acetyl-N-nitrosotryptophan after the addition
of ascorbate was about 5-fold higher than from
S-nitrosoglutathione. In contrast, a "simple"
N-nitrosamine like N-nitrosomorpholine did not
liberate nitric oxide under similar conditions (data not shown). The
low ·NO-releasing efficiency of S-nitrosoglutathione
is explained by the rigorous depletion of copper ions in the applied
buffer solution. In the presence of 10 µM
Cu2+, however, S-nitrosoglutathione as well as
N-acetyl-N-nitrosotryptophan did
release nitric oxide with nearly the same yield in the presence of
ascorbate (data not shown). In the absence of vitamin C only negligible
amounts of nitric oxide were released from either
N-acetyl-N-nitrosotryptophan or
S-nitrosoglutathione on decomposition. These results
demonstrate unequivocally that
N-acetyl-N-nitrosotryptophan has nitric
oxide-releasing capabilities similar to those of
S-nitrosocysteine.

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Fig. 5.
Time course of ascorbic acid-induced
·NO liberation from
N-acetyl-N-nitrosotryptophan and
S-nitrosoglutathione. ·NO release from
N-acetyl-N-nitrosotryptophan and
S-nitrosoglutathione (100 µM each) in
potassium phosphate buffer (50 mM, pH 7.4, 37 °C) in the
presence of 400 µM ascorbate as monitored
electrochemically with the ·NO electrode.
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 |
DISCUSSION |
The results presented above clearly show that
N2O3 nitrosates the secondary amine function at
the indole ring of N-blocked tryptophan with high reactivity at
physiological pH values. It is known that indole and indole
derivatives are easily attacked by N2O3 under
acidic conditions, however, with exclusive C-nitrosation, thus yielding 3-nitroso products (30, 41). Such products
were not observed for tryptophan because here the C-3 position is
blocked by the alanine residue, rendering nitrosation at position N-1 (Nindole) more feasible. Nitrosation at C-2 is possible
only for derivatives carrying powerful electron donors at the C-3
position (30). Our data indicate that N-acetyltryptophan,
melatonin, and the tripeptide lysine-tryptophan-lysine are nitrosated
directly by N2O3 because these nitrosation
reactions could be inhibited effectively by the
N2O3 scavengers morpholine and piperazine, respectively. In contrast, Turjanski et al. (42) reported
that melatonin is nitrosated mainly by a combined attack of the
radicals ·NO2 and ·NO. It should be noted
that such a mechanism has never been proven for ordinary amines
and that a variety of side products, i.e. N-nitro, C-nitro, and C-nitroso
compounds, had to be produced by an operating radical mechanism.
Noticeably, the 15N NMR data demonstrated that reaction of
15N2O3 with N-blocked tryptophan
exclusively yields 15N-nitrosotryptophan.
Recently, the reaction between albumin and N2O3
generated from NaNO2 at low pH has been monitored by UV
spectroscopy. On the basis of these measurements it was assumed that
tryptophan would be nitrosated also (21, 23). However, this conclusion
has not been generally accepted (22, 43). As there is presently no
specific test for N-nitrosotryptophan available and because N-nitrosotryptophan derivatives and
S-nitrosocysteine have almost identical UV-visible
absorption spectra (
max ~335 (24) and ~340 nm
(30), respectively), the efficiency of nitrosation of both tryptophan
and cysteine in proteins is hard to verify experimentally by UV-visible
spectroscopy. On the other hand, the effectiveness of these reactions
can reasonably be estimated on the basis of the experimental rate
constants and the concentrations of both amino acids in proteins. At
physiological pH values, tryptophan exists practically exclusively in
the reactive nonprotonated form. Hence, the rate constant of
N2O3 with protein-bound tryptophan can be
assumed to be k = 4.4 × 107
M
1s
1 (see "Results"). In
contrast, thiols are only marginally deprotonated at pH 7.4. From the
average pKa values of ~8.2 of thiolates in proteins (22), it can be deduced that only 13.5% of the
cysteine residues are viable targets for N2O3.
Thus, as the rate constants for reaction of thiolates
(RS
) with N2O3 is about
k = 2 × 108
M
1s
1 (2), protein-bound
cysteine should react at pH 7.4 with N2O3 with
a rate constant of k (cysteine + N2O3) = 2 × 108
M
1s
1 × 0.135 = 2.7 × 107 M
1s
1. Thus,
the rate constant of the reaction of protein-bound tryptophan with
N2O3 is, somewhat unexpectedly, estimated to be
about 63% higher than the rate constant of protein-bound cysteine with
N2O3. To verify that this conclusion can be
extended to the reaction rate, the amounts of both tryptophan and
cysteine residues in proteins were verified by searching the
RCSB Protein Data Bank (44) for those proteins that are believed to be
S-nitrosated (Table II). From
the data in Table II it can be deduced that in the selected proteins
the total amount of cysteine is higher than the total amount of
tryptophan. With regard to the differences in the rate constants (see
above), one can now estimate that N2O3 reacts
primarily (40-90%) with tryptophan. Thus, we hypothesized that
protein-bound tryptophan should be preferentially nitrosated under
physiological conditions. To verify this prediction, we are currently
developing highly sensitive protocols for the detection of both
N-nitrosotryptophan and S-nitrosothiols in
proteins.
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Table II
Tryptophan and cysteine residues in proteins which may be subject to
S-nitrosation in physiological regulation mechanisms
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|
In this paper we have demonstrated additionally that
N-nitrosotryptophan has the capability of releasing nitric
oxide in the presence of ascorbate. This implies that
N-nitrosotryptophan might operate as a nitric oxide carrier,
a property that to date has been attributed more or less exclusively to
S-nitrosothiols (7, 11, 45, 46). However, as the chemistry
of N-nitrosotryptophan derivatives in physiological
environment is not very well developed, it is as yet too early to
consider N-nitrosotryptophan derivatives as "harmless"
compounds in biological systems. It might be assumed that potentially
harmful reactive nitrogen species like peroxynitrite (40) or
N2O3 are deactivated by their reaction with
tryptophan residues and that this may them give some antioxidative
functions. However, the observation of Venitt et al. (26)
that N-acetyl-N-nitrosotryptophan at 5-15
mM induces mutagenicity in bacteria is, in our view, easily explained by its capability of releasing nitric oxide, which is known
to induce such effects at unphysiologically high concentrations (47).
In order not to be misunderstood, at present harmful effects of
N-nitrosotryptophan derivatives cannot be ruled out. It
should be remembered that harmful reactions are also known for
S-nitrosothiols. For example, it has been reported that
S-nitrosothiols react with thiols to yield nitroxyl (48),
which generates hydroxyl radicals and/or peroxynitrite in the absence
and presence of oxygen, respectively (49).
Because N-nitrosotryptophan derivatives are rather
long-lived and yet not "indefinitely" stable compounds (the
hydrolysis of N-acetyl-N-nitrosotryptophan is
expected to yield the harmless products tryptophan and nitrite (28)),
one may speculate that this could further decrease the putative
mutagenic potential of N-nitrosotryptophan. On the other
hand, the observed half-life (t1/2 > 2 h) is
so long that N-nitrosotryptophan derivatives
(NindoleNO) may participate in physiological processes, e.g. in the transport and release of nitric oxide. In fact,
Zhang et al. (21) observed that peptide-bound
N-nitrosotryptophan induces vasorelaxation of rabbit aortic
rings, a function that is typical for freely diffusing nitric oxide
(45). In conclusion, we hypothesize that a putative
physiological potential of N-nitrosotryptophan should be
more important than its pathophysiological one. This feature remains to
be clarified in the near future. In any case, we have identified, in
addition to cysteine, a second major target for
N2O3 in proteins. This fact will strongly
influence the general understanding of protein nitrosation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. H.-G. Korth for a series of
clarifying discussions pertaining to the nature of the underlying
chemistry and for useful comments on this manuscript. We also thank H. Bandmann for advice on the NMR technique. The present investigation
would have been impossible without the technical assistance of
E. Heimeshoff, M. Holzhauser, and A. Wensing.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
These authors contributed equally to this work.
§
To whom correspondence should be addressed. Tel.: 49-201-723-4107;
Fax: 49-201-723-5943, E-mail: michael.kirsch@uni-essen.de.
Published, JBC Papers in Press, January 23, 2003, DOI 10.1074/jbc.M300237200
 |
ABBREVIATIONS |
The abbreviations used are:
N-blocked, N-terminal-blocked;
MAMA, NONOate,
(Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate;
spermine NONOate, (Z)-1-{N-(3-aminopropyl)-N-[-4-(3-aminopropylammonio)butyl]-amino}diazen-1-ium-1,2diolate.
 |
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