regiospecific nitrosation of n-(terminal) blocked … · - 3 - n 2o 3-induced tryptophan...

Regiospecific nitrosation of N-(terminal) blocked tryptophan derivatives by N2O3 at

physiological pH

Michael Kirsch*§, Anke Fuchs§, and Herbert de Groot

¥Institut für Physiologische Chemie, Universitätsklinikum, Hufelandstrasse 55, D-45122 Essen

*To whom correspondence should be addressed:

Dr. M. Kirsch

Institut für Physiologische Chemie

Universitätsklinikum Essen

Hufelandstr. 55

D-45122 Essen

Germany

Tel.: 49 - 201 / 723 - 4107

Fax: 49 - 201 / 723 - 5943

E-Mail: [email protected]

§ M. Kirsch and A. Fuchs contributed equally to the work presented here.

RUNNING TITLE: N2O3-induced tryptophan nitrosation

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on January 27, 2003 as Manuscript M300237200 by guest on O

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N2O3-induced tryptophan nitrosation

SUMMARY

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 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-acetyl-tryptophan and lysine-

tryptophan-lysine, respectively, was unequivocally proven by UV-Vis spectroscopy as well as by 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 addition of ascorbate (vitamin C) induced release of nitric oxide from

N-acetyl-N-nitrosotryptophan as polarographically monitored with a NO-electrode. Theoretical

considerations strongly suggested that the reactivity of protein-bound tryptophan would be high

enough to effectively compete with protein-bound cysteine for N2O3. Conclusively, our data clearly

demonstrate that N2O3 nitrosates the secondary amine function (NIndole) at the indole ring of N-blocked

tryptophan with high reactivity at physiological pH values.

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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 substantially be increased 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)).

2 •NO + O2

→ 2 •NO2 k1 = 2.9 x 106 M-2 s-1 (Eq. 1)

•NO + •NO2 N2O3 k2 = 1.1 x 109 M-1 s-1 (Eq. 2)

k-2 = 8.03 x 104 s-1

Since N2O3 is highly effective in nitrosating sulfhydryl groups (7) (equations 3 (8) and 4 (2)),

RSH + N2O3 → RSNO + (H+) + NO2- k3 = 6.4 x 106 M-1 s-1 (Eq. 3)

RS- + N2O3 → RSNO + NO2- k4 = 1.8 x 108 M-1 s-1 (Eq. 4)

the putative formation of S-nitrosothiols is now generally believed to be of high physiological

importance in vivo ((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).

RSNO + ascorbate / Cu2+ → RSNO + products + Cu1+ → •NO + RS— + Cu2+ (Eq. 5)

Further, S-nitrosation of thiols is postulated to induce several functional protein modifications in vivo

(13-18). Beside thiols, secondary amines also react rapidly with N2O3 (7). Consequently, the question

arose whether (protein-bound) tryptophan can be nitrosated at the nitrogen atom of the indole ring at

physiological pH 7.4. It has recently been demonstrated 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 for tryptophan at pH 7.4 so far. However, there are

indications that the secondary amine of tryptophan (NIndole) can be nitrosated when the primary amine

function (NAlanine) is N-blocked. Consequently, albumin (3,21-23) or, more simply, N-acetyltryptophan


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(24) were N-nitrosated by N2O3 generated in situ by HCl/NaNO2 (Scheme 1).

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 further developed probably because thiols are now believed to be the main physiological

targets for N2O3 in proteins.

In contrast to this opinion we here unequivocally demonstrate 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 fast 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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EXPERIMENTAL PROCEDURES

L-tryptophan, N-acetyl-tryptophan, melatonin, lysine-tryptophan-lysine, L-cysteine, morpholine,

piperazine and 15N-labeled sodium nitrite were obtained from Aldrich-Sigma (Deisenhofen, Germany).

MAMA NONOate and spermine NONOate were purchased from Situs (Düsseldorf, Germany).

Experimental conditions - Since 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) were daily

prepared 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:

K4 [Fe(CN)6] + Na15NO2 + 2 CH3COOH →

K3[Fe(CN)6] + CH3COOK + CH3COONa + H2O + 15•NO (Eq. 6)

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 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 (100 mM) 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 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 (δ) are

given in ppm relative to neat nitromethane (δ=0) as external standard.


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Reactivity of N-acetyl-tryptophan towards 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 spectrophotometrically

analyzed 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 competitor.

Control experiments demonstrated that transnitrosation reactions did not proceed at this pH between

either nitrosomorpholine + N-acetyl-tryptophan, nitrosopiperazine + N-acetyl-tryptophan, 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-Vis absorption at λMax= 335 nm (ε335 = 6100 M-1cm-1, (24)) at 12 – 30 time steps. In between the

recordings the samples were protected from light. The temperature control was maintained at ± 0.1°C.


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RESULTS

Formation of N-nitrosotryptophan in phosphate buffer pH 7.4 - Evidence exists that at acidic conditions

similar to that in the stomach the NaNO2-dependent nitrosation of either HSA, BSA 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 has strongly to be

expected to nitrosate N-terminal-blocked tryptophan-derivatives, like N-acetyl-tryptophan and peptide-

associated tryptophan (lysine-tryptophan-lysine), too.

In order 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 1,2 (see Introduction). Since

the formation of N-acetyl-N-nitrosotryptophan from the reaction of N-acetyl-tryptophan with NaNO2 at

pH 3.5 has been described in detail (24,30) we used this reaction as a reference system. In analogy to

data of Bonnet et al. (24) we recorded the UV-Vis spectrum of N-acetyl-N-nitrosotryptophan with an

absorption maximum at 335 nm (Fig.1A, trace b). Interestingly, an almost identical UV-Vis spectrum

was observed when N-acetyl-tryptophan was allowed to react with preformed nitric oxide in the

presence of oxygen at physiological pH 7.4 (Fig. 1A, trace e). This very strongly indicated that N2O3

can indeed nitrosate the secondary amine of the indole system in tryptophan. Likewise, the UV-Vis

spectrum of N-acetyl-N-nitrosotryptophan was also detected during reaction of N-acetyl-tryptophan

with the above mentioned nitric oxide donating compounds in the presence of air (Fig. 1A, traces c

and d).

Since in vivo N-terminal-blocked tryptophan is primarily present 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-acetyl-

tryptophan (see above) all applied N2O3 generating systems were able to effectively nitrosate peptide-

bound tryptophan at the selected pH-values (Fig. 1B). In contrast, the nitrosation of N-unprotected L-

tryptophan by the N2O3-generating systems was much less effective (data not shown) in line with data


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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-nitroso-tryptophan by 15N-NMR spectrometry - Since UV-Vis 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.

recorded the 15N-NMR spectrum of 15N-labeled N-acetyl-15N-nitrosotryptophan from reaction of N-

acetyl-tryptophan with Na15NO2 at pH 4 (31). As commonly observed for N-nitroso compounds (32) the

15N-NMR spectrum exhibited two resonances, at 184.6 ppm and 169.6 ppm relative to neat

nitromethane, of the Z- and E-conformer of N-acetyl-N-nitrosotryptophan, respectively. Figure 2A

shows the 15N-NMR spectrum of preformed (authentic) N-acetyl-15N-nitrosotryptophan at pH 7.4,

exhibiting two resonance lines at 180.8 ppm and 166.3 ppm, respectively. The small shift difference of

approximately 3 ppm compared to the data of Dorie et al. 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-acetyl-tryptophan was reacted with

authentic 15NO in the presence of oxygen, only the two new 15N-NMR resonances at 180.1 ppm 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.

Analogous to N-acetyl-tryptophan the tripeptide lysine-tryptophan-lysine was nitrosated at pH 7.4

by preformed 15NO as proven by the 1 5N-NMR resonance lines at 181.1 ppm and 166.7 ppm,

respectively (Fig. 2C).

Reactivity of N-blocked tryptophan-derivatives towards 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-acetyl-tryptophan 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-


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nitrosation of N-blocked tryptophan derivatives with N2O3 by a competition method, employing

morpholine as N2O3 scavenger (2,36,37). The reaction of 2 mM N-acetyl-tryptophan with 0.5 mM

MAMA NONOate yielded about 180 µM N-acetyl-N-nitrosotryptophan. Morpholine competitively

inhibited the MAMA NONOate-induced nitrosation of N-acetyl-tryptophan in a non-linear 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. Taken this fraction into account, the true IC50 value is given by

13.4 mM x 0.157 = 2.1 mM. Conclusively, as 2 mM N-acetyl-tryptophan was used (see above), N2O3

reacts at 37°C with virtually identical rate constant with both N-acetyl-tryptophan and morpholine. In

order 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-acetyl-tryptophan (Fig. 3). From inspection of Figure 3 it can be deduced that

MAMA NONOate-induced nitrosation of N-acetyl-tryptophan 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 unexpected, the reactivity of other N-

terminal blocked tryptophan derivatives towards N2O3 were found to be similar (Table I). In order to

compare the rate constant of these nitrosation reactions with other nitrosation reactions reported in the

literature, one experiment with N-acetyl-tryptophan was performed at 25°C and a rate constant of 4.4 x

107 M-1s-1 was obtained. Thus, N-blocked tryptophan reacts rather fast with N2O3 (see Discussion).

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). In order 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

(Figure 4A). Similarly, first-order decay kinetics were also observed at other temperatures (15°C -

45°C, data not shown). From the excellent Arrhenius plot of the rate data (Figure 4B) an activation

barrier of Ea = 13.2 ± 0.1 kcal mol-1 and an A-factor of 1.7 ± 0.3 x 105 s-1 was extracted. This A-factor


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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.

Since 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 with release of 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 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 unequivocally demonstrate that N-acetyl-N-

nitrosotryptophan has nitric oxide-releasing capabilities similar to S-nitrosocysteine.


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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 has been known

before 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 the position N-1 (NIndole) more feasible. Nitrosation at C-2 is only possible for derivatives

carrying powerful electron donors at the C-3 position (30). Our data indicate that N-acetyl-tryptophan,

melatonin, and the tripeptide lysine-tryptophan-lysine are directly nitrosated by N2O3 because these

nitrosation reactions could effectively be inhibited by the N2O3 scavengers morpholine and piperazine,

respectively. Contrary, Turjanski et al. (42) reported that melatonin is mainly nitrosated by a combined

attack of the radicals •NO2 and •NO. It should be noted that such a mechanism has never been proven

to proceed 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 1 5N-

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 too (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-Vis absorptions (λmax ca. 335 nm (24) and

ca. 340 nm (30), respectively), the efficiency of nitrosation of both tryptophan and cysteine in proteins

is experimentally hard to verify by UV-Vis spectroscopy. On the other hand, the effectiveness of these

reactions can be reasonably estimated on the basis of the experimental rate constants and

concentrations of both amino acids in proteins. At physiological pH-values tryptophan exists practically

exclusively in the reactive non-protonated form. Hence, the rate constant of N2O3 with protein-bound

tryptophan can be assumed to be k = 4.4 x 107 M-1s-1 (see Results). Contrary, thiols are only

marginally deprotonated at pH 7.4. From the average pKa values of ca. 8.2 of thiolates in proteins (22)

it can be deduced that only a fraction of 13.5% of the cysteine-residues are viable targets for N2O3.


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Thus, as the rate constants for reaction of thiolates (RS-) with N2O3 is about k = 2 x 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

x 108 M-1s-1 x 0.135 = 2.7 x 107 M-1s-1. Thus, the rate constant of reaction of protein-bound tryptophan

with N2O3 is, somewhat unexpected, estimated to be about 63% higher than the rate constant of

protein-bound cysteine with N2O3. In order 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 using

the RCSB Protein Data Bank (44) for those proteins which are believed to be S-nitrosated (Table II).

From the data of 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

hypothesize that protein-bound tryptophan should be preferentially nitrosated under physiological

conditions. In order to verify this prediction, we are currently developing highly sensitive protocols for

the detection of both N-nitrosotryptophan and S-nitrosothiols in proteins.

In this paper we additionally demonstrate 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 which to date has been more or less exclusively attributed 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 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 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 to release nitric oxide which is known to induce such effects

at unphysiological high concentrations (47). In order not to be misunderstood: 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 in the

presence of oxygen, respectively (49).


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Because of the fact that N-nitrosotryptophan-derivatives are rather long-lived yet not “indefinitely”

stable compounds in physiological environment (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 (t 1/2 > 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 which is typical for freely diffusing nitric oxide (45). Conclusively we hypothesize that a

putative physiological potential of N-nitrosotryptophan should be more important than its

pathophysiological one. This feature has to be clarified in the near future. In any case, we identified,

beside cysteine, a second major target for N2O3 in proteins. This fact will strongly influence the

understanding of protein nitrosation in general.

Acknowledgments - 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 would also like to

thank Dipl. Ing. H. Bandmann for advice on the NMR technique. The present investigation would have

been impossible without the technical assistance of Ms. Heimeshoff, Ms. Holzhauser, and Ms.

Wensing.


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FIGURE LEGENDS

Fig. 1. UV-Vis absorption spectra of N-nitroso-tryptophan residues generated from the

reactions of both N-acetyl tryptophan and lysine-tryptophan-lysine with various NO-sources.

A, spectra of N-acetyl-tryptophan (trace a) and of authentic N-acetyl-N-nitrosotryptophan (trace b) in

air-saturated potassium phosphate buffer/NaHCO3 at pH 7.4. Spectra observed after reaction of N-

acetyl-tryptophan (1 mM) with either 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 (5mM) (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). Spectra observed after reaction of lysine-tryptophan-lysine (5 mM) with

either 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 mM/25 mM, pH 8.0, 37°C) (trace e,

dilution 1:10). In the latter buffer the pH decreased to 7.4 during experiment.

Fig. 2. 15N-NMR spectra of 15N-labeled N-nitrosotryptophan derivatives.

A, 15N-labeled N-acetyl-N-nitrosotryptophan prepared from reaction of N-acetyl-tryptophan 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 addition of 1N NaOH). B, 15N-labeled N-acetyl-N-nitrosotryptophan

and C, 15N-labeled lysine-15N-nitrosotryptophan-lysine were produced from reaction of either 100 mM

N-acetyl-tryptophan or 200 mM lysine-tryptophan-lysine with preformed 1 5•NO in oxygenated

potassium phosphate/triglycine buffer (250 mM/25 mM, pH 8.0, 37°C). Under the conditions of B and C

the pH decreased to 7.4 during experiment.

Fig. 3. Influence of morpholine and piperazine on MAMA NONOate-induced nitrosation of N-

acetyl-tryptophan.

MAMA NONOate (0.5 mM) was incubated for 30 min with 2 mM N-acetyl-tryptophan in potassium

phosphate buffer (50 mM, pH 7.5, 37°C) in the presence of various concentrations of A morpholine or


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B piperazine, respectively. N-acetyl-N-nitrosotryptophan formation was monitored at 335 nm. Each

value represents the mean +/- SD of 3 determinations.

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 +/- SD of 3 determinations.

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 electrochemically

monitored with the •NO-electrode.


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Table I

Rate data for the reaction of tryptophan derivatives with N2O3

N2O3-target competitor T IC50 IC50 k x 107

(apparent) (correcteda) (apparent)

2 mM °C mM mM M-1s-1

N-acetyl-tryptophan morpholine 37 13.4 2.1 6.4

N-acetyl-tryptophan piperazine 37 2.7 2.7 n.c.b

tripeptide morpholine 37 14.8 2.3 6.4

N-acetyl-tryptophan morpholine 25 15.0 1.4 4.4

melatonin morpholine 37 23.4 3.0 9.2

a, corrected for the concentration of the operating amine.

b, n.c.: not calculable.


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Table II

Tryptophan and cysteine residues in proteins which may be subject to S-nitrosation in

physiological regulation mechanisms

proteina tryptophan cysteine references

tissue transglutaminase 30 17 (50)

caspase 3 8 21 (51)

aldehyde dehydrogenase 5 5 (52)

tissue plasminogen activator 10 24 (46)

creatine kinase 4 4 (53-56)

ryanodine receptor 56 89 (13)

Ca 2+ ATPase 12 25 (14)

mouse adenylyl cyclase type I 15 28 (18)

c-jun N-terminal kinase 4 8 (15)

a, randomly selected from the RCSB Protein Data Bank (44).


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FIGURE 1A


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FIGURE 1B


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Figure 2


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Figure 3


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Figure 4A


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Figure 4B


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Figure 5


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Michael Kirsch, Anke Fuchs and Herbert de Grootat physiological pH

Regiospecific nitrosation of N-(terminal) blocked tryptophan derivatives by N2O3

published online January 27, 2003J. Biol. Chem.

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regiospecific nitrosation of n-(terminal) blocked … · - 3 - n 2o 3-induced tryptophan...

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