Generation, basic chemistry, and detection of N-nitrosotryptophan derivatives

Article abstract of DOI:10.1039/b713377b

N-Terminal blocked tryptophan derivatives like melatonin or tryptophan residues in peptides are easily nitrosated at the nitrogen atom of the indole ring to give the corresponding N-nitrosotryptophan derivatives. This article provides a comprehensive view of the synthesis, chemical properties, and detection methods of this class of N-nitroso compounds of potential importance in biological systems. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b713377b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Generation, basic chemistry, and detection of N-nitrosotryptophan derivatives
a
b
Michael Kirsch* and Hans-Gert Korth
Received 30th August 2007, Accepted 1st October 2007
First published as an Advance Article on the web 17th October 2007
DOI: 10.1039/b713377b
N-Terminal blocked tryptophan derivatives like melatonin or tryptophan residues in peptides are easily
nitrosated at the nitrogen atom of the indole ring to give the corresponding N-nitrosotryptophan
derivatives. This article provides a comprehensive view of the synthesis, chemical properties, and
detection methods of this class of N-nitroso compounds of potential importance in biological systems.
7
Introduction
S-nitrosothiols. It is currently often proposed that the nitrosation
of cysteine residues in proteins should be an important posttrans-
lational modification event in signal transduction mechanisms as
well as in the storage and transport of nitric oxide. Since N
reacts at physiological pH with N-terminal blocked tryptophan
1
In 1901 Hopkins and Cole were the first to isolate tryptophan,
the structure of which was established in 1908 by Ellinger and
8–10
2
O
3
2
Flamand. Although several other amino acids had been described
before, tryptophan was the first one that was demonstrated to be
6
derivatives as fast as with cysteine residues, N-nitrosotryptophan
3,4
indispensible. Tryptophan is probably the indole derivative most
widely distributed in nature. It is converted into many compounds
of important biological significance, e.g. neurohormones (sero-
tonin and melatonin), the phytohormone indoleacetic acid, some
residues must also be expected to be produced endogenously. It has
been demonstrated in experimental systems that pharmacological
effects of N-nitrosotryptophan compounds like vasorelaxation
and platelet inhibiting activity are comparable to the ones induced
5
pigments found in the eyes of insects, and a variety of alkaloids.
11
by S-nitrosocysteine derivatives. In contrast to S-nitrosothiols,
In chemical systems, N-terminal blocked tryptophan derivatives
like N-acetyltryptophan (but not tryptophan itself) can be easily
nitrosated at the nitrogen atom of the indole ring even at
physiological pH values. This capability is very similar to the
nitrosation of cysteine and its derivatives to give the corresponding
N-nitrosotryptophan derivatives react spontaneously with various
biologically important molecules at physiological pH. During
such reactions nitric oxide is often released. The strongly un-
derestimated capability to release nitric oxide suggests that N-
nitrosotryptophan derivatives act as special nitric oxide storage
compounds. Generally, the understanding of the biological impact
of a certain compound or class of compounds can only be
developed when its basic chemistry is well understood, when
reproducible protocols for its synthesis exist, and when sensitive
and versatile detection procedures are available. In order to address
6
a
Institut f u¨ r Physiologische Chemie, Universit a¨ tsklinikum Essen, Hufeland-
strasse 55, 45122 Essen, Germany. E-mail: michael.kirsch@uni-duisburg-
essen.de; Fax: (49) 201-723 5943; Tel: (49) 201-723 4108
b
Institut f u¨ r Organische Chemie, Universit a¨ t Duisburg-Essen, Univer-
sit a¨ tsstrasse 5, 45117 Essen, Germany
Michael Kirsch received a Ph.D. in 1996 in physical chemistry from the Heinrich-Heine University of D u¨ sseldorf, Germany, with Prof.
E. Baumgarten. Since 1994 he has worked in the group of Prof. de Groot at the University Hospital of Essen. In 2003 he received a
postdoctoral lecture qualification in medical biochemistry. His research interests are redox chemistry and posttranslational modifications in
general, with current focus on action and detection of nitroso compounds in biological systems.
Hans-Gert Korth received a Ph.D. in 1980 from the University
of M u¨ nster, Germany, with Prof. Reiner Sustmann. Since 1980 he has
held a position as research scientist in the group of Reiner Sustmann at
the University of Essen, now University of Duisburg-Essen. In 1987/88
he spent a research year at the NRC of Canada in Ottawa with Prof.
Keith Ingold. His research interests are radical chemistry and ESR
spectroscopy in general, with current focus on reactive oxygen and
nitrogen species in biological systems.
Michael Kirsch
Hans-Gert Korth
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these issues for N-nitrosotryptophan derivatives, we studied their
reactions with biologically important molecules intensively. The
present findings are summarized in this report.
Table 1 Calculated charges of the heavy atoms of the indole ring in N-
acetyltryptophan
a
Charge
Position
Mulliken
Electrostatic
Natural
Preparation of N-nitrosotryptophan compounds
N-1
C-2
C-3
C-4
C-5
C-6
C-7
C-7a
C-3a
−0.704
−0.017
0.070
−0.212
−0.140
−0.152
−0.164
0.318
−0.459
−0.083
−0.116
−0.219
−0.167
−0.085
−0.326
0.270
−0.559
−0.026
−0.121
−0.222
−0.258
−0.242
−0.265
0.158
The traditional synthetic route to N-terminal blocked N-
nitrosotryptophan derivatives is the reaction between tryptophan
12
derivatives and nitrite in acidic media. At acidic conditions
pH ≤ 3), nitrite is expected to yield an electrophilic nitrosating
intermediate (NO , or its equivalent, e.g., H NO , N ) which
(
+
+
7
2
2
2
O
3
attacks the five-membered ring of the indole moiety. In indoles
with the 3-position unsubstituted, nitrosation takes place very
rapidly at C-3 and the thus formed C-nitroso compound tautomer-
0.075
0.092
−0.088
a
N-Acetyltryptophan was optimized at the B3LYP/6-31G(d) level of
theory. Charges were calculated at this level by using the Spartan 06
software package.
13
izes quickly (where possible) to the oxime form. In 3-substituted
tryptophan derivatives the corresponding N-nitroso compounds
are produced almost quantitatively to give mixtures of the Z-
and E-conformers with respect to the N–N=O group (ca. 35%
Thus, for the nitrosation process (eqn (1)) there is no need to
assume another nucleophilic center for primary attack other than
the secondary nitrogen atom of the indole ring itself.
Interestingly, N-terminal blocked tryptophan derivatives can
also be smoothly nitrosated at physiological pH values. For
this, three nitrosating entities have been suggested. The first
nitrosating species is N
dependently that melatonin is nitrosated by nitric oxide under
aerobic conditions.
1
5
6,14–16
and 65%, respectively), as proven by N NMR spectrometry.
1
5
The discrimination of the conformers by N NMR at room
temperature in solution is possible because the rotational barrier of
the N–NO bond is relatively high (high level quantum mechanical
calculations performed at the CBS-Q3 level of theory predict
−
1
a barrier of 17.7 kcal mol in aqueous solution (calculations
not shown) which is about 62% of the N–NO bond dissociation
2
O . Recently, two groups reported in-
3
17
energy ). Detailed protocols for the preparation of both N-acetyl-
N-nitrosotryptophan (NANT) and N-nitrosomelatonin are given
22,23
The titles of these studies, “Nitrosation
22
of melatonin by nitric oxide” and “Scavenging of NO by
13,15,18
in the literature.
23
melatonin”, respectively, appeared to be somewhat misleading
Some ambiguities exist about the precise mechanism of the
nitrosation process. From chemical experience one would expect
that electrophilic nitrosating intermediates directly attack the
nitrogen atom of the indole ring (eqn (1)):
with respect to the nitrosating entity because N-terminal blocked
tryptophan derivatives do not react with nitric oxide in the
24
strict absence of oxygen. However, two well-known N
2
3
O -
7
scavengers, morpholine and piperazine, which do not react
with N-nitrosotryptophan compounds, are highly effective in
inhibiting such nitric oxide-dependent nitrosations of N-terminal
6
blocked tryptophan derivatives, clearly indicating that under
the conditions employed by the two cited papers, N
2
O is the
3
(
Eqn 1)
actual nitrosating species. From competition experiments with
7
−1
−1
morpholine, rate constants of 4.4 × 10
M
s
and 9.2 ×
7
−1 −1
1
0 M
s
were evaluated for the N
2
O
3
-mediated nitrosation of
N-acetyltryptophan and melatonin, respectively.
6
The second compound able to nitrosate N-terminal blocked
1
8,22,25,26
Somewhat surprisingly, however, with consideration of the
molecular structure of 3-substituted indoles, it has been concluded
that the most nucleophilic center of the molecule is the C-3
tryptophan derivatives at physiological pH is peroxynitrite.
This reaction does not seem to have a decisive impact under physi-
ological situations because in comparison to N -generating sys-
tems peroxynitrite levels are very low in the (ubiquitous) presence
2
O
3
19,20
position,
because in 18 M sulfuric acid protonation occurs at
21
18,27
28
this position. Therefore, it was proposed that N-terminal blocked
tryptophan derivatives were initially nitrosated at the C-3 position
and that the final N-nitroso product is formed via an internal
transfer of the NO function.
Hinman and Whipple noted that hydrogen–deuterium exchange
of carbon dioxide.
Recently, we confirmed a hypothesis of
Williams that the peroxynitrite-induced nitrosation of various
29
compounds proceeds via N
an electrophilic, amine-nitrosating agent than N
2
O
4
, which is somewhat less effective as
+
19,20
30
In contrast to this proposal,
2
O
3
. In addition,
21
peroxynitrite nitrates melatonin at various positions (N-1, C-4
and C-6). However, nitration at positions C-4 and C-6, but
not at N-1, proceeds via radical (NO ) pathways. Thus, the
+
31
of the indole NH (via intermediate NHD ) is strongly accelerated
32
already in 20 lM D
2
SO
4
, i.e., at a proton concentration that does
2
not lead to protonation at the C-3 position. This fact strongly
suggests that the C-3 position does not represent the primary
nucleophilic center. In line with this observation, DFT calculations
for N-acetyltryptophan predict that the largest fraction of negative
charge on the indole ring (Mulliken, electrostatic, and natural
charge) is located at the N-1 position (Table 1).
low efficiency of peroxynitrite to nitrosate N-terminal blocked
tryptophan derivatives can be satisfactorily explained with its
nitrating capabilities.
The third nitrogen–oxygen species capable of producing
3
−
25,33
N-nitrosotryptophan compounds is nitroxyl (HNO/ NO ).
Mechanistic details of this nitrosation process are so far not fully
3
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This journal is © The Royal Society of Chemistry 2007

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understood because of the highly complex chemistry of nitroxyl.
Evidence has been found that depending on the actual reaction
ambient temperature. Noticeably, from reaction of NANT with
thiols, hydrogen peroxide anion, or melatonin, the corresponding
nitroso compounds, i.e., S-nitrosothiols, peroxynitrite, and N-
•
•
conditions, a variety of reactive intermediates, such as HO , NO,
•
−
−
•
34–36
15
O
2
, ONOO , and HN
2
O
2
, may be produced from nitroxyl.
nitrosomelatonin, respectively, could be identified by N-NMR
15,16,43
spectrometry.
These observations clearly support a direct
reaction as outlined in eqn (2b).
Basic chemistry of N-nitrosotryptophan derivatives
Since the chemical properties of YNO are strongly dependent
on the nucleophile part (Y), a variety of products can be generated
from N-nitrosotryptophan derivatives, depending on the pH
value. For example, at pH 4–7 NANT nitrosates halides to
yield N-acetyltryptophan and the corresponding nitrosyl halides
In aqueous solution, N-nitrosotryptophan derivatives can ni-
trosate (“transnitrosation”) a variety of nucleophiles (Y /YH),
−
3
7
37
37
7,38
such as water, halides, pseudo-halides, aliphatic alcohols,
39
40,41
42
6
nucleotides, activated phenols,
catechols, vitamin C, hydro-
4
3
16
18
37
gen peroxide, thiols, and primary aromatic amines (but not
according to eqn (2a). The latter intermediates readily hydrolyze
6
45
aliphatic secondary ones) at virtually all pH values (0–14) to yield
to give nitrous acid (pK (HNO ) = 3.1) and the parent halide
a
2
7
the parent tryptophan derivative and a nitroso compound (YNO).
ion. Thus, in the presence of halides and pseudohalides NANT
operates as a clean source for nitrite. A completely different
situation is encountered when NANT is reacted with Trolox, a
water soluble vitamin E derivative, at pH 7.4–11. In this case the
product YNO, an aryl nitrite, is a short-lived species that does
not readily hydrolyze but undergoes rapid homolytic dissociation
to yield quantitatively nitric oxide and the corresponding Trolox
radical. The latter subsequently dismutates (disproportionates) to
Trolox and to the related quinone. As no other products were
formed in this reaction, NANT now operates as a clean source of
nitric oxide. To generalize these observations, homolytic cleavage
of the transnitrosation product YNO to produce nitric oxide must
+
The simplest transnitrosation, i.e., the exchange of the NO
function between two N-terminal blocked tryptophan derivatives,
15
proceeds even at physiological pH and in non-aqueous solution.
The hydrolysis reaction (YH = H O) of N-nitrosotryptophan
derivatives at pH 1–7 is a proton-catalyzed process (eqn (2a)).
2
37
•
(
Eqn 2a)
always be expected in cases where a stabilized Y radical will be
formed (eqn (3)).
The pH-rate profile of this denitrosation reflects two proton-
catalyzed reactions, from which the one dominating in the pH
range 4–7 is associated with a halide/pseudohalide nucleophilic
37
catalysis. Although there is no doubt that eqn (2a) is basically
correct, it fails to describe the action of some nucleophiles which
have been found to denitrosate N-nitrosotryptophan derivatives
effectively at alkaline pH values. Thus, besides the two proton-
catalyzed denitrosation pathways there must be a third, un-
catalyzed one in which N-nitrosotryptophan derivatives act as
electrophilic nitrosating agents, resulting in heterolytic N–NO
bond fission (eqn (2b)).
(
Eqn 3)
However, the foregoing examples in which the products were
generated stoichiometrically are specific cases. More often sig-
nificantly lower yields of such products are found because the
transnitrosation product YNO may react with the educt, as is the
case with thiols, or the formed YNO decomposes by various
competitive pathways, as is the case with vitamin C, or the N-
16
24
nitrosotryptophan derivative can trap the intermediate reactant
(
Eqn 2b)
24,42
radical, as is the case with catechols and vitamin C.
The
most complex situation is encountered when a radical precursor
46
like peroxynitrite is the initial product, as is the case in the
4
3
In accord, it has been shown that various biologically important
molecules, for instance, thiols, phenols, and some vitamins, in
NANT–H
2
O reaction. Other products from reactions of N-
2
nitrosotryptophan derivatives are listed in Table 2.
16,24,40–42
part react via eqn (2b).
Since the thus formed nitroso
compounds YNO are often very short-lived (preferably because of
rapid hydrolysis) and, therefore, may easily escape detection, the
relevance of eqn (2b) might be questioned. In fact, de Biase et al.
Quantitative determination of N-nitrosotryptophan
derivatives
44
proposed that all processes which finally lead to denitrosation
of N-nitrosotryptophan-derivatives, even the hydrolysis reaction,
should proceed via initial homolysis of the N–NO bond. However,
this assumption cannot be correct, because the aminyl radicals
which are initial products of N–NO homolysis are highly unlikely
to yield the corresponding amine in stoichiometric amounts in
the presence of molecular oxygen. Further, the N–NO bond
Several procedures have been developed for the determination of
N-nitrosotryptophan derivatives. In the absence of residues having
similar UV–Vis absorptions, the maximums of the electronic
−
1
−1
12
absorption around 340 nm (e335 = 6100 M cm for NANT and
6
−1
−1
N-nitrosotryptophan residues in proteins, e346 = 7070 M cm
23
for N-nitrosomelatonin ) have been used to determine concen-
trations of N-nitrosotryptophan and its derivatives. The moderate
extinction coefficients allow the detection of 5–100 lM N-nitroso
compound in a standard 1 cm cuvette.
−
1 17
dissociation energy (28.7 kcal mol ) is too high for a significant
contribution of a rate-determining, spontaneous dissociation at
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Table 2 Products from reaction of N-nitrosotryptophan derivatives with nucleophiles
Reaction
YNO intermediate
Products (yields)
Recommended for detection
Reference
NANT + water
NANT + halide
NANT + R
NANT + serotonin
NOMela + serotonin
NANT + vitamin C
NANT + catechols
Nitrous acid
Nitrosyl halide
Alkylnitrite
Arylnitrite
Arylnitrite
Alkylnitrite
Arylnitrite
Nitrite (100%)
Nitrite (100%)
Alkylnitrite (n.d.)
Nitric oxide (100%)
Nitric oxide (100%)
Nitric oxide (60%), nitroxyl (13%)
Various products
Hydroxyl radical, nitrogen dioxide,
nitrate, nitrite (pH dependent)
No
No
No
No
No
No
No
No
37
37
7,38
40
40
6
aliphatic
a
OH
b
b
42
43
NANT + H
2
O
2
Peroxynitrite
c
NANT + Thiol
S-Nitrosothiol
None
Primary aromatic nitrosamines
N-Nitroso-hydrazine
N-Nitroso-hydrazine derivative
N-Nitroso-hydrazine derivative
Arylnitrite
S-Nitrosothiol (pH dependent)
None
Triazole (55%), deamination (13%)
No
No
Yes, Yes
No
Yes
Yes
Yes
Yes
16
6
18
aliphatic
NANT + R
NANT + R
NANT + N
2
NH
NH
aromatic
d
2
2
H
4
N
2
, NH
3
, N
2
O (n.d.)
This work
This work
This work
41
NANT + MNBDH
MNBDA (80%)
MNBDA (80%)
Nitric oxide (100%)
Nitric oxide (100%)
e
NOW + MNBDH
NANT + Trolox
e
NOW + Trolox
Arylnitrite
41
a
b
c
n.d. = not determined. This yield can only be observed at the beginning of the reaction. The yield is decreased by a reaction of the educt with the
d
aromatic
e
final product.
R
NH
2
= aminophenyl fluorescein and 4,5-diaminofluorescein. NOW = N-nitrosotryptophan residues in proteins.
In the presence of compounds which absorb in the same wave-
dyes were produced in relatively low yields (55% and 13% in the
case of DAF-2 and APF, respectively), the detection limits drop
to 100 nM (DAF-2) and 25 nM (APF), respectively. It should be
noted, however, that at pH 7.4 the pseudo-first order hydrolysis
reaction of N-nitrosotryptophan derivatives at concentrations
lower than 25 nM is faster than any other transnitrosation
length range, similar levels can be detected by separating the N-
15
nitrosotryptophan compounds by capillary zone electrophoresis.
Somewhat lower detection limits can be achieved (around 1 lM)
25,33
when the separation process is performed with HPLC.
The detection limits can be significantly reduced by employing
indirect detection procedures in which the N-nitrosotryptophan
derivatives, but not S-nitrosothiols, react with a suitable com-
pound to produce an easily detectable product. Aromatic amines
16
reaction. This fact limits the quantification of lower levels of
N-nitrosotryptophan residues. Unfortunately, the fluorescence of
DAF-2T and fluorescein is quenched by present proteins to about
38
18
can be nitrosated by N-nitrosotryptophan derivatives to give
50% and 99%, respectively, so that N-nitrosotryptophan residues
7,47
chemically labile aromatic N-nitrosamines.
Accordingly, the
in proteins cannot be quantified by these methods. In order to
improve the detection procedure, other probe systems are required.
N-nitrosamines of aminophenyl fluorescein (APF) and 4,5-
diaminofluorescein (DAF-2) decay to give the fluorescent dyes
fluorescein and triazolfluorescein (DAF-2T), respectively (eqn (4)
and (5)).
48
In 1996 the Stedman group reported that arylhydrazines react
49
effectively with nitrous acid, and in 1999 B u¨ ldt and Karst applied
the nonfluorescent hydrazine derivative N-methyl-4-hydrazino-7-
nitrobenzofurazan (MNBDH) for the quantification of nitrite as
low as 50 nM in acidic solution. The subsequently generated flu-
orescent dye N-methyl-4-amino-7-nitrobenzofurazan (MNBDA)
has a sensitivity limit of 5 nM in non-aqueous solution. A similar
reaction between N-nitrosotryptophan derivatives and hydrazine
compounds, however, has not been reported so far (eqn (6)).
(
Eqn 6)
(
Eqn 4)
1
5
In Fig. 1A it is demonstrated that N-acetyl- N-nitro-
1
5
sotryptophan indeed nitrosates
N
2
H because expected reaction
4
1
5
15
15
50
15
products (i.e. NH
3
, N
2
O, N ) could be detected by N NMR
2
spectrometry.
From this observation it is conceivable that an activated
hydrazine compound should react as well, and, in fact, the
reaction of NANT with MNBDH generated the fluorescent dye
MNBDA with a yield of 80% (Fig. 1B). The major improvement
(
Eqn 5)
Thus, these aromatic amines can be used as reporter molecules
for N-nitrosotryptophan derivatives. Although the fluorescent
18
3
892 | Org. Biomol. Chem., 2007, 5, 3889–3894
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has a quantification limit of 250 nM nitrosotryptophan residues in
the c-globulin fraction (insert in Fig. 1C). This is the first example
that N-nitrosotryptophan residues in proteins can be quantified
by means of a relatively economical fluorescence method.
Although N-nitrosotryptophan residues in other proteins may
also be detected, the problem with the hydrolysis reaction still
exists for the MNBDH assay, and the detection limit is much too
high for a useful quantification in biological samples.
As noted above, stoichiometric amounts of nitric oxide are
released from the reaction of Trolox with N-nitrosotryptophan
41
derivatives. The reaction proceeds with low molecular N-
nitrosotryptophan compounds like NANT as well as with N-
nitrosotryptophan residues in proteins. S-Nitrosothiols do not
release any NO under such conditions because the experiments
are performed in the presence of EDTA (which complexes the cat-
alytically active transition metal ions required for decomposition
of S-nitroso compounds). The influence of the always competing
hydrolysis reaction can be reduced by working at pH 11 where
the rate of the hydrolysis reaction is about 8-fold lower than at
pH 7.4. Further, the rate of the reaction is increased at excess
concentrations of Trolox. Since the Trolox-N-nitrosotryptophan
reaction proceeds even under oxygen-free conditions, the liberated
nitric oxide can be quantified by transporting nitric oxide into
the gas phase where it may be detected with the very sensitive
7
(
5 pM) ozone-chemiluminescence method. Considering all these
facts, the Trolox assay is expected to reach a detection limit of
about 5 nM for N-nitrosotryptophan compounds and is, therefore,
recommended as a standard detection procedure.
Conclusions
N-Nitrosotryptophan derivatives can be both generated and
denitrosated at physiological pH. Under such conditions, the den-
itrosation process starts with a so-called direct transnitrosation,
+
i.e., the NO function is transferred from the N-nitrostryptophan
derivative to various substrates without the formation of other
intermediates, such as, e.g., N
2
3
O . Since the thus formed substrate-
derived nitroso compound YNO often acts as a spontaneous
nitric oxide releasing entity (for example, with catechols, serotonin,
vitamin E, or vitamin C as substrates) or as a nitric oxide transfer
agent (with, for instance, thiols as substrates), N-terminal blocked
tryptophan derivatives feature all chemical capabilities necessary
for action as an endogenous nitric oxide transporter. So far,
the lack of a reliable detection procedure had prevented the
accurate analysis of endogenous or applied N-nitrosotryptophan
derivatives in biological samples. The methods suggested in this
paper are believed to fill this gap.
Fig. 1 Evidence that N-nitrosotryptophan derivatives can be quan-
tified by using an activated hydrazine derivative. (A) N-Acetyl- N-
15
1
5
nitrosotryptophan (100 mM) and
N
2 4
H (200 mM) were mixed in 100 mM
◦
phosphate buffer at pH 7.4 and 25 C. After 2 h, products were identified
by N NMR spectrometry. Solutions of NANT (5–60 lM) (Fig. 1B) or
of N-nitrosotryptophan residues in c-globulin (0–10 lM) (Fig. 1C) in
15
the presence of N-methyl-4-hydrazino-7-nitrobenzofurazan (MNBDH)
◦
References
(
20 lM) were used in 2 mL phosphate buffer (50 mM, pH 7.4, 37 C,
1
00 lM EDTA). The concentrations around the detection limit are
additionally shown in the insert in Fig. 1C. After a reaction period of
h, ethanol (0.5 mL) was added to each sample and the formation of
the fluorescence dye MNBDA was fluorimetrically determined at kem.
1
2
F. G. Hopkins and S. W. Cole, J. Physiol., 1901, 27, 418–431.
A. Ellinger and C. Flamand, Hoppe-Seyler’s Z. Physiol. Chem., 1908,
55, 8–24.
2
=
3 E. G. Willcock and F. G. Hopkins, J. Physiol., 1906, 35, 88–103.
4
5
T. B. Osborne and L. B. Mendel, J. Biol. Chem., 1914, 17, 325–349.
H. Sidransky, Tryptophan. Biochemical and health implications, CRC
Press, Boca Raton, 2002.
5
36 nm (kexc. = 484 nm). The data shown are representative for three
experiments performed independently (Fig. 1A) or are means of three
experiments independently performed in duplicate (Fig. 1B and 1C).
6
7
M. Kirsch, A. Fuchs and H. de Groot, J. Biol. Chem., 2003, 278,
1
1931–11936.
of the MNBDH assay is given by the fact that the fluorescence of
MNBDA is not quenched by proteins (Fig. 1C). The novel assay
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oxide, Elsevier, Amsterdam, 2004.
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