From the Indian Institute of Chemical Biology, Jadavpur, Calcutta, India 700032
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ABSTRACT |
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Intramolecular aromatic interactions in aqueous
solution often lead to stacked conformation for model organic
molecules. This designing principle was used to develop stacked and
folded uridine nucleotide analogs that showed highly quenched
fluoroscence in aqueous solution by attaching the fluorophore
1-aminonaphthalene-5-sulfonate (AmNS) to the terminal phosphate via a
phosphoramidate bond. Severalfold enhancement of
fluorescence could be observed by destacking the molecules in
organic solvents, such as isopropanol and dimethylsulfoxide or by
enzymatic cleavage of the pyrophosphate bond. Stacking and destacking
were confirmed by 1-H NMR spectroscopy. The extent of
quenching of the uridine derivatives correlated very well with the
extent of stacking. Taking 5-H as the monitor, temperature-variable NMR
studies demonstrated the presence of a rapid interconversionary
equilibrium between the stacked and open forms for
uridine-5'-diphosphoro- Since the seminal work of Weber and Laurence (1) with
1-anilinonaphthalene-8-sulfonic acid, extrinsic fluorescent
probes have been extensively used to monitor various aspects of
protein-ligand or enzyme-substrate interactions. Among others, these
probes have been used (i) to establish the degree of polarity or
hydrophobicity of a particular region of a protein, (ii) to measure the
distance between groups on protein surface, (iii) to measure the extent of flexibility of protein in solution, (iv) to measure the rate of very
rapid conformational transitions, and (v) to measure kinetic constants
of interaction between protein and ligand (2). To facilitate these
studies, a variety of derivatized fluorescent ligands or substrate
analogs have been synthesized over the years without any serious
attention being given to their solution conformations or their
transitions to new conformations on interaction with the target
proteins. Such conformational transitions are often crucial steps in
biological interactions as typically exemplified by NAD, the common
cofactor for a very large number of dehydrogenases. The molecule
exhibits reversible stacking between the adenine and pyridine moiety
with both the open and the closed forms in rapid interconversionary
equilibrium in aqueous solutions (3, 4). During catalysis, NAD takes a
totally extended conformation on the enzyme surface; the conserved
tertiary structure of the pyridine nucleotide binding site being a very
characteristic feature of all these oxidoreductases (5).
Etheno-ATP was originally synthesized as a fluorescent analog of ATP.
It had a high quantum yield and a long fluorescence lifetime and could
be used to follow ATP interactions primarily by polarization studies
(6-8). In contrast, as in case of NAD, significant population of
etheno-NAD was in a folded conformation in aqueous solution as a result
of aromatic interactions between the pyridine and the modified adenine
moiety, leading to dynamic collisional quenching of fluorescence and
short fluorescence life-time (9). This stacked and quenched fluorophore
was brilliantly used to establish negative co-operativity for
glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle for the
binding of the tetrameric apoenzyme to the coenzyme. The conformational
transition of etheno-NAD from folded to stretched conformation as
reflected by its enhanced fluorescence on interaction with the target
protein was the monitoring parameter for this purpose (10). Although
stacked fluorophore with quenched fluorescence can be of immense use in
protein-ligand binding studies, as is exemplified by etheno-NAD, it is
surprising to note that no deliberate effort has so far been made to
design such compounds taking advantage of the potential aromatic
interaction between the attached fluorophore and a suitable moiety of
the desired biomolecule.
The interaction between aromatic rings is of wide chemical and
biological interest, because it plays important roles in vital biological processes, such as stabilization of protein and nucleic acid
structure and recognition of mRNA cap-binding proteins, and in the
biological reduction by NADH (11-14). Studies with model systems such
as benzene, naphthalene, and their fluorinated derivatives have shown
formation of both T-type and parallel stacking in the gas and solution
phases. The energetics of such interactions have been calculated
(15-19). In general, the interaction between two nonpolar aromatic
ring systems is so weak that it is easily compensated by the entropy
factor. This is expected to be predominant only in concentrated
solutions or when the interacting groups are brought together by some
other interactions such as coulombic interactions or hydrogen bonds as
in the designing of devices for molecular recognition, catalysis, and
development of self-replicating molecules and molecular clips (20-24).
Such interactions are facilitated when the aromatic moieties are
brought close to each other, making them substituents of the same
molecule (25-28). In this report, we demonstrate that following this
designing principle, stacked nucleotide fluorophores that are in rapid
equilibrium with the stretched forms can be synthesized quite easily.
More importantly, rapid collision between the fluorophore and the
heterocyclic base often leads to dramatic quenching of fluorescence
that can be used to monitor conformational changes of the ligand on
interaction with its target protein.
In search of a suitable fluorescent nucleotide substrate for the
DNA-dependent RNA polymerase, Yarbrough and colleagues
synthesized a new class of fluorescent nucleostide triphosphate analogs
that contained the fluorophore 1-aminonaphthalene-5-sulfonate
(AmNS)1 attached via a
All common chemicals such as buffering salts and solvents were
purchased from SD Fine Chemicals, SRL, Qualigen, or Merck. The
spectrograde solvents were purchased from Spectrochem. AmNS was
purchased from Fluka. The nucleotides 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, DEAE-cellulose, and all other chemicals unless otherwise mentioned were purchased from Sigma.
Spectrophotometric analyses were carried out in a Hitachi U3200
spectrophotometer. Fluorimetric measurements were done in a Hitachi
F4010 spectrofluorimeter. 1H NMR experiments were done in
either a 100-MHz Jeol or a 200-MHz Bruker NMR spectrometer.
Temperature-variable NMR was done in a 100-MHz Jeol NMR spectrometer.
Synthesis and Purification of Uridine Nucleotide-1-(5-sulfonic
acid) Naphthylamidates--
The nucleotide-AmNS derivatives were
synthesized according to the procedure of Yarbrough et al.
(29) with some modifications. 223.5 mg (1 mmol) of AmNS was added to 10 ml of water, and the pH was adjusted to 5.8 with 0.1 N
NaOH. Any insoluble material was removed by centrifugation. 4 ml of
12.5 mM nucleotide and 2 ml of 1 M EDC at pH
5.8 were added to a reaction vessel maintained at 20 °C. The
reaction was initiated by adding 10 ml of AmNS solution and allowed to
continue for 2.5-3 h. The pH was kept between 5.65 and 5.75 by
periodic addition of 0.1 N HCl. After the completion of the
reaction, the mixture was neutralized with 0.1 N NaOH and made 0.05 M in ammonium bicarbonate. This was then
centrifuged to remove any insoluble material. The clear supernatant was
then loaded onto a 40-ml DEAE-cellulose column equilibrated with 0.05 M ammonium bicarbonate and washed with 60 ml of 0.05 M ammonium bicarbonate, followed by a 600-ml gradient of
ammonium bicarbonate (0.05-0.5 M) with a flow rate of 25 ml/h. The fluorescent analog eluted out after the unreacted AmNS and
showed a brilliant blue fluorescence. 6-ml fractions were collected,
and the absorbance was monitored at 260 and 320 nm. The fractions for
which the ratio of A260 and
A320 fell within 1.75-1.85 were pooled. The
value for unreacted AmNS was ~0.8. The pooled fractions were then
subjected to repeated evaporation with water under reduced pressure at
35 °C to drive out ammonium bicarbonate. The purified material was dissolved in 0.5-2 ml of water. Purity was assessed by TLC on cellulose plates developed with absolute alcohol and 0.5 M
ammonium acetate, pH 7.5, in the ratio 7:3. If traces of free AmNS were detected, the purified fluorescent nucleotide was rechromatographed on
a 15-ml DEAE-cellulose column using a 300-ml gradient of ammonium bicarbonate (0.05-0.25 M). All other nucleotide
derivatives were synthesized essentially following the same protocol.
The yield was between 30 and 40% of the starting nucleotide.
The pyrophosphate adduct of AmNS (PPAmNS) was prepared by reacting
sodium pyrophosphate with AmNS and the water-soluble
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide under the same
conditions described above for the synthesis of uridine nucleotide
analogs. It was purified by chromatography on a DEAE-cellulose column
and was shown to be homogeneous on TLC as described earlier.
The uridine nucleotide derivatives had a absorbance peak at 320 nm
compared with PPAmNS and AmNS, both of which had peaks at 330 nm. To
take advantage of this peak shift and also to compare our results with
that of earlier published values by Yarbrough et al. (29),
all fluorimetric experiments were carried out by exciting the
fluorophores at 360 nm.
Acid hydrolysis and and phosphodiesterase digestion of the
phosphoramidates were performed according to the procedure of Yarbrough et al. (29) and Yarbrough (33).
1H NMR Spectroscopy--
The 1H NMR
spectra of the samples were usually taken in the 200-MHz Bruker NMR
spectrometer or the 100-MHz Jeol NMR spectrometer. In all cases,
~5-10-mg samples were taken in 250-300 µl D2O or d6-Me2SO. The acetone peak at 2.20 ppm was
taken as the internal standard for experiments in D2O, and
for experiments in d6-Me2SO, the signal
attributable to the residual protons of the solvent at 2.49 ppm was
taken as the internal standard. The temperature-variable 1H
NMR in D2O was done in the 100-MHz Jeol NMR spectrometer
using an acetone signal at 2.20 ppm as standard throughout the
experiment. The NMR tube was tightly capped to avoid escape of acetone
at high temperature (maximum 80 °C). An external thermocouple sensor fitted with the instrument was used as a temperature probe.
Enhancement of Fluorescence in Absence of Phosphodiester Bond
Cleavage--
Fig. 1A clearly
shows that phosphodiester bond cleavage is not a prerequisite for
fluorescence enhancement of uridine-5'-diphosphoro- NMR Evidence for Stacking Interaction--
When the 1H
NMR spectrum of UDPAmNS in D2O was compared with that of
free UDP, a large upfield shift of the signals (~0.5 ppm) was
observed for both the uracil protons at the 5 and 6 positions (Fig.
2, A and B). The
signal for the 6-H, which overlaps with the naphthalene proton signals,
was confirmed by the position of the cross-peaks between the 5-H and
6-H in the two-dimensional COSY spectrum and from proton-decoupling
studies (data not shown). This type of symmetrical upfield shift can
best be interpreted if the uracil ring comes very close to the
naphthalene ring with the two protons positioned symmetrically over the
two naphthalene ring centers. The proximity of the two rings in aqueous
solutions was also indicated by the nuclear Overhauser effect
spectroscopy spectrum, which showed faint but clear cross-peaks between
the 5-H of the uracil and the naphthalene proton signals (data not shown).
The chemical shifts in D2O were found to be independent of
concentration, which ruled out any major intermolecular stacking interactions. When the 1H NMR spectrum was taken in
d6-Me2SO, the signals for 5-H and 6-H shifted
downfield, and their chemical shifts were almost identical to those of
UDP, confirming destacking of UDPAmNS under this condition (Fig.
2C). Finally, the extent of stacking interaction as
indicated by the extent of the peak shift for 5-H was found to vary
with the intermediate phosphoryl chain length. Excellent correlation was observed with the corresponding enhancement of fluorescence in
100% isopropanol. Of the three uridine nucleotide analogs, the UDP
analog was found to have the maximum stacking interaction and is the
most quenched (Fig. 2, inset).
Energetics of Stacking--
To study the temperature dependence of
the stacking phenomenon, the 5-H peak was monitored because it was
distinct and isolated. Fig. 3 shows that
after increasing the temperature from 30 to 80 °C, a gradual
downfield shift of 5-H peak was observed, which approached the chemical
shift for the proton for UDP. This indicates that the equilibrium
constant is changing with rising temperature to favor the extended
form. Because no separate signals for the folded and unfolded forms
were recorded, a rapid interconversion between the two forms could be
assumed. The interconversion is faster with respect to the NMR time
scale, and the observed value is actually a weighted average of the two
forms. In their classic conformational study of NAD in aqueous solution
using NMR techniques, Jardetzky and Wade-Jardetzky (4) observed a
similar gradual shift of the proton signals on increasing the
temperature. Using similar calculations as done by Jardetzky and
Wade-Jardetzky (4) for NAD, the thermodynamic parameters at 30 °C
were calculated to be Fluorescence Lifetime Measurements--
To understand the
molecular basis of quenching, fluorescence lifetime measurements were
carried out at 25 °C. The lifetime of UDPAmNS in water was found to
be 2.5 ns, which increased to 16 ns on phosphodiesterase treatment. The
fluorescence lifetime of PPAmNS in water was determined to be 15 ns. The lifetime of UDPAmNS in Me2SO was 11 ns. Such a
greatly reduced lifetime of UDPAmNS in aqueous solution strongly
suggests collisional interaction between the two stacked rings to be
the cause for the quenching of fluorescence.
Although a significant amount of both theoretical and experimental
work has been done in recent years, the relative contribution of forces
that drive aromatic-aromatic interactions in aqueous solutions is not
clear yet. Thermodynamic signatures for self-association of purine and
pyrimidine derivatives in aqueous solution (enthalpically favorable but
entropically unfavorable) have been interpreted to imply that these
associations are driven by intrinsic attractions between the
heterocyclic rings, rather than by their mutual exclusion from water.
The nature of the attraction between the heterocycles is uncertain;
both dispersion forces and interactions between partial charges within
the adjacent rings have been assumed (36, 37). In a recent study,
involving naphthyl and adenine moieties connected through a
carboxylated propylene linker, Newcomb and Gellman (26) suggested that
attractive interactions between partial positive and negative charges
along with a "nonclassical" hydrophobic effect may be the main
driving forces for stacking in aqueous solutions. Whatever may be the
driving force for stacking, our work shows that all the uridine
nucleotides have a significant population of molecules in stacked
conformation that is in equilibrium with the relaxed conformation in
aqueous solution. ATPAmNS does not undergo quenching of fluorescence in
aqueous solution and has a long fluoroscence lifetime of 20 ns in
water. But contrary to our expectation, it was found to assume a
stacked conformation in aqueous solution as evidenced from NMR
spectroscopy.2 Clearly,
stacking is a necessary but not a sufficient condition for the
designing of such quenched fluorophores. Dynamic fluorescence quenching
as evidenced by very significant reduction in lifetime for UDPAmNS
compared with that of free PPAmNS in aqueous solution suggests
collisional interaction between the two rings. It is likely that
replacement of the smaller pyrimidine ring by the more extended purine
ring as in ATPAmNS introduces steric problems that still result in
stacking, but the desired orientation and proximity of groups involved
in quenching are not achieved. Aromatic stacking can take various
conformations ranging from parallel to T type, although theoretical
calculations suggest the T type to be energetically the preferred
conformation in case of a benzene-naphthalene type interaction similar
to our system (35). This remains an unanswered aspect of our present
work. Needless to say, much more work needs to be done with different
fluorophores and different nucleotides before the principles governing
stacking that leads also to quenching can be understood. Our present
work is progressing in that direction.
The usefulness of such stacked and quenched nucleotide fluorophores is
easy to visualize. The quenched ligand can be conveniently used as a
probe to study its interaction with the target protein provided the
fluorophore takes a stretched or unstacked conformation on the protein
surface. Kinetic manipulations can then provide information regarding
bindng affinity, nunber of binding sites, and nature of cooperativity,
if any. This was, in fact, essentially done for
glycerldehyde-3-phosphate dehydrogenase from rabbit muscle with
etheno-NAD as the probe (10). Folding studies can also be facilitated
by using these probes to monitor the generation of ligand binding site
during the folding process. In the following paper usefulness of
UDPAmNS as a quenched fluorophore will be demonstrated taking
UDPglucose-4-epimerase from Escherichia coli as the target
enzyme (32).
-1-(5-sulfonic acid) naphthylamidate
(UDPAmNS) in aqueous solution.
H was calculated to be
2.3
Kcal/mol, with 43-50% of the population in stacked conformation.
Fluorescence lifetime for UDPAmNS in water was determined to be 2.5 ns as against 11 ns in dimethyl sulfoxide or 15 ns for the
pyrophosphate adduct of AmNS in water. Such a greatly reduced lifetime
for UDPAmNS in water suggests collisional interaction between the
pyrimidine and thefluorophore moieties to be responsible for quenching.
The potential usefulness of such stacked and quenched nucleotide
fluorophores as probes for protein-ligand interaction studies has been
briefly discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-phosphoramidate bond (29, 30). A linear increase of fluorescence
was observed during RNA synthesis with UTPAmNS as one of the
substrates. This was assumed to be attributable to the
pyrophosphorolysis of UTPAmNS during catalysis, because independent
enzymatic cleavage of the analog with phosphodiesterase also led to
severalfold enhancement of fluorescence. The possibility of stacking
was implied but was not systematically explored. UTPAmNS has since been
regularly used for studies on the topology of the RNA polymerase active
site without actually realizing the significance of the solution
conformation of the synthesized fluorophore (31). We have now
reinvestigated the problem to understand the reason for this intense
quenching of fluorescence. For this purpose we synthesized all the
three AmNS derivatives of uridine nucleotides with appropriate
controls. The extent of quenching of fluorescence of the uridine
phosphates could be clearly correlated with their degree of stacking
interactions. The molecules showed reversible intramolecular stacking
between uracil and naphthalene moieties with both the open and the
closed forms in rapid interconversionary equilibrium in aqueous medium.
Phosphodiester bond cleavage is not a prerequisite for fluorescence
quenching. Such enhancement can also be brought about by destacking the
molecules in nonaqueous solvents or on binding to a protein in a
stretched conformation. In the following paper we shall demonstrate the
usefulness of one of these stacked and quenched fluorophores to follow
its conformational transition from stacked to the stretched form as it
lands on the substrate binding site of UDP-galactose-4-epimerase as a
substrate analog (32). It is anticipated that designing of such stacked fluorophores for other nucleotides will greatly facilitate analysis of
many aspects of nucleotide-protein interactions of biological importance and also for development of monitors for high throughput screening systems.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-1-(5-sulfonic acid) naphthylamidate (UDPAmNS), because decrease of medium polarity alone can lead to complete dequenching of fluorescence. A 8-fold enhancement of fluorescence could be observed in 100% isopropanol. Similar results were also obtained with Me2SO. For
comparison, the relative fluorescence values of free AmNS, PPAmNS, and
UDPAmNS, are presented in Fig. 1B. Formation of a
phosphoramidate bond leads to a 5-fold increase in fluorescence
compared with AmNS, but introduction of uridine moiety dramatically
quenches the fluorescence (Fig. 1B, trace c), which becomes
much lower than that of the free fluorophore. In a reverse experiment,
the quenched fluorescence of UDPAmNS was fully released on
phosphodiesterase treatment (Fig. 1B, trace d), which
drastically reduced as the free fluorophore (AmNS) was generated by
further treatment with alkaline phosphatase (Fig. 1B, trace
e) or by acid hydrolysis of UDPAmNS (Fig. 1B, trace
f). The phoshodiesterase-treated product is presumably PAmNS. The
difference in fluorescence emission maxima between traces b
and d probably reflects the difference in spectrum of PPAmNS and PAmNS. Taken together, these results suggested stacking interaction as the possible basis for quenching of fluorescence for UDPAmNS. As in
the case of UDPAmNS, enhanced fluorescence could also be observed both
for UMPAmNS and UTPAmNS in 100% isopropanol, the extent being 7- and
5-fold compared with that in water. ATPAmNS, GTPAmNS, and PPAmNS failed
to show any significant enhancement under identical conditions. Most of
the subsequent spectroscopic work reported here was carried out with
UDPAmNS, because this molecule showed maximum enhancement of
fluorescence and was also used as the probe for our subsequent study
with UDPgalactose-4-epimerase, as reported in the following paper
(32).
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Fig. 1.
A, dependence of fluorescence of UDPAmNS
on solvent polarity. Equal amounts of UDPAmNS (9.3 µM)
were taken in water and in varying concentrations of isopropanol and
the fluorescence emission scanned. a, water; b,
25%; c, 50%; d, 75%; and e, 100%
isopropanol. Excitation wavelength is 360 nm. B, relative
fluorescence diagram. a, AmNS (9.3 µM);
b, PPAmNS (9.3 µM); c, UDPAmNS (9.3 µM); d, after adding snake venom
phosphodiesterase (2 µg) to c and incubation for 10 min;
e, after adding alkaline phosphatase (10 µg) to
d and incubation for 20 min; f, after incubating
UDPAmNS (9.3 µM) in 0.5 N HCl at 37 °C for
3 h. All incubations were done in 50 mM Tris-HCl, pH
8.0, containing 10 mM MgCl2 and 0.1 mM dithiothreitol. The excitation wavelength used was 360 nm.
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Fig. 2.
1H NMR spectra of UDP and
UDPAmNS. A, UDP in D2O; B,
UDPAmNS in D2O; C, UDPAmNS in
d6-Me2SO. The signals for 5-H, 6-H and 1'-H are
indicated by arrows. The position of the 6-H signal in
UDPAmNS was confirmed by the 1H,1H-COSY
spectrum from the position of the cross-peaks between 5-H and 6-H.
Inset, correlation between the 5-H NMR signal shift in
D2O for the uridine nucleotide derivatives on stacking and
the enhancement of fluorescence in 100% isopropanol compared with that
in water. and
, 5-H signal shift and fluorescence ratio,
respectively.
H =
2.3 Kcal/mol,
S =
7.7 entropy units, and
Keq = 0.75-1.0, and the percentage of stacked
form was 43-50%. These values are close to those obtained
experimentally for other stacked systems such as NAD (4), etheno-NAD
(9), and FAD (34). The theoretical value for stabilization energy in
the benzene-naphthalene system as recently calculated by Jorgensen and
Severence (35) is
3.2 to
3.5 Kcal/mol, which is sufficiently close
to that obtained for our system.
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Fig. 3.
Dependence of chemical shift on
temperature. 1H NMR UDPAmNS was taken in D2O at
different temperatures from 30 to 80 °C. The chemical shift for the
proton at the 5 position of the uracil ring was observed for this
purpose.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Eshak Ali and Dr. Anup Bhattacharjya (Indian Institute of Chemical Biology, Calcutta, India) for lively discussions and suggestion throughout the course of this work. We are also grateful to Dr. Amit Basak (Department of Chemistry, Indian Institute of Technology, Kharagpur, India) for providing us the 200-MHz NMR facility.
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FOOTNOTES |
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* This work was supported in part by the Council for Scientific and Industrial Research (CSIR), India.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.
Research Associate for the CSIR. Present address: Dept. of
Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90024.
§ Emeritus Scientist for CSIR. To whom correspondence should be addressed: Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Rd., Jadavpur, Calcutta, India 700032. Tel. and Fax: 91-33-4735197; E-mail: IICHBIO{at}GIASCL01.VSNL.NET.IN.
2 G. Dhar and A. Bhaduri, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
AmNS, 1-aminonaphthalene-5-sulfonate;
PPAmNS, pyrophosphate adduct of
AmNS;
UDPAmNS, uridine-5'-diphosphoro--1-(5-sulfonic acid)
naphthylamidate;
UTPAmNS, uridine-5'-triphosphors-
-1-(5-sulfonic
acid)naphthylamidate.
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