A bifunctional molecule as an artificial flavin mononucleotide cyclase and a chemosensor for selective fluorescent detection of flavins

Article abstract of DOI:10.1021/ja9018012

Flavins, comprising flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin (RF, vitamin B₂), play important roles in numerous redox reactions such as those taking place in the electron-transfer chains of mitochondria in

Full text of DOI:10.1021/ja9018012

Published on Web 07/01/2009
A Bifunctional Molecule as an Artificial Flavin Mononucleotide
Cyclase and a Chemosensor for Selective Fluorescent
Detection of Flavins
Hyun-Woo Rhee,† So Jung Choi,† Sang Ho Yoo,† Yong Oh Jang,† Hun Hee Park,‡
Rosa Mar´ıa Pinto,§ Jose´ Carlos Cameselle,§ Francisco J. Sandoval,| Sanja Roje,|
Kyungja Han,‡ Doo Soo Chung,† Junghun Suh,*,† and Jong-In Hong*,†
Department of Chemistry, College of Natural Sciences, Seoul National UniVersity, Seoul
151-747, Korea, Department of Clinical Pathology, Catholic UniVersity Medical College, Seoul,
Korea, Grupo de Enzimolog´ıa, Departamento de Bioqu´ımica y Biolog´ıa Molecular y Gene´tica,
Facultad de Medicina, UniVersidad de Extremadura, 06080 Badajoz, Spain, and Institute of
Biological Chemistry, Washington State UniVersity, Pullman, Washington 99164
Received March 8, 2009; E-mail: jihong@snu.ac.kr; jhsuh@snu.ac.kr
Abstract: Flavins, comprising flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin
(RF, vitamin B2), play important roles in numerous redox reactions such as those taking place in the electron-
transfer chains of mitochondria in all eukaryotes and of plastids in plants. A selective chemosensor for
flavins would be useful not only in the investigation of metabolic processes but also in the diagnosis of
diseases related to flavins; such a sensor is presently unavailable. Herein, we report the first bifunctional
chemosensor (PTZ-DPA) for flavins. PTZ-DPA consists of bis(Zn2+-dipicolylamine) and phenothiazine.
Bis(Zn2+-dipicolylamine) (referred to here as XyDPA) was found to be an excellent catalyst in the conversion
of FAD into cyclic FMN (riboflavin 4′,5′-cyclic phosphate, cFMN) under physiological conditions, even at
pH 7.4 and 27 °C, with less than 1 mol % of substrate. Utilizing XyDPA’s superior function as an artificial
FMN cyclase and phenothiazine as an electron donor able to quench the fluorescence of an isoalloxazine
ring, PTZ-DPA enabled selective fluorescent discrimination of flavins (FMN, FAD, and RF): FAD shows
ON(+), FMN shows OFF(-), and RF shows NO(0) fluorescence changes upon the addition of PTZ-DPA.
With this selective sensing property, PTZ-DPA is applicable to real-time fluorescent monitoring of riboflavin
kinase (RF to FMN), alkaline phosphatase (FMN to RF), and FAD synthetase (FMN to FAD).
Introduction
However, many artificial metalloenzymes operate efficiently
only at high temperatures over 50 °C, requiring a relatively large
Development of an artificial enzyme with activity comparable
to that of natural enzymes is an important goal in chemical
biology.1-3 Artificial metalloenzymes2,3 have been developed
to mimic the catalytic metallic core of natural enzymes.
amount of enzyme compared to that of substrate such as nucleic
acids,2e,3a proteins,2b or dinucleotides.2f,3c,e Reports of artificial
enzymes working promptly in physiological conditions with less
than 1 mol % of a substrate are rare.
Recently, fluorescent chemosensors have been intensively
developed to detect specific biomolecules or monitor biological
† Seoul National University.
‡ Catholic University Medical College.
events.4,5 In particular, many chemosensors based on bis(Zn2+
-
§ Universidad de Extremadura.
| Washington State University.
DPA) (DPA ) dipicolylamine) complex5 have been synthesized
(1) (a) Breslow, R. Artificial Enzymes; Wiley-VCH: Weinheim, 2005. (b)
Purse, B. W.; Rebek, J., Jr. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
10777. (c) Lehn, J.-M. Science 1985, 227, 4689.
(4) (a) Anslyn, E. V. J. Org. Chem. 2007, 72, 687. (b) Que, E. L.;
Domaille, D. W.; Chang, C. J. Chem. ReV. 2008, 108, 1517. (c) Zhang,
J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Nat. ReV. Mol. Cell.
Biol. 2002, 3, 906. (d) Yee, D. J.; Balsanek, V.; Bauman, D. R.;
Penning, T. M.; Sames, D. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
13304. (e) Dale, T. J.; Rebek, J., Jr. J. Am. Chem. Soc. 2006, 128,
4500.
(2) (a) Breslow, R.; Huang, D. L.; Anslyn, E. Proc. Natl. Acad. Sci. U.S.A.
1989, 86, 1746. (b) Suh, J.; Chei, W. S. Curr. Opin. Chem. Biol. 2008,
12, 207. (c) Chin, J. Curr. Opin. Chem. Biol. 1995, 1, 514. (d)
Williams, N. H.; Takasaki, B.; Wall, M.; Chin, J. Acc. Chem. Res.
1999, 32, 485. (e) Komiyama, M.; Sumaoka, J. Curr. Opin. Chem.
Biol. 1998, 2, 751. (f) Komiyama, M.; Kina, S.; Matsumura, K.;
Sumaoka, J.; Tobey, S.; Lynch, V. M.; Anslyn, E. J. Am. Chem. Soc.
2002, 124, 13731. (g) Letondor, C.; Humbert, N.; Ward, T. R. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 4683. (h) Scarso, A.; Scheffer, U.;
Go¨bel, M.; Broxterman, Q. B.; Kaptein, B.; Formaggio, F.; Toniolo,
C.; Scrimin, P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5144.
(3) (a) Delehanty, J. B.; Stuart, T. C.; Knight, D. A.; Goldman, E. R.;
Thach, D. C.; Bongard, J. E.; Chang, E. L. RNA 2005, 11, 831. (b)
Jang, S. W.; Suh, J. Org. Lett. 2008, 10, 481. (c) Kawahara, S.;
Uchimaru, T. Eur. J. Inorg. Chem. 2001, 2437. (d) Yashiro, M.;
Kaneiwa, H.; Onaka, K.; Komiyama, M. Dalton Trans. 2004, 605.
(e) Young, M. J.; Chin, J. J. Am. Chem. Soc. 1995, 117, 10577.
(5) (a) Ojida, A.; Nonaka, H.; Miyahara, Y.; Tamaru, S.; Sada, K.;
Hamachi, I. Angew. Chem., Int. Ed. 2006, 45, 5518. (b) Leevy, W. M.;
Gammon, S. T.; Jiang, H.; Johnson, J. R.; Maxwell, D. J.; Jackson,
E. N.; Marquez, M.; Piwnica-Worms, D.; Smith, B. D. J. Am. Chem.
Soc. 2006, 128, 16476. (c) Lee, H. N.; Xu, Z.; Kim, S. K.; Swamy,
K. M. K.; Kim, Y.; Kim, S. J.; Yoon, J. J. Am. Chem. Soc. 2007, 129,
3828. (d) Lee, D. H.; Kim, S. Y.; Hong, J. I. Angew. Chem., Int. Ed.
2004, 43, 4777. (e) Rhee, H. W.; Choi, H. Y.; Han, K.; Hong, J. I.
J. Am. Chem. Soc. 2007, 129, 4524. (f) Rhee, H. W.; Lee, C. R.; Cho,
S. H.; Song, M. R.; Cashel, M.; Choy, H. E.; Seok, Y. J.; Hong, J. I.
J. Am. Chem. Soc. 2008, 130, 784.
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Rhee et al.
to detect phosphate-containing biomolecules because this com-
plex binds strongly to phosphate or diphosphate groups in water.
There are many other kinds of phosphate-containing biomol-
ecules that may play critical roles in a living system. However,
many of them have not yet been identified. Therefore, there is
a real need to develop selective chemosensors, not only for the
detection of phosphate-containing biomolecules but also for
biological applications and disease diagnosis.
Riboflavin (RF), also known as vitamin B2, is the central
component of the cofactors flavin adenine dinucleotide (FAD)
and flavin mononucleotide (FMN) and plays a key role in energy
metabolism. FMN and FAD are essential coenzymes in the
electron-transfer chains of mitochondria in all eukaryotes and
of plastids in plants.6a FMN is a prosthetic group of various
oxidoreductases, including NADH dehydrogenase, alcohol oxi-
doreductases, and amino acid oxidoreductases. FAD participates
as an initial electron acceptor in glucose oxidation by glucose
oxidase (GOx). FMN, FAD, and RF structures share an
isoalloxazine ring. The isoalloxazine ring is the key catalytic
component of flavins. It exhibits green fluorescence (quantum
yield, 0.26; excitation wavelength, 445 nm; emission wave-
length, 525 nm) in water.6b Due to the presence of the
isoalloxazine ring, FMN and RF exhibit strong green emissions;
FAD shows only weak fluorescence (quantum yield, 0.03) owing
to its stacked conformation between adenine and the isoallox-
azine ring in water.7
Figure 1. Catalytic splitting of FAD to cyclic FMN by XyDPA. (A)
Fluorescence enhancement at 525 nm in the mixture of FAD (50 µM) and
XyDPA (5 µM) with time. Inset graph: Y-axis, fluorescence intensity at
525 nm; X-axis, elapsed time. (B) Capillary electrophoresis electrophero-
grams of a reaction mixture of FAD (100 µM) and XyDPA (10 µM) after
10 min (upper) and 1 h (lower) for FAD splitting catalyzed by XyDPA (10
µM) at pH 7.4 and 27 °C. (C) Proposed mechanism of FAD splitting to
cFMN and AMP by XyDPA.
Flavins (FAD, FMN, and RF) are not de noVo synthesized
in vertebrates. These organisms thus have well-developed
salvage pathways, consisting of RF uptake and rapid conversion
to FMN or FAD on the mitochondrial membrane.8 RF kinase
(flavokinase, EC 2.7.1.26) phosphorylates RF to FMN, and FAD
synthetase (FAD adenylyltransferase, EC 2.7.7.2) adenylylates
FMN to FAD. Altered levels of RF kinase or FAD synthetase
activities were directly associated with flavin deficiency, which
is responsible for glossitis, cheilosis, organic acidurias, visual
impairment, and growth retardation.9a,b Therefore, sensitive and
selective detection of each flavin and its enzymatic conversion
are critical for diagnosis of these diseases.9
there has been no report on the development of selective
fluorescent chemosensors that can discriminate flavins.
We have developed a bifunctional molecule that can act as
both an artificial enzyme and a chemosensor. Herein, we report
an artificial FMN cyclase that shows superior catalytic FAD
splitting activity, even at pH 7.4 and 27 °C and with less than
1 mol % of a substrate, when compared with many of the
artificial metalloenzymes that operate efficiently only at high
temperatures over 50 °C and with a relatively large amount of
enzyme. Furthermore, we show that this artificial enzyme can
also act as a fluorescent sensor for discriminating among flavins
and monitoring their enzymatic conversions.
Methods to discriminate flavins using high-performance liquid
chromatography (HPLC)10a or capillary electrophoresis (CE)10b
are available. However, these methods lack selectivity and are
not applicable to the real-time assay of enzymatic conversion
of flavins. Therefore, there is a pressing need to develop
selective fluorescent chemosensors for flavins, as such chemosen-
sors would enable the selective and real-time assay of flavin
conversion. An RNA aptamer was developed previously, but it
showed no fluorescence selectivity among flavins; it quenched
fluorescence of all the flavins.11 To the best of our knowledge,
Results and Discussion
FAD Splitting to Cyclic FMN by XyDPA. Recently, we
reported the selective chemosensing of FAD with bis(Zn2+
-
DPA) (XyDPA, Figure 1C).5e XyDPA bound strongly to the
diphosphate group of FAD and caused a change in the in-
tramolecularly stacked conformation between adenine and the
isoalloxazine ring in aqueous solution. As a result, XyDPA
induced 7-fold enhancement in FAD fluorescence in water.
Further studies revealed that XyDPA not only bound to the
diphosphate group of FAD but also split FAD to cyclic
riboflavin 4′,5′-cyclic phosphate (cFMN) and adenosine 5′-
monophosphate (AMP) in 100 mM HEPES buffer solution (pH
7.4) at 27 °C. At first, we observed increasing emission intensity
at 525 nm in the mixture of FAD (50 µM) and XyDPA (5 µM)
with time (Figure 1A). The products of FAD splitting by
XyDPA (cFMN and AMP) were confirmed by CE (Figure 1B).
On the CE electropherogram, the FAD peak decreased and its
(6) (a) Walsh, C. Acc. Chem. Res. 1980, 13, 148. (b) Massey, V. Biochem.
Soc. Trans. 2000, 28, 283.
(7) Raszka, M.; Kaplan, N. O. Proc. Natl. Acad. Sci. U.S.A. 1974, 71,
4546.
(8) (a) Aw, T. Y.; Jones, D. P.; McCormick, D. B. J. Nutr. 1983, 113,
1249. (b) Gastaldi, G.; Ferrari, G.; Verri, A.; Casirola, D.; Orsenigo,
M. N.; Laforenza, U. J. Nutr. 2000, 130, 2556.
(9) (a) Capo-Chichi, C. D.; Feillet, F.; Gueant, J. L.; Amouzou, K.; Zonon,
N.; Sanni, A.; Lefebvre, E.; Assimadi, K.; Vidailhet, M. Am. J. Clin.
Nutr. 2000, 71, 978. (b) Said, H. M.; Ortiz, A.; Moyer, M. P.;
Yanagawa, N. Am. Physiol. Soc. 2000, 278, 270. (c) Rivlin, R. S.
N. Engl. J. Med. 1970, 283, 463. (d) Rivlin, R. S.; Langdon, R. G.
Endocrinology 1969, 84, 584.
(10) (a) Light, D. R.; Walsh, C.; Marletta, M. A. Anal. Biochem. 1980,
109, 87. (b) Hustad, S.; Ueland, P. M.; Schneede, J. Clin. Chem. 1999,
45, 862.
(12) Suh, J.; Cho, W.; Chung, S. J. Am. Chem. Soc. 1985, 107, 4530–
(11) Anderson, P. C.; Mecozzi, S. Nucleic Acids Res. 2005, 33, 6992.
4535.
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product peaks emerged as time elapsed. The migration time of
the peak at 5.3 min was different from that of FMN but identical
to that of cFMN. The peak at 9.3 min matched AMP. We also
detected the high-resolution mass spectrum of cFMN in the
reaction mixture (m/e calculated mass for C17H18N4O8P- [M]+
437.0868, found 437.0873).
The splitting of FAD to cFMN by XyDPA can be easily
inferred from the known mechanism of artificial phosphodi-
esterases (Figure 1C).2c,d,h FAD’s diphosphate diester group,
activated by two Zn ions of XyDPA, is attacked by the nearest
4′-hydroxyl group of the ribityl chain of FAD, and the activated
AMP is detached from FAD as a good leaving group to generate
cFMN. Intramolecular nucleophilic attack by the nearest hy-
droxyl group and the good leaving ability of the activated AMP
unit by the Lewis acidic Zn ions make the splitting of FAD
much more favorable, even at pH 7.4 and 27 °C, than hydrolysis
of other dinucleotides, which usually occurs at g50 °C with a
large excess of catalysts compared to substrates.3c,d
In order to obtain kinetic data for the splitting of FAD to
cFMN catalyzed by XyDPA (10 µM), we analyzed the
increasing fluorescence emission intensity of cFMN with time
(Figure 2A). From the transformed pseudo-first-order equation
(Figure 2B) and a modified Michaelis-Menten scheme (Figure
2C), we were able to analyze the kinetics of FAD splitting: kcat
) (6.0 ( 0.2) × 10-3 s-1 and Km ) (1.0 ( 0.1) × 10-5 M.
Details are described in the Supporting Information.
Catalytic FAD splitting by XyDPA was also detected in
eosinophils, which have a considerable amount of FAD in their
granules.13 At each time point in eosinophil incubation in 10
µM XyDPA saline solution (NaCl 100 mM, pH 7.4, 25 °C),
enhanced eosinophil emission intensity was recorded over time
by a fluorescence activated cell sorter (see the Supporting
Information). These experimental results show that XyDPA can
efficiently catalyze the splitting of FAD to cFMN, even inside
cells.
It is interesting to compare the activity of XyDPA to that of
natural FMN cyclase, which accelerates the conversion of FAD
into cFMN and AMP, one of the two reaction types catalyzed
by the dual-activity enzyme, FAD-AMP lyase (dihydroxyac-
etone kinase/FMN cyclase).14 FMN cyclase (or FAD-AMP
lyase, EC 4.6.1.15) catalyzes FAD splitting to produce cFMN.
Divalent transition metal cations (Mn2+ or Co2+) are known to
be required for this reaction.The kcat/Km of natural FMN cyclase
was reported14a to be 32.9 µM-1 min-1 (using Mn2+ as the
activating cation) or 5.6 µM-1 min-1 (using Co2+ as the ac-
tivating cation), which is ∼910- or 160-fold higher than the
kcat/Km of XyDPA (0.036 µM-1 min-1). However, considering
that XyDPA (631.46 g/mol) is much smaller than FMN cyclase
(120 kDa), and that most likely XyDPA does not catalyze the
phosphorylation of DHA by ATP, XyDPA may be a mimetic
of the cyclizing lyase part of the active site(s) of dihydroxy-
acetone kinase/FMN cyclase.
Figure 2. Splitting of FAD catalyzed by XyDPA (10 µM) at pH 7.4 and
27 °C. (A) Plot of [P] (concentration of cyclic FMN) against time. (B) Plot
of ln(St - [P])/St against time. (C) Plot of V0 (initial rate of FAD splitting
reaction) against St (initial concentration of FAD).
Supporting Information). The differentiating ability of PTZ-
DPA with regard to flavins is made possible not only because
PTZ-DPA has the same catalytic core as XyDPA does, but
also because the phenothiazine group, as an electron donor,
quenches the emission of the isoalloxazine ring in the complex
by photoinduced electron transfer (PET).15
Selective Flavin Chemosensing Using Artificial FMN Cyclase.
XyDPA’s superior function as an artificial FMN cyclase can
be further elaborated to generate a selective fluorescent chemosen-
sor (PTZ-DPA) for flavins (FMN, FAD, and RF) by attaching
a phenothiazine group to XyDPA (for synthesis, see the
As expected, PTZ-DPA split FAD into cFMN and AMP.
Consequently, the emission intensity at 525 nm increased as
time elapsed, because of the released cFMN (Figures 3 and 4A).
(13) Mayeno, A. N.; Hamann, K. J.; Gleich, G. J. J. Leukocyte Biol. 1992,
51, 172.
(14) (a) Cabezas, A.; Pinto, R. M.; Fraiz, F.; Canales, J.; Gonzalez-Santiago,
S.; Cameselle, J. C. Biochemistry 2001, 40, 13710. (b) Cabezas, A.;
Costas, M. J.; Pinto, R. M.; Couto, A.; Cameselle, J. C. Biochem.
Biophys. Res. Commun. 2005, 338, 1682.
(15) Ko¨nig, B.; Pelka, M.; Zieg, H.; Ritter, T.; Bouas-Laurent, H.; Bonneau,
R.; Desvergne, J. P. J. Am. Chem. Soc. 1999, 121, 1681.
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Figure 3. FAD fluorescence enhancement by PTZ-DPA. (A) Mechanism
of FAD splitting to cFMN by PTZ-DPA. (B) FAD (50 µM) fluorescence
emission changes by PTZ-DPA (10 µM) with time (excitation wavelength,
445 nm). Inset graph: Y-axis, fluorescence emission intensity at 525 nm;
X-axis, elapsed time. Inset picture: (left) only FAD (10 µM) fluorescence
and (right) increased FAD (10 µM) fluorescence after 30 min from addition
of 1 equiv of PTZ-DPA.
The initial rates (V0) of the splitting of FAD catalyzed by PTZ-
DPA (10 µM), which could be gathered from a transformed
pseudo-first-order equation (Figure 4B), are plotted against the
initial concentration of FAD (St), as shown in Figure 4C.
Analysis of the kinetic data by a modified Michaelis-Menten
scheme led to kcat ) (2.8 ( 0.1) × 10-3 s-1, Km < 1 × 10-5 M,
and Ki ) (1.1 ( 0.2) × 10-4 M. In terms of kcat, Km, or kcat/Km,
the catalytic activity of PTZ-DPA is comparable to that of
XyDPA. For PTZ-DPA, however, the reaction is slowed as St
is increased, which is accounted for by assuming substrate
inhibition due to the formation of a 1:2-type complex between
PTZ-DPA and FAD. The details are described in the Supporting
Information.
Figure 4. Splitting of FAD catalyzed by PTZ-DPA (10 µM) at pH 7.4
(10 mM HEPES) and 27 °C. (A) Plot of [P] (concentration of cyclic FMN)
against time. (B) Plot of ln(St - [P])/St against time. (C) Plot of V0 against
St. The dotted line indicates the kinetic data predicted at low St concentrations.
It is intriguing that substrate inhibition is shown in the kinetics
of PTZ-DPA. In fact, substrate inhibition typically occurs with
20% of known natural enzymes. In the presence of excess
substrate, substrate inhibition happens when two identical
substrate molecules bind to the active site of the enzyme at the
same time: One molecule may inhibit another molecule’s correct
position for reaction at the active site. Although PTZ-DPA and
XyDPA have the same catalytic unit, the extra pendant attached
to PTZ-DPA appears to facilitate complexation of two FAD
molecules to one PTZ-DPA molecule at high St concentrations,
leading to deactivation of the catalyst. In the possible structure
of (FAD)2(PTZ-DPA), each terminal phosphate group of two
FADs seems to be bound to the mono(Zn2+-DPA) unit of PTZ-
DPA. When St e 30 µM, however, deactivation of PTZ-DPA
due to substrate inhibition can be neglected.
FMN fluorescence was completely quenched upon the addi-
tion of PTZ-DPA. The phosphate group of FMN was bound to
bis(Zn2+-DPA) of PTZ-DPA (Ka ) 9.4 × 105 M-1), and the
emission of FMN was quenched by the PET from the electron
donor, phenothiazine of PTZ-DPA (Figure 5A,B). RF has no
phosphate group in it and thus cannot form a complex with
PTZ-DPA. Therefore, RF fluorescence showed a negligible
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Selective Fluorescent Chemosensor for Flavins
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RF), and FAD synthetase (FMN to FAD).16a As shown in Figure
6, the results matched our expectations well. In riboflavin kinase
assay (Figure 6A), the production of FMN from RF gradually
increased with incubation time. This was supported by the
decrease in the emission intensity of a flavin and enzyme
mixture in PTZ-DPA (100 µM) solution with time. In alkaline
phosphatase assay (Figure 6B), RF was generated from FMN
with time. Therefore, the fluorescence of a flavin and enzyme
mixture was enhanced with time in PTZ-DPA (100 µM)
solution. In FAD synthetase, FAD was synthesized from FMN
over time (Figure 6C). Consequently, the fluorescence of a flavin
and enzyme mixture in PTZ-DPA (100 µM) solution was
enhanced gradually. The detailed enzymatic experimental condi-
tions and procedure are depicted in Figure 6 and described in
Materials and Methods.
Conclusion
We found that XyDPA efficiently split FAD to cFMN and
AMP, even at pH 7.4 and 27 °C, with less than 1 mol % of a
substrate, like natural FMN cyclase. Using XyDPA’s superior
catalytic function and the PET quenching of phenothiazine as
an electron donor, PTZ-DPA enabled the first selective
fluorescent detection of FAD (fluorescence-ON) by a splitting
mechanism and FMN (fluorescence-OFF) by a PET mechanism.
We were also able to successfully apply PTZ-DPA to the real-
time fluorescent assay of various enzymatic reactions related
to flavin conversions: riboflavin kinase, alkaline phosphatase,
and FAD synthetase.
Figure 5. Selective fluorescent detection of FMN and riboflavin with PTZ-
DPA. (A) FMN fluorescence quenching mechanism by the photoinduced
electron transfer (PET) of PTZ-DPA. (B) FMN (10 µM) fluorescence
emission titration graph upon addition of PTZ-DPA (excitation wavelength,
445 nm). Inset picture: (left) only FMN (10 µM) fluorescence and (right)
decreased FMN (10 µM) fluorescence after addition of PTZ-DPA (20 µM).
(C) Graphs of RF (10 µM) fluorescence emission by PTZ-DPA (excitation
wavelength, 445 nm). Inset picture: (left) only RF (10 µM) fluorescence
and (right) RF (10 µM) fluorescence after addition of 2 equiv of PTZ-
DPA. All inset pictures were taken under a UV lamp (excitation wavelength,
365 nm).
Materials and Methods
Kinetics of FAD Splitting by XyDPA and PTZ-DPA. Increas-
ing fluorescence intensity (Ft) of the FAD splitting reaction (FAD
) 10-1000 µM) by XyDPA (10 µM) or PTZ-DPA (10 µM) was
measured with time after dilution (final flavin (FAD + cFMN)
concentration ) 1 µM) in 10 mM inorganic pyrophosphate buffer
solution (100 mM HEPES, 27 °C, pH 7.4) because FAD (also
cFMN) fluorescence was self-quenched at high concentrations. To
distinguish the fluorescence of cFMN from that of FAD bound
to XyDPA, excess inorganic pyrophosphate (10 mM) was added
into the solution during each incubation time. Because excess
inorganic pyrophosphate bound to XyDPA more strongly than FAD
did,5d,e only conformationally changed FAD (i.e., bound to XyDPA)
would return to its original stacked conformation upon treatment
with excess pyrophosphate, but the FAD splitting product, cFMN,
would not be affected by adding inorganic pyrophosphate. From
the fluorescence intensity (Ft) at the specific time point, we could
get the concentration of product ([cFMN] ) [P]), through the
following equation:
Figure 6. Fluorescent real-time enzyme monitoring with PTZ-DPA. (Left)
Riboflavin kinase fluorescent real-time assay: 50 µM riboflavin, 100 µM
ATP, 15 mM MgCl2, 0.02% Tween-20, 10 mM Na2SO3, 1 mM dithio-
threitol, 100 mM Tris-HCl (pH 8.5, 37 °C), enzyme 0.25 µg/mL or 0.75
µg/mL, fluorescence detection after dilution (×50) in PTZ-DPA 100 µM
solution. (Middle) Alkaline phosphatase fluorescent real-time assay: 100
µM FMN, 20 mM MgCl2, 100 mM Tris-HCl (pH 8.5, 37 °C), enzyme 0.1
µg/mL, 0.3 µg/mL, or 0.5 µg/mL, fluorescence detection after dilution
(×100) in PTZ-DPA 100 µM solution. (Right) FAD synthetase assay: 20
µM FMN, 100 µM ATP, 15 mM MgCl2, 1 mM tris(hydroxypropyl)phos-
phine, 0.01% CHAPS, 100 mM Tris-HCl (pH 8.5, 37 °C), enzyme 0.1 µg/
mL or 6.7 µg/mL, fluorescence detection after dilution (×20) in PTZ-DPA
100 µM solution.
[P] ) St{(Ft - FFAD)/(FcFMN - FFAD)}
change upon the addition of PTZ-DPA (Figure 5C). In
summary, FAD shows ON(+), FMN shows OFF(-), and
riboflavin shows NO(0) fluorescence changes upon the addition
of PTZ-DPA. Thus, PTZ-DPA is the first fluorescent sensor
that can distinguish among FAD, FMN, and RF.
where FFAD is the fluorescence of FAD (1 µM), FcFMN is the
fluorescence of cFMN (1 µM), and St is the initial FAD concentration.
For pseudo-first-order reactions, the plots of ln(St - [P])/St against
time (t) form straight lines (eq 1). For reactions proceeding through
the formation of catalyst-substrate complexes, such as enzymatic
reactions following the Michaelis-Menten scheme, the plots of ln(St
- [P])/St against time (t) deviate from straight lines.12 The simplest
equation fitting the kinetic data obtained in the present study is eq
2, which contains one additional parameter compared with eq 1.
From eq 2, the dependence of [P] on t is described as eq 3.
Real-Time Fluorescent Detection of Enzymatic Conversion
of Flavins with PTZ-DPA. With this selective sensing property,
PTZ-DPA is applicable to real-time fluorescent monitoring of
riboflavin kinase (RF to FMN), alkaline phosphatase (FMN to
(16) (a) Sandoval, F. J.; Roje, S. J. Biol. Chem. 2005, 280, 38337. (b)
Sandoval, F. J.; Zhang, Y.; Roje, S. J. Biol. Chem. 2008, 283, 30890.
ln(St - [P])/St ) -Rt
(1)
9
J. AM. CHEM. SOC. VOL. 131, NO. 29, 2009 10111

A R T I C L E S
Rhee et al.
(600 s) for FAD splitting to cFMN before the fluorescence
emission of the reaction mixture was recorded. Riboflavin kinase
was purchased from Abnova Corp. and alkaline phosphatase from
Sigma-Aldrich.
ln(St-[P])/St ) -Rt/(1 + ꢀt)
[P] ) St{1 - e[-Rt/(1+ꢀt)]
(2)
(3)
}
The initial velocity (V0, defined as d[P]/dt at t ) 0) is derived as
RSt from both eqs 1 and 2. Kinetic data obtained for the splitting
of FAD were analyzed according to eq 2 by nonlinear regression
to obtain the values of R, ꢀ, and V0. Figures S1-S28 (Supporting
Information) illustrate examples of the plots of ln(St - [P])/St against
time or of [P] against time analyzed by eq 2 or eq 3, respectively.
In these figures, the solid lines are obtained by linear regression.
Catalytic turnover for the splitting of FAD by XyDPA is clearly
seen in Figure S2. The values of V0 thus estimated are summarized
in Tables S1 and S2. Further kinetic data (kcat, Km, and Ki) for the
FAD splitting by XyDPA or PTZ-DPA are also reported in the
Supporting Information.
Real-Time Monitoring of Enzymatic Conversion of Flavins
with PTZ-DPA. At each enzymatic reaction time point, we re-
corded the fluorescence emission intensity of the reaction mixture
after dilution in 100 µM PTZ-DPA solution of riboflavin kinase
(×50),16a alkaline phosphatase (×100), or FAD synthetase
(×20).16b The final concentration of flavin in each enzymatic
reaction mixture would be 1 µM in PTZ-DPA (100 µM) solution.
In the case of the FAD synthetase assay, after dilution in 100
µM PTZ-DPA solution, there was additional incubation time
Acknowledgment. This work was supported by the KOSEF
grant funded by the MEST (Grant No. 2009-0080734) and Seoul
R&BD. S.R. thanks the USDANRI grant (2007-03559). J.C.C. and
R.M.P. thank the Ministerio de Educacio´n y Ciencia (Grant No.
BFU2006-00510) and the Consejer´ıa de Econom´ıa, Comercio e
Innovacio´n, Junta de Extremadura (Grants GRU08043 and 09153)
for funding cofinanced by FEDER and FSE. H.-W.R. is the recipient
of the Seoul Science Fellowship. H.-W.R. and S.J.C. thank the
Ministry of Education for the BK fellowship.
Supporting Information Available: Synthesis of PTZ-DPA,
analysis of kinetic data of FAD hydrolysis by XyDPA and PTZ-
DPA, capillary electrophoresis experimental data, titration of
FMN and cFMN with PTZ-DPA, Job’s plot between FMN and
PTZ-DPA, and FAD hydrolysis by XyDPA in eosinophils. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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10112 J. AM. CHEM. SOC. VOL. 131, NO. 29, 2009