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Fluorescent Imaging of Citrate and Other Intermediates in the Citric Acid Cycle.

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Angewandte
Chemie
Citric Acid Cycle
Fluorescent Imaging of Citrate and Other
Intermediates in the Citric Acid Cycle**
Zhihong Lin, Meng Wu, Michael Schferling, and
Otto S. Wolfbeis*
Citrate is an important intermediate in the citric acid cycle,[1]
which is the central metabolic hub in the cell for harvesting
chemical energy and building many biomolecules. Citrate is a
chelating agent that assists in the elimination of heavy-metal
ions (which are taken up and biotransformed by bacteria),[2] it
is used as an anticoagulant[3] to prevent blood clotting, and it
is an additive in the food and pharmaceutical industry.[4] In
addition, the concentration of citrate in urine can be
diagnostic for certain diseases.[5]
Fluorescent imaging is a powerful means for visualizing
the distribution of species in a sample, but is it possible only if
one the following prerequisites is met: 1) If the species of
interest has a fluorescence of its own (such as NADH, many
flavins, and porphyrins);[6] 2) if the species of interest can be
rendered fluorescent by attaching a label;[7] or 3) if probes are
available for the species of interest (e.g. pH, oxygen concentration).[8] Unfortunately, citrate and the other intermediates
in the citric acid cycle do not have significant physical and
chemical properties suitable for direct determination, so that
for detection they must be transformed by enzymes into
“visible products” or recognized by synthetic receptors.[9] The
development of a sensitive fluorescent probe (sensor) for
citrate would be an elegant alternative.
We report here on a europium(iii) complex that serves as a
fluorescence sensor for citrate. The visualization of citrate and
other intermediates in the citric acid cycle is based on the
finding that the weakly fluorescent europium(iii) tetracycline
complex (EuTc) reversibly associates with citrate to form the
strongly fluorescent europium–tetracycline–citrate complex
(EuTc-Cit). Both EuTc and EuTc-Cit have the typical merits
of europium complexes,[10] namely, a large Stokes' shift (~
210 nm), a linelike emission band, a decay time in the ms
range, and an excitation wavelength that is compatible with
the blue diode laser (405 nm). In addition, it is simple to
prepare and photostable both in solution and in the solid
state.
The fluorescence properties of EuTc have been described
previously,[11] including its application for the detection of
hydrogen peroxide.[12] The absorbance of EuTc-Cit peaks at
l = 381 to 408 nm, similar to that of EuTc.[13] In contrast, the
[*] Dr. Z. Lin, Dr. M. Wu, Dr. M. Sch7ferling, Prof. O. S. Wolfbeis
Institute of Analytical Chemistry, Chemo- and Biosensors
University of Regensburg
93040 Regensburg (Germany)
Fax: (+ 49) 941-943-4064
E-mail: otto.wolfbeis@chemie.uni-regensburg.de
[**] We thank Henrik Bauer (Picoquant GmbH, Berlin) for assistance in
determining decay times. Z. Lin and M. Wu thank Chromeon GmbH
(Regensburg) for financial support. M. Wu acknowledges support
from the NSFC (20005004).
Angew. Chem. Int. Ed. 2004, 43, 1735 –1738
fluorescence intensity of the 615-nm band of EuTc-Cit is 22
times stronger than that of EuTc. The linelike main emission
band of EuTc-Cit is due to the 5D0 !7F2 electronic transition.
Side bands are found at l = 580, 590, 651, and 697 nm.
The stoichiometry of EuTc-Cit is 1:1:2 (Eu:Tc:Cit) as
determined by both the continuous variation (Job's) method
and the mole-ratio method,[14] and is thus similar to that of
other tetracycline/metal ion complexes.[11a, 15] Citrate, a polydentate ligand, can chelate the Eu3+ ion through the oxygen
atoms of the carboxy and hydroxy groups.[15a,b] It is assumed
that citrate displaces water molecules that occupy the eight to
nine coordination sites of the Eu3+ ion and quench its
fluorescence. We estimated the dissociation constants of the
(EuTc)(citrate)2 system based on Benesi–Hildebrand-type
equations[16] and obtained pKd values in the range of 4.2–4.9.
Fluorescence quantum yields (QYs) were determined[17]
to be 0.4 % for EuTc and increased to 3.2 % for EuTc-Cit
(Table 1). This indicates that the total quantum yield does not
Table 1: Average luminescence decay times tav [ms] and quantum yields
QY of EuTc-L complexes.[a]
Ligand L
tav
QY[b]
Ligand L
tav
QY[b]
EuTc
EuTc citrate
EuTc isocitrate
EuTc ketoglutarate
44
83
66
37
0.004
0.032
0.007
0.004
EuTc
EuTc
EuTc
EuTc
38
63
77
56
0.004
0.005
0.008
0.014
succinate
fumarate
malate
oxalacetate
[a] L is the respective intermediate in the citric acid cycle, which is here
the ligand in the EuIII complex. EuTc: 50 mmol L1, L: 150 mmol L1. It
should be noted that at this concentration of ligands L, EuTc is fully
complexes by citrate only, but not by the other ligands. [b] Quantum yield
relative to that of ruthenium(ii) tris(2,2’-dipyridyl) dichloride hexahydrate.[17]
increase as much as the intensity of the emission at l =
615 nm. All the Eu3+ complexes have long fluorescence
decay times (Table 1). The decay profile data can be fitted to a
three-component model, in which the average decay times are
83 ms for EuTc-Cit and 44 ms for EuTc. This large difference in
average decay times was exploited in the time-resolved
detection and imaging methods described here.
Unlike Tc, EuTc, and other ternary tetracycline complexes whose emission is sensitive to pH,[11b, 12a] the fluorescence of the EuTc-Cit system is pH-independent between
pH 7.4 and 9.2. The best results were obtained with
10 mmol L1 HEPES buffer at pH 8.0. We also studied
possible interferences by about 40 common cations, anions,
gases, and some biomolecules occurring in biological fluids.
Most of them affect the emission of EuTc-Cit by < 10 %.
However, Ni2+, Co2+, Cu2+, phosphate, and ATP do interfere
when their concentrations exceed 16 (Ni, Co), 2 (Cu), 280
(phosphate), and 8 (ATP) mmol L1. The interference by
phosphate is particularly annoying since phosphate is present
in many biofluids and is often used as a buffer.
It is noted that hydrogen peroxide, which is the analyte in
fluorescence studies with the EuTc (3:1) complex,[11] does not
notably affect the fluorescence of EuTc-Cit. This can be
explained by the much weaker coordinating ability of H2O2 to
Eu3+ and the different stoichiometry of the EuTc-Cit system.
DOI: 10.1002/anie.200353169
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1735
Communications
By using time-resolved detection (using a lag time of
100 ms), we could detect citrate in a concentration range
between 1.6 G 107 and 5.6 G 105 mol L1, with a detection
limit (3s/slope) as low as 6.0 G 108 mol L1.[18] The long decay
time of the EuTc-Cit system facilitates not only gated optical
sensing of citrate but also imaging.[19] Among the methods
known to be useful for fluorescence imaging, we have
preferably
applied
fluorescence
lifetime
imaging
(FLIM)[8b–c, 12b] since it has advantages in terms of signal
generation and suppression of artifacts such as local inhomogeneities. Specifically, we employed rapid lifetime imaging
(RLI)[20] to obtain the images shown in Figure 1 a. The steady-
Figure 1. Fluorescence imaging of citrate in a 96-well microtiter plate
with EuTc as the fluorescent probe. a) Rapid lifetime imaging of
increasing concentrations of citrate. b) Intensity-based fluorescence
images of increasing concentrations of citrate. The concentration of
EuTc is 50 mmol L1 throughout, citrate concentrations (from left to
right) are 0, 0.16, 0.4, 1.0, 1.6, 4.0, 10.0, 16.0, 20.0, 40.0, 60.0 and
80.0 mmol L1.
state (intensity-based) images in Figure 1 b are included for
comparison. The latter, in contrast to RLI, display substantial
heterogeneity due to fluctuations of the light source and light
scattering.
We also studied other intermediates in the citric acid
cycle—isocitrate, a-ketoglutarate, succinate, fumarate, lmalate, and oxaloacetate. The large differences in fluorescence intensity on addition of EuTc is apparent in Figure 2 a.
These intermediates can act as polydentate ligands similar to
citrate and form ternary complexes with EuTc.[15a] As aketoglutarate and succinate cannot effectively coordinate
with Eu3+, significant fluorescence enhancement was not
expected nor indeed observed.
The fluorescence decay profiles and the quantum yields of
these complexes are summarized in Table 1. Furthermore, by
choosing different lag times for “gated” detections, we could
detect different intermediates in different time windows.
Gating obviously can be used to fine-tune between selectivity
and sensitivity. However, on increasing the gating time from 0
to 100 ms, the normalized intensity [defined as (FFo)/Fo] of
all species except a-ketoglutarate is increased (Figure 2 b).
On increasing the gating time to 250 ms, the signal of
oxaloacetate is almost completely suppressed and that of
citrate is reduced by 40 %, while the signal intensities of
isocitrate, fumarate, and malate are much less affected.
Obviously, l-malate and oxaloacetate, and citrate and iso-
1736
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. a) Two-dimensional fluorescence images of intermediates of
the citric acid cycle; b) relative fluorescence intensities of the EuTc-L
complexes at different lag times. EuTc: 50 mmol L1, L (citrate, isocitrate, a-ketoglutarate, succinate, fumarate, l-malate, and oxaloacetate):
150 mmol L1. Fo and F are the fluorescence intensities of EuTc and the
EuTc-L complex, respectively.
citrate can be nicely discerned by means of different delay
times.
It should be emphasized that this scheme does not require
enzymes or multienzyme systems. However, in assays for
other intermediates in the citric acid cycles, the samples
should not contain citrate. The application, specificity, and
reversibility of the EuTc-Cit system can be best exemplified
by the stepwise EuTc-based visualization of oxaloacetate, lmalate, and fumarate, which are formed in the enzymatic
reactions (1)—(3). In these reactions CL stands for citrate
CL
citrate ƒ!oxaloacetate þ acetate
ð1Þ
MDH
oxaloacetate þ NADH þ Hþ ƒƒ!
ƒƒ l-malate þ NADþ
FM
l-malate ƒ!fumarate þ H2 O
ð2Þ
ð3Þ
lyase, MDH for malic dehydrogenase, and FM for fumarase.[21]
Figure 3 depicts the resulting stepwise changes in the
fluorescence of the EuTc system. The fluorescence of a blank
solution, composed of EuTc and NADH only, is stable over
time. After addition of citrate, fluorescence increases due to
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Angew. Chem. Int. Ed. 2004, 43, 1735 –1738
Angewandte
Chemie
Figure 3. Kinetics of the formation and stepwise decomposition of the
EuTc-Cit. The blank solution: 200 mL EuTc (0.5 mmol L1), and 60 mL
NADH (4.8 mmol L1) in 1.7 mL of HEPES buffer. Then, 40 mL citrate
solution (2 mmol L1), 70 mL citrate lyase (CL) solution (3.4 U mL1),
50 mL malate dehydrogenase (MDH) solution (1588 U mL1), and
80 mL (618 U mL1) fumarase (FM) solution (1588 U mL1) were
added, and the time course of fluorescence recorded.
formation of the EuTc-Cit complex. Then, CL, MDH, and
FM were added consecutively. The signal intensity decreases
stepwise, thereby indicating the complete consumption of
citrate and formation of EuTc-oxaloacetate, EuTc-malate,
and EuTc-fumarate, respectively, complexes with different
fluorescence intensities. This experiment clearly shows that
EuTc-Cit is formed in a reversible manner and that EuTc can
be used to probe the sequence of reactions.
In conclusion, we have demonstrated both the direct
sensing and the time-resolved imaging of citrate and other
intermediates in the citric acid cycle. The EuTc has great
potential as a fluorescent probe for monitoring citrate-based
bioprocesses in vitro and in vivo.
Received: October 28, 2003 [Z53169]
.
Keywords: citric acid cycle · europium · fluorescence
spectroscopy · imaging
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[13] a) Reagent solution: The EuTc solution was obtained by
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[18] Protocol for the time-resolved assay of citrate: Aqueous samples
(100 mL containing 0.32 to 112 mmol L1 of citrate) were added to
100 mL of a 100 mmol L1 of EuTc in each well of a microtiter
plate. After a reaction time of 30 min, fluorescence (excitation
wavelength = 405 nm) at a filter wavelength of l = 612 nm was
recorded with a lag time of 100 ms and an integration time of
40 ms. The experiments were conducted with a Tecan GENios +
microplate reader (Tecan, Salzburg-Groedig, Austria).
[19] It should be noted that the term imaging has different meanings.
Initially, imaging was used to describe the visual presentation of
spatially resolved concentrations of (bio)chemicals or of physical
parameters such as temperature. More recently, it also has been
used for visual presentation of whole sets of spots (such as in
arrays or proteomics and opposed to sequential scanning of each
data point). While wells of microplates are imaged here, we see
no reason that the method may not be applied to spatially
resolved imaging as well.
[20] Imaging was performed as previously described.[8b] In the steadystate intensity mode, images were acquired within one window
between 0 and 50 ms. In the RLI scheme, two windows were used
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2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1737
Communications
(100 to 180 ms, and 200 to 240 ms). Data processing such as the
rotation and crop of images, subtraction of dark images from the
raw images, and the ratioing of images were performed by a selfdeveloped program based on Matlab (6.1, Mathwork, Natick,
MA, USA).
[21] Citrate lyase (EC: 4.1.3.6, from Enterobacter aerogenes), mitochondrial malic dehydrogenase (EC: 1.1.1.37, from porcine
heart), and fumarase (EC: 4.2.1.2 from porcine heart) were from
Sigma and used without further purification.
1738
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Angew. Chem. Int. Ed. 2004, 43, 1735 –1738
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