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Studying zinc biology with fluorescence: ain’t we got fun?Richard B Thompson
Zinc has emerged as a metal ion of substantial interest in
biology and medicine, especially in neuroscience, gene
transcription, the immune response, and mammalian
reproduction. Fueling these advances in understanding has
been the development of new fluorescence-based indicator
systems for zinc with unprecedented sensitivity and selectivity.
This review summarizes recent progress in the development of
fluorescence-based sensors and biosensors for zinc, with a
view to evaluating their suitability for use with biologically
derived specimens, especially in vivo and in situ.
Addresses
Department of Biochemistry and Molecular Biology, University of
Maryland School of Medicine, Baltimore, MD 21201, USA
Corresponding author: Thompson, Richard B
(rthompso@umaryland.edu)
Current Opinion in Chemical Biology 2005, 9:526–532
This review comes from a themed issue on
Analytical techniques
Edited by Chris D Geddes and Ramachandram Badugu
Available online 29th August 2005
1367-5931/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.cbpa.2005.08.020
IntroductionThis is certainly the best of times to study the biology of
zinc. After decades of obscurity, zinc has emerged from
the chorus of elements in biochemistry and is elbowing its
way towards center stage. Especially since Berg’s mani-
festo appeared nearly ten years ago [1], the importance of
zinc in an extraordinary range of biological processes has
been revealed. These include brain function and pathol-
ogy, gene transcription, immune function, and mamma-
lian reproduction [2–4], as well as a host of disease
processes, such as Alzheimer’s disease, epilepsy, ischemic
stroke, and infantile diarrhea [5��,6,7]. Yet some of the
most basic questions about zinc function remain unan-
swered, or the subjects of vigorous dispute. For instance,
there is no consensus on the in vivo role of the vesicular
zinc found in the hippocampus and throughout the cortex
[8], nor the granular zinc found in Paneth cells of the
intestine, nor that in granulocytes. What role(s) zinc may
play in apoptosis remains controversial, with some saying
that zinc is apoptogenic and others claiming it inhibits
apoptosis [9]. Similarly, whether zinc is a bystander or
prime mover in excitotoxicity in the brain is far from
Current Opinion in Chemical Biology 2005, 9:526–532
settled. We do not know how zinc is allocated among its
hundreds of functional niches in enzymes and transcrip-
tion factors. Until very recently we only had estimates
[10,11] of the level of free (rapidly exchangeable) zinc ion
in the cytoplasm of typical cells. Of course, a lack of
consensus and understanding makes the most fertile
ground for the scientist to till, and this is very fertile
ground indeed.
All of which is not to say that we don’t understand a great
deal more about the biology of zinc than a decade ago [2].
Many of the advances since then can be attributed to the
development of molecular biological techniques and their
application in, for instance, knockout mice that do not
express genes for certain zinc transporters [12]. However,
the bulk of what we now understand probably comes from
the application of zinc-sensitive fluorescent dyes, begin-
ning with TSQ ((N-(6-methoxy-8-quinolyl)-p-toluenesul-
fonamide)) [13]. As demonstrated in the cases of calcium,
pH, and now zinc, fluorescent indicators elucidate the
biology of these substances (and the biochemicals that
interact with them) by combining chemical information
with the spatial information obtained by observing the
target cell in the microscope. Although TSQ and its
congeners TflZn (N-(2-methyl-6-methoxy-8-quinolyl)-
p-carboxylbenzenesulfonamide) and zinquin (N-(2-
methyl-6-(O-(2-acetate))-8-quinolyl-p-toluenesulfona-
mide) revealed much, their shortcomings were also
manifest, and only recently have several new approaches
been developed that address the very difficult issues of
measuring a trace metal like zinc in situ; these are the
focus of this essay. Space limitations herein unfortunately
do not permit a more comprehensive review [14–16,17�].
Compared with most other biological metals like calcium,
analysis of zinc is a tougher nut to crack, simply because it
is less abundant, especially in the free form. Thus,
although Ca indicators are useful if they can quantify
nanomolar to micromolar concentrations, free Zn levels in
most cell types appear to be in the picomolar to nanomolar
range. However, concentrations of zinc that may be of
biological importance potentially range from femtomolar
to millimolar, suggesting that a very broad dynamic range
(or a range of indicators) is necessary. The generally lower
concentrations of Zn also put a premium on selectivity in
its determination, since even species at low (micromolar)
levels can potentially interfere. Other desiderata include
facile quantitation, preferably by wavelength ratio, aniso-
tropy, or lifetime; high quantum efficiency, high extinc-
tion coefficient, photostability, and long emission and
excitation wavelengths, which contribute to brightness
and the ability to discern faint signals over background;
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Studying zinc biology with fluorescence Thompson 527
water solubility; pH-sensitivity; and the ability to get
them into the cell or (for protein-based indicators) have
them expressed therein.
Small-molecule zinc indicatorsAmong the earliest and most prominent contributors of
fluorescent tools for the study of zinc have been the
laboratories of Aoki, Hirano, Kikuchi, Kimura, Nagano,
and their colleagues [16]. Most recently, Aoki, et al. have
introduced fluorophores having twisted intramolecular
charge transfer states (TICT) to fluorescent zinc sensing.
Although the photophysics of TICT has been extensively
studied for decades, this is the first example we are aware
of where analyte binding has been used to perturb the
twisting (and therefore the population of states), leading
to large fluorescence changes [18]. In this case, the
different rotamers of the indicator exhibit different emis-
sions; binding of metal favors one rotamer and shifts the
emission (Figure 1). In terms of several criteria above, the
new indicators are not really competitive, but from the
standpoint of offering a really powerful and flexible
transduction approach they represent a significant
advance. Perhaps more prosaic but very useful indeed
are the new chelators developed by Kawabata et al. [19].
Chelators are valuable tools for reducing extracellular or
intracellular free zinc. Among the most common extra-
cellular Zn chelators has been CaEDTA, wherein the Ca
is exchanged for free Zn (the latter binds 50 000-fold
tighter); unfortunately, the kinetics of the process are not
as fast as necessary (ideally, microseconds) to block
neurotransmission [20]. Kawabata and colleagues synthe-
sized non-cell penetrant chelators with much higher
selectivity for Zn (8–10 orders of magnitude), which
would have much reduced Ca bound, and therefore less
need for Zn to displace it with potentially faster kinetics.
The recent, unexpected discovery (Bozym and Thomp-
son, unpublished results) that the 1:1 Zn complex of
TPEN (like TPEN itself) is quite apoptogenic suggests
Figure 1
TICT-based Zn indicator. Absent Zn the pyridinyl moiety rotates relatively fr
the pyridinyl nitrogen bound to the zinc and emission at 450 nm predomina
360 nm. Redrawn from [18] with permission. Copyright 2004, The American
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that there will be a need for selective intracellular Zn
chelators as well.
The Lippard group has for some time been developing
fluorescent indicators for zinc based on a fluorescein
fluorophore platform and homologs of TPEN for Zn
recognition [17�]. Most recently, they have described a
clever approach for intracellular excitation-ratiometric
zinc indicators, the coumazin family [21]. This indicator
comprises a fluorescein-chelate moiety whose emission
responds to zinc, and a (more or less) inert coumarin
moiety coupled to it by an ester linkage. The fused
molecule (which exhibits little fluorescence) enters the
cell and the ester linkage is hydrolyzed (albeit slowly) by
esterases, whereupon it separates. The fluorescein moiety
intensity reflects the zinc concentration, whereas the
coumarin fluorescence is proportional to the amount of
indicator present, providing an excitation ratiometric
response. The two fluorophores appear unlikely to dis-
tribute themselves differently within the cell. Presum-
ably the sensitivity and selectivity of the indicator is
comparable to the parent ZinPyr-1 (now itself commer-
cially available), but a calibration curve would have been
welcome.
The O’Halloran group have been leaders in the field of
metallobiochemistry for some years, and zinc remains an
abiding interest. Most recently [22] they have described
an emission ratiometric indicator based on a fluorescent
benzoxazole moiety coupled with the pervasive amino-
methylpyridine chelating moiety, providing an apparent
Kd in the nanomolar range. The UV excitability and
asymmetry of the fluorophore make it well suited for
two-photon excitation fluorescence microscopy with the
mode-locked titanium sapphire laser. Although a two
photon-excited fluorescence polarization zinc indicator
had been demonstrated previously [23], it was not wave-
length ratiometric and thus less convenient to implement
eely (left panel); when zinc binds, the twisted conformer (right) with
tes compared with the planar form (center) having emission at
Chemical Society.
Current Opinion in Chemical Biology 2005, 9:526–532
528 Analytical techniques
in most microscopes. Key issues left outstanding with this
promising approach are whether it is only usable on fixed
cells, and the very small ratio span in the images com-
pared with the calibration curve.
Molecular Probes (now part of Invitrogen) has offered
fluorescent zinc indicators for more than a decade. Some
developed in-house remain among the most important,
such as Newport Green (now Newport Green DCF),
which employs the now frequently used TPEN moiety
for selective recognition. Gee has been instrumental in
this regard, working in collaboration with the Weiss,
Sensi, and Kennedy groups. Of particular interest has
been their recent introduction of a rhodamine-based zinc
indicator (RhodZin-3) that tends (like Rhodamine 123
and others) to localize in the mitochondrion [24]. Interest
in mitochondrial zinc levels and their relationship to
oxidative stress and apoptosis has grown dramatically,
and this indicator (or its successors) offers an appealing
approach to resolving these issues. The indicator is not
ratiometric but exhibits a Kd of 65 nM, suggesting that it
will be well suited for determining free Zn levels under
pathological conditions. Like FluoZin-3 and other
BAPTA-type structures this indicator is interfered with
by calcium at micromolar levels, compromising its use for
Zn quantitation extracellularly [25�,26,27�].
Fahrni and colleagues have also introduced a new trans-
duction approach for zinc sensing, called ESIPT (excited
state intramolecular proton transfer). In this case, the
fluorophore is a phenolic benzimidazole derivative
(Figure 2), which exhibits the intramolecular transfer
of a proton during the time the probe is in the excited
state; the transfer results in a substantial reduction in
excited state energy and consequent red shift in the
emission. This process had been well studied; the inno-
vation of Henary et al. [28] is to have zinc binding inhibit
the proton transfer, such that the zinc-bound form emits
Figure 2
ESIPT-based zinc indicator. The ground state tautomer of the indicator (cen
proton shifts to the benzimidazole nitrogen (left) with a substantial redshift i
blueshifted emission: fractional occupancy of the binding site with zinc (and
at the two wavelengths. Redrawn from [28] with permission. Copyright 2004
Current Opinion in Chemical Biology 2005, 9:526–532
in the blue, providing a ratiometric signal. This is a very
attractive transduction approach, and may be extendable
to other fluorophores with more desirable emission prop-
erties. Moreover, one of the indicators they developed is
the first small-molecule indicator to match the carbonic
anhydrase-based indicators (see below) in having pico-
molar affinity with high selectivity. Although these indi-
cators have not yet been applied in a biological system,
nor are they excitable at convenient wavelengths, this
approach has much to offer.
Zinc sensing using biological/biomimeticmacromolecules: biosensorsSince the first reports [29,30], several groups have worked
to adapt the high selectivity and affinity of zinc binding
found in biological or biomimetic systems to fluorescent
zinc measurement. The Imperiali group [31] has contin-
ued their work in this vein by introducing a fluorescent,
artificial amino acid (abbreviated Sox) to a series of
synthetic peptides designed to bind zinc. By choosing
different amino acids to serve as zinc ligands they were
able to modulate the values of the apparent Kd by more
than 1000-fold over a useful range, with affinities as tight
as 10 nM, making them among the best of the de novobiomimetic indicator systems (but see below). Although a
large intensity change was observed in some cases upon
Zn binding, the shifts were too small to be useful for
ratiometric measurements, and other accurate transduc-
tion modes (such as lifetimes) were not measured.
Several groups have attempted to adapt existing proteins
for Zn measurement by fluorescence [32,33], or design
them de novo [34,35]. Some investigators employed clever
variants of the now-classic approach of Miyawaki et al.,whereby metal ion binding induces a large conformational
change in a polypeptide linking (or induces a crosslinking
of) two different GFP variants that represent a Forster
energy transfer pair: metal binding results in improved
ter) has the proton bound to the aniline nitrogen; after excitation the
n the emission. Binding of zinc prevents the proton shift, resulting in
thus its concentration) can be determined from the ratio of emission
, Chemistry.
www.sciencedirect.com
Studying zinc biology with fluorescence Thompson 529
energy transfer efficiency, which can be quantitated
ratiometrically. A key advantage of these approaches is
that the protein can be expressed in vivo (in principle, in
the desired cell type), avoiding altogether the problem of
how to insert the indicator into the cell. Unfortunately,
these approaches resulted in rather modest signal changes
and the affinities were only suitable for the upper end
(tens of micromolar) of the zinc concentration range of
interest. An interesting variation on this theme has been
the Maret group’s development of metallothionein deri-
vatives, which report the zinc occupancy of this important
intracellular zinc carrier [36]. Recently, Dwyer et al. [37]
designed a Zn receptor based on a ribose-binding protein
for the purpose of controlling bacterial gene expression;
collaterally, they measured the zinc affinity using an
attached fluorescent label. Transducing Zn levels by gene
expression is a novel and potentially powerful approach to
sensing Zn in whole organisms, as well as a clever means
of inducing gene expression. This approach will require
improvement, however, because the intracellular free
zinc concentrations required by the current version
(micromolar) are typically toxic.
Among the biologically derived indicator systems,
perhaps the most successful in terms of demonstrated
sensitivity and selectivity has been the carbonic anhy-
drase-based indicators of Thompson and Fierke
(reviewed in [38�]). The wild-type protein binds zinc
Figure 3
Excitation ratiometric zinc biosensor. In the presence of zinc Dapoxyl sulfon
anhydrase (right), and its UV-excited green fluorescence is efficiently transfe
fluorescence. In the absence of zinc, Dapoxyl sulfonamide does not bind to
the Alexa Fluor, resulting in weak UV-excited orange emission. The UV exci
which is proportional to the amount of protein present. Reproduced with pe
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with high affinity (Kd in the picomolar range) without
interference by Ca and Mg at 10 mM and 50 mM, respec-
tively. In most (but not all [39]) cases, zinc binding to
apocarbonic anhydrase is specifically transduced by a
change in emission of a fluorescent ligand whose binding
is strongly zinc-dependent. Binding occupancy (a func-
tion of the free Zn concentration) is transduced as
changes in excitation or emission ratios, anisotropy and/
or lifetime to maintain accuracy. A key advantage is that
the affinity, selectivity and kinetics of metal ion binding
have all been improved by subtle changes in the protein
structure; thus, variants are available with zinc affinities
ranging from picomolar to micromolar, with association
rate constants for zinc binding up to 1000-fold faster than
the wild type, and with relative affinities for Cu(II) and
Zn(II) ranging over seven orders of magnitude. Contrary
to an earlier report, carbonic anhydrase’s demonstrated
selectivity for Zn(II) over Cd(II) (>200-fold in Kd [40]) is
greater than that reported for other indicators. Taken
together, this range of capabilities represents a powerful
toolbox for zinc study.
Recently, Bozym, et al. [41] used an excitation ratio-
metric-based carbonic anhydrase system [42] (Figure 3)
to measure free zinc inside a ‘typical’ resting eukaryotic
cell. Many workers had measured free zinc levels in
different cell types (notably neurons) known to be rich
in free zinc, but most indicators described heretofore
amide binds tightly to the Alexa Fluor 594-labeled holocarbonic
rred to the Alexa Fluor, whence it is emitted as strong orange
the protein and the weak free Dapoxyl emission does not transfer to
ted emission is ratioed with the directly excited Alexa Fluor emission,
rmission from [41]. Copyright 2004 SPIE.
Current Opinion in Chemical Biology 2005, 9:526–532
530 Analytical techniques
Figure 4
Excitation ratiometric determination of free zinc in PC-12 cells. PC-12 cells in Neurobasal medium plus supplement were stained with
TAT-L198C-Alexa Fluor 594-apocarbonic anhydrase and Dapoxyl sulfonamide, and fluorescence micrographs with green excitation (lower left)
and UV excitation (lower right), as well as brightfield (upper left) were obtained. The false color ratio image at the upper right indicates the free
zinc concentrations on the scale at right.
displayed insufficient sensitivity for typical cell types.
The protein was introduced into the cell by fusing a TAT
peptide to it [43], avoiding any need for microinjection.
The false color images obtained in the microscope indi-
cated (Figure 4) that resting levels in the cytoplasm are in
the range of five picomolar. While higher than the fem-
tomolar levels predicted by Outten and O’Halloran for
prokaryotic cells [11], the levels are still very low indeed.
Confirmation of this value was obtained by use of a variant
(E117A) having slightly reduced affinity but much faster
kinetics (Bozym et al., unpublished data). The equilibra-
tion of the wild-type protein in particular was much
faster than expected (see below), and (contrary to an
earlier suggestion) evidently quite capable of responding
quickly.
The key advantage of Miyawaki’s approach for calcium
measurement is that the indicator protein is expressible
(in principle) inside any cell of interest. The excitation
ratiometric approach depicted in Figure 3 [42] can be
made expressible by replacing the covalently attached
fluorescent label with a fused GFP variant having the
appropriate spectral properties. In the event, the fused
protein gives quite a usable twofold intensity ratio change
with the same picomolar sensitivity being conferred by
the carbonic anhydrase active site [44] (Figure 5). By
comparison, the holoprotein is insensitive to variations in
zinc concentration, as expected.
An emission ratiometric carbonic anhydrase system was
also used to measure free zinc release in vivo and in situ
Current Opinion in Chemical Biology 2005, 9:526–532
in the mammalian brain. These measurements were
obtained using the well-known dialysis probe in a rabbit
ischemia model (CJ Frederickson, unpublished data), and
using a new fiber optic sensor in a dog global ischemia
model [44]. Both groups found that resting extracellular
zinc in the brain was approximately 5 nM but increased
abruptly to upwards of 100 nM following ischemia. The
fiber optic sensor offers significantly higher temporal
resolution than the dialysis probe, providing real-time
measurements with sub-minute resolution; importantly, a
different carbonic anhydrase variant with reduced affinity
and rapid kinetics (H94N) was used because of the
nanomolar concentrations present [45].
A key issue just emerging is consideration of the biolo-
gical importance of the kinetics of zinc (or any molecule at
low concentration) binding to saturable sites in the cell. In
particular, it is evident from simple kinetics that if the
association rate constant of zinc binding to a site is
diffusion-controlled (e.g. as fast as possible) and free zinc
is present at picomolar levels, it will take some hours for
the system to arrive at a steady state. It seems counter-
intuitive that an apoprotein would ‘wait around’ (in a
more labile state) for hours following protein synthesis for
zinc to arrive and bind to it. O’Halloran’s group has
proposed [46] that zinc addition to proteins is under
kinetic control rather than thermodynamic control, based
on the extremely high (femtomolar) zinc affinity of pro-
karyotic metalloregulatory proteins, a concentration that
implies that free zinc comprises less than one atom in a
bacterial cell on the average. Yet the results of Bozym
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Studying zinc biology with fluorescence Thompson 531
Figure 5
Zinc response of expressible carbonic anhydrase-based fluorescence
indicator. The ratio of fluorescence intensities at 617 nm excited at
366 to 546 nm is plotted as a function of free zinc ion concentration
for the apoprotein (blue circles) and the holoprotein (green circles).
Adapted with permission from [44]. Copyright 2005, SPIE.
et al. show that the picomolar affinity carbonic anhydrase-
based indicator comes to a steady state in tens of minutes
in a eukaryotic cell, implying some level of catalysis of the
process. A large set of zinc chaperones akin to the dedi-
cated copper chaperones that serve individual enzymes
[47] seems unlikely because of the great diversity of zinc-
containing enzymes and transcription factors. Rather, it
seems likely that for zinc one or more small molecules
may serve as zinc chaperones to catalyze this process for
many different proteins, in a manner analogous to the
catalysis by dipicolinate of zinc binding to carbonic anhy-
drase [48]. Alternatively, several zinc chaperones with
overlapping selectivities may catalyze zinc insertion into
proteins.
Finally, it has been encouraging that an increasing num-
ber of workers in the field now recognize that total zinc (or
added zinc) ion concentration typically is orders of mag-
nitude greater than free (or rapidly exchangeable) zinc in
many matrices such as culture medium, serum and blood,
and that binding of free zinc ion by (for instance) serum
albumin may substantially reduce its apparent potency as
biological effector. As in the cases of pH or calcium, ion
buffers are usually needed to ‘clamp’ free zinc ions at low
concentrations; these are becoming commercially avail-
able. It has also been heartening to see investigators
testing selectivity of indicators by the ability of potential
interferents to compete with zinc binding itself, not just
produce fluorescent responses.
ConclusionsOwing to very substantial creative effort by several inves-
tigators, the palette of fluorescent zinc indicators has
www.sciencedirect.com
expanded many-fold and contributed enormously to
our understanding of the biology of this ‘trace’ element.
Many of these indicators, both the small molecules and
protein-based, are now available commercially from Invi-
trogen (Molecular Probes, http://www.invitrogen.com),
NeuroBioTex (http://www.neurobiotex.com) and Tef-
Labs (http://www.teflabs.com). Many important questions
remain to be resolved, including very basic functional and
mechanistic issues regarding zinc in several organs and
organelles. The answers to these questions are likely to be
far-reaching in their importance in view of the putative
roles of zinc in many diseases. It truly is the best of times in
which to be studying the biology of zinc.
AcknowledgementsThe author wishes to thank his colleagues at Maryland for their effortsand many stimulating discussions, Carol Fierke and Chris Fredericksonfor frequent and invaluable guidance, and Krystyna Gryczynska forpreparing some of the figures. This work was supported by the NationalInstitute of Biomedical Imaging and Bioengineering grant.
References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:
� of special interest�� of outstanding interest
1. Berg JM, Shi Y: The galvanization of biology: a growingappreciation for the roles of zinc. Science 1996, 271:1081-1085.
2. Frederickson CJ, Koh J-Y, Bush AI: The neurobiology of zinc inhealth and disease. Nat Rev Neurosci 2005, 6:449-462.
3. Fraker PJ, King LE: Reprogramming of the immune systemduring zinc deficiency. Annu Rev Nutr 2004, 24:277-298.
4. Bertrand G, Vladesco R: Intervention probable du zinc dans lesphenomenes de fecondation chez les animaux vertebres.Comptes Rendus de l’Academie des Sciences (Paris)1921:173-176. [Title translation : Probable intervention ofzinc in the reproduction of vertebrate animals.]
5. Bush AI, Pettingell WH, Multhaup G, Paradis Md, Vonsattel J-P,Gusella JF, Beyreuther K, Masters CL, Tanzi RE: Rapid inductionof Alzheimer AB amyloid formation by zinc. Science 1994,265:1464-1467.
6. Koh JY, Suh SW, Gwag BJ, He YY, Hsu CY, Choi DW: The role ofzinc in selective neuronal death after transient global cerebralischemia. Science 1996, 272:1013-1016.
7. Walker CF, Black RE: Zinc and the risk for infectious disease.Annu Rev Nutr 2004, 24:255-275.
8. Frederickson CJ: The neurobiology of zinc and of zinc-containing neurons. Int Rev Neurobiol 1989, 31:145-238.
9. Truong-Tran AQ, Carter J, Ruffin RE, Zalewski PD: The role of zincin caspase activation and apoptotic cell death. Biometals 2001,14:315-330.
10. Peck EJ, Ray WJ: Metal complexes of phosphoglucomutasein vivo: alterations induced by insulin. J Biol Chem 1971,246:1160-1167.
11. Outten CE, O’Halloran TV: Femtomolar sensitivity ofmetalloregulatory proteins controlling zinc homeostasis.Science 2001, 292:2488-2492.
12. Cole TB, Wenzel HJ, Kafer KA, Schwartzkroin PA, Palmiter RD:Elimination of zinc from synaptic vesicles in the intact mousebrain by disruption of the ZnT3 gene. Proc Natl Acad Sci USA1999, 96:1716-1721.
13. Frederickson CJ, Kasarskis EJ, Ringo D, Frederickson RE:A quinoline fluorescence method for visualizing and assaying
Current Opinion in Chemical Biology 2005, 9:526–532
532 Analytical techniques
histochemically reactive zinc (bouton zinc) in the brain.J Neurosci Methods 1987, 20:91-103.
14. Jiang P, Guo Z: Fluorescent detection of zinc in biologicalsystems: recent development on the design of chemosensorsand biosensors. Coord Chem Rev 2004, 248:205-229.
15. Kimura E, Aoki S: Chemistry of zinc(II) fluorophore sensors.Biometals 2001, 14:191-204.
16. Kikuchi K, Komatsu K, Nagano T: Zinc sensing for cellularapplication. Curr Opin Chem Biol 2004, 8:182-191.
17.�
Burdette SC, Lippard SJ: ICCC34-golden edition ofcoordination chemistry reviews. Coordination chemistry frothe neurosciences. Coordination Chemistry Reviews 2001,216-217:333-361.
See [14–16,17�] for current reviews on fluorescent zinc indicators.
18. Aoki S, Kagata D, Shiro M, Takeda K, Kimura E: Metal chelation-controlled twisted intramolecular charge transfer and itsapplication to fluorescent sensing of metal ions and anions.J Am Chem Soc 2004, 126:13377-13390.
19. Kawabata E, Kikuchi K, Urano Y, Kojima H, Odani A, Nagano T:Design and synthesis of zinc-selective chelators forextracellular applications. J Am Chem Soc 2005,127:818-819.
20. Xue H, Sigg L, Kari FG: Speciation of EDTA in natural waters:Exchange kinetics of Fe-EDTA in river water. Environ SciTechnol 1995, 29:59-68.
21. Woodroofe CC, Lippard SJ: A novel two-fluorophore approachto ratiometric sensing of Zn(2+). J Am Chem Soc 2003,125:11458-11459.
22. Taki M,Wolford JL, O’Halloran TV: Emission ratiometric imagingof intracellular zinc: Design of a benzoxazole fluorescentsensor and its application in two-photon microscopy.J Am Chem Soc 2004, 126:712-713.
23. Thompson RB, Maliwal BP, Zeng HH: Zinc biosensing withmultiphoton excitation using carbonic anhydrase andimproved fluorophores. J Biomed Opt 2000, 5:17-22.
24. Sensi SL, Ton-That D, Weiss JH, Rothe A, Gee KR: A newmitochondrial fluorescent zinc sensor. Cell Calcium 2003,34:281-284.
25.�
Gee KR, Zhou Z-L, Qian WJ, Kennedy R: Detection and imagingof zinc secretion from pancreatic beta-cells using a newfluorescent zinc indicator. J Am Chem Soc 2002,124:776-778.
Kennedy’s group has made wonderful images and movies of zinc releaseaccompanying insulin release from pancreatic b cells.
26. Kay AR: Evidence for chelatable zinc in the extracellular spaceof the hippocampus, but little evidence for synaptic release ofZn. J Neurosci 2003, 23:6847-6855.
27.�
Haugland RP: The Handbook: A Guide To Fluorescent ProbesAnd Labeling Technologies, 10th Edn. Invitrogen Corp.; 2005.
Tenth edition of Haugland’s handbook on fluorescent probes: an essen-tial reference, and free for the asking.
28. Henary MM, Wu Y, Fahrni CJ: Zinc(II)-selective ratiometricfluorescent sensors based on inhibition of excited stateintramolecular proton transfer. Chemistry 2004, 10:3015-3025.
29. Thompson RB, Jones ER: Enzyme-based fiber optic zincbiosensor. Anal Chem 1993, 65:730-734.
30. Godwin HA, Berg JM: A fluorescent zinc probe based onmetal induced peptide folding. J Am Chem Soc 1996,118:6514-6515.
31. Shults MD, Pearce DA, Imperiali B: Modular and tunablechemosensor scaffold for divalent zinc. J Am Chem Soc 2003,125:10591-10597.
Current Opinion in Chemical Biology 2005, 9:526–532
32. Barondeau DP, Kassman CJ, Tainer JA, Getzoff ED: Structuralchemistry of a green fluorescent protein Zn biosensor.J Am Chem Soc 2002, 124:3522-3524.
33. Pearce LL, Gandley RE, Han W, Wasserloos K, Stitt M, Kanai AJ,McLaughlin MK, Pitt BR, Levitan ES: Role of metallothionein innitric oxide signaling as revealed by a green fluorescent fusionprotein. Proc Natl Acad Sci USA 2000, 97:477-482.
34. Marvin JS, Hellinga HW:Conversion of amaltose receptor into azinc biosensor by computational design. Proc Natl Acad SciUSA 2001, 98:4955-4960.
35. Regan L, Clarke ND: A tetrahedral zinc(II)-binding siteintroduced into a designed protein. Biochemistry 1990,29:10878-10883.
36. Hong S-H, Maret W: A fluorescence resonance energy transfersensor for the beta-domain of metallothionein. Proc Natl AcadSci USA 2003, 100:2255-2260.
37. Dwyer MA, Looger LL, Hellinga HW: Computational design ofa Zn2+ receptor that controls bacterial gene expression.Proc Natl Acad Sci USA 2003, 100:11255-11260.
38.�
Fierke CA, Thompson RB: Fluorescence-based biosensing ofzinc using carbonic anhydrase. Biometals 2001, 14:205-222.
Review of carbonic anhydrase-based fluorescent zinc measurement.
39. Thompson RB, Maliwal BP, Feliccia VL, Fierke CA, McCall K:Determination of picomolar concentrations of metal ionsusing fluorescence anisotropy: biosensing with a‘‘reagentless’’ enzyme transducer. Anal Chem 1998,70:4717-4723.
40. Thompson RB, Maliwal BP, Fierke CA: Selectivity and sensitivityof fluorescence lifetime-based metal ion biosensing using acarbonic anhydrase transducer. Anal Biochem 1999,267:185-195.
41. Bozym RA, Zeng HH, Cramer M, Stoddard A, Fierke CA,Thompson RB: In vivo and intracellular sensing and imaging offree zinc ion. In Proceedings of the SPIE Conference on AdvancedBiomedical and Clinical Diagnostic Systems II. Edited by Cohn GE,Grundfest WS, Benaron DA, Vo-Dinh T: SPIE; 2004. vol5318:34-38.
42. Thompson RB, Cramer ML, Bozym R, Fierke CA: Excitationratiometric fluorescent biosensor for zinc ion at picomolarlevels. J Biomed Opt 2002, 7:555-560.
43. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF: In vivo proteintransduction: Delivery of a biologically active protein into themouse. Science 1999, 285:1569-1572.
44. Zeng H-H, Bozym RA, Rosenthal RE, Fiskum G, Cotto-Cumba C,Westerberg N, Fierke CA, Stoddard A, Cramer ML, FredericksonCJ et al.: In situ measurement of free zinc in an ischemiamodeland cell culture using a ratiometric fluorescence-basedbiosensor. In SPIE Conference on Advanced Biomedical andClinical Diagnostic Systems III; San Jose, CA. Edited by Vo-Dinh T,Grundfest WS, Benaron DA, Cohn GE: SPIE: 2005:51-59.
45. McCall KA, Fierke CA: Probing determinants of the metal ionselectivity in carbonic anhydrase using mutagenesis.Biochemistry 2004, 43:3979-3986.
46. Hitomi Y, Outten CE, O’Halloran TV: Extreme zinc-bindingthermodynamics of the metal sensor/regulator protein, ZntR.J Am Chem Soc 2001, 123:8614-8615.
47. Rae TD, Schmidt PJ, Pufahl RA, Culotta VC, O’Halloran TV:Undetectable intracellular free copper: the requirement of acopper chaperone for superoxide dismutase. Science 1999,284:805-808.
48. Pocker Y, Fong CTO: Inactivation of bovine carbonic anhydraseby dipicolinate: Kinetic studies and mechanistic implications.Biochemistry 1983, 22:813-818.
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