studying zinc biology with fluorescence: ain’t we got fun?

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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 ([email protected]) 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 Introduction This 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 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; Current Opinion in Chemical Biology 2005, 9:526–532 www.sciencedirect.com

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Page 1: Studying zinc biology with fluorescence: ain’t we got fun?

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

([email protected])

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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Page 2: Studying zinc biology with fluorescence: ain’t we got fun?

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

www.sciencedirect.com

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

Page 3: Studying zinc biology with fluorescence: ain’t we got fun?

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.

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Page 4: Studying zinc biology with fluorescence: ain’t we got fun?

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

www.sciencedirect.com

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

Page 5: Studying zinc biology with fluorescence: ain’t we got fun?

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

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532 Analytical techniques

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25.�

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Tenth edition of Haugland’s handbook on fluorescent probes: an essen-tial reference, and free for the asking.

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