Selectivity and Sensitivity of Fluorescence Lifetime-Based Metal Ion Biosensing Using a Carbonic Anhydrase Transducer

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Analytical Biochemistry 267, 185–195 (1999)Article ID abio.1998.2991, available online at http://www.idealibrary.com on

electivity and Sensitivity of Fluorescence Lifetime-Basedetal Ion Biosensing Using a Carbonic

nhydrase Transducer

ichard B. Thompson,*,1 Badri P. Maliwal,* and Carol A. Fierke†Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street,altimore, Maryland 21201; and †Department of Biochemistry, Duke University Medical Center,urham, North Carolina 27710

eceived September 1, 1998

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A key performance criterion for metal ion determina-ions in complex media like serum, cytoplasm of the cell,nd sea water is selectivity: the ability to determine thenalyte(s) of interest, in the presence of relatively highoncentrations of interferents. Cu(II), Zn(II), Cd(II),o(II), and Ni(II) may be determined by changes they

nduce in the fluorescence lifetime and intensity of site-pecifically labeled fluorescent variants of apocarbonicnhydrase II. Free metal ion concentrations in the pico-olar range (for Cu(II) and Zn(II)) and the nanomolar

ange (for Cd(II), Co(II), and Ni(II)) were determined,ased on the affinity of the apoenzyme for these ions.g(II) at 50 mM and Ca(II) at 10 mM produced no effect.y the use of different fluorescent labels, transducersere made which responded well to Cu(II), Co(II), andi(II), but not to Zn(II) and Cd(II), and vice versa. © 1999

cademic Press

The determination of metal ion concentrations inomplex media like serum, sea water, and the cyto-lasm of cells remains an important analytical task.ields as diverse as chemical oceanography, clinicaledicine, and wastewater monitoring have stimulated

he development of fluorescence-based optical sensorsapable of continuous measurement of key analytesver long periods of time. Recently, the study of metalon fluxes such as Ca(II), Zn(II), and Cu(II), both withinnd between cells, has taken on new importance (1–3).hese research questions may be addressed, as in thease of Ca(II), by fluorescent indicator systems whichermit imaging of free metal ion concentrations (4). In

1

ZTo whom correspondence should be addressed. Fax: (410) 708-

122. E-mail: [email protected]

003-2697/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

he cases of Cu(II) and Zn(II), the low concentrations ofree metal ion compared with the high concentration ofotential interferents such as Mg(II) and Ca(II) sug-est that very selective fluorescent indicator systemsill be required.Our laboratory has developed a new approach to

uorescence-based determination of metal ions, onemploying an enzyme as the sensor transducer. Thenzyme chosen was human carbonic anhydrase II fromrythrocytes. This well-characterized enzyme has ainc ion bound to three histidinyl residues in a tetra-edral geometry, which catalyzes the hydration of car-on dioxide to form bicarbonate (5). From a sensingerspective apo-carbonic anhydrase has high selectiv-ty for zinc, and we have developed several means toeport the presence of various metal ions in the activeite as changes in fluorescence. Thus we have deter-ined free zinc ion concentrations in solution by

hanges in fluorescence intensity ratio (6), lifetime (7,), and anisotropy (9–11) as well as concentrations ofopper and other metals by changes in lifetime andntensity (12). While apo-carbonic anhydrase’s high af-nity and selectivity for zinc have long been known13), only more recently has the enzyme’s relativelyigh selectivity for Zn (and a few other metals) beenxploited. The relative affinities for Zn(II) and otherivalent cations of apocarbonic anhydrase, as well asther fluorescent and nonfluorescent chelators, and anon-selective electrode, are depicted in Fig. 1. It is ap-arent that apocarbonic anhydrase has higher affinityor Zn(II) (Kd 5 4 pM at pH 7.5, 25°C.) than metal-ofluorescent indicators suggested for use as zinc indi-ators, including BTC-5N (100 nM), Mag-fura-2 (20M), and Fura-2 (3 nM) (14, 15). Indeed, while Fura-2

s best known as a calcium probe (16), in fact it binds
n(II) more tightly than Ca(II), and the only reason
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186 THOMPSON, MALIWAL, AND FIERKE

n(II) doesn’t interfere with Ca(II) measurements isue to the low levels of free Zn inside cells. Other,lassical metallofluorescent indicators such as hy-roxyquinoline derivatives, morin, and anthraquinoneerivatives are generally less selective than the newerndicators. Morin, for instance, exhibits significanthanges in fluorescence in the presence of no less than5 different metal ions (17, 18). Similarly, the chelatoritrilotriacetic acid has low cation selectivity, although

t binds Zn(II) quite tightly (19). By comparison, car-onic anhydrase binds only two metal ions, Cu(II) (KD

0.4 pM) and Hg(II) (KD , femtomolar), with higherffinity (Fig. 1). Particularly noteworthy are the factshat Ca(II) at 10 mM and Mg(II) at 50 mM do notompete with higher affinity metals for binding to apo-arbonic anhydrase. Finally, we note that although theon-selective electrode (Fig. 1) is somewhat more selec-ive for Cd(II) than Zn(II), it has significant shortcom-ngs in that it has poor discrimination for Zn(II) com-ared to Na, and as described is insensitive tooncentrations lower than micromolar (20); however,ecent results promise a significant improvement inon-selective electrode sensitivity (21). Taken together,hese results indicate that sensors employing biologicalor biologically derived) molecules can offer enhancedensitivity and selectivity over simple organic mole-ules.

Several of the transduction schemes for carbonic an-ydrase-based Zn(II) sensing are themselves highlyelective for Zn(II), in that they rely on the uniquehemical properties of Zn(II) bound in the active site to

IG. 1. Selectivity of sensor transducers. Affinities for various meura-2 and BTC-5N, wild-type human carbonic anhydrase II, andarameter is shown for the Cd-selective ionophore, ETH 1062, in a

ransduce the signal. Thus the Zn-dependent binding n

f aryl sulfonamides to carbonic anhydrase (the basis oflinically useful inhibitors such as acetazolamide) maye transduced as changes in fluorescence intensity ra-ios (6), lifetimes (8, 22), or anisotropy (9, 10). Butubstitution of other metal ions for Zn(II) (with thexception of Co(II) (23, 24) substantially decreases theffinity for aryl sulfonamides.Alternatively, we have shown that other metals that

ind to carbonic anhydrase and exhibit weak d-d ab-orbance bands will induce proximity-dependentuenching of a fluorescent label with suitable spectralroperties, mainly by the Forster energy transferechanism. Thus Cu(II), Co(II), and Ni(II) may be

etermined in this fashion by changes in fluorescencentensity or, preferably, by changes in anisotropy (11)r lifetime (12). We have subsequently demonstratedhat metal ions, including Hg(II) and Cd(II), would alsouench certain fluorescent labels on the enzyme if theabel were in close proximity by other mechanismsuch as electron transfer. However, nearly all the flu-rescent-labeled apocarbonic anhydrases sensitive tohese other metals exhibited little or no response ton(II) binding. Consequently, we sought a labeled apo-arbonic anhydrase variant that would respond to theresence of Zn(II) and other metals as well, hopefullyn a differential fashion to enhance the resolution of

ixtures of metal ions. Ultimately, we developed twoifferent labeled carbonic anhydrases which exhibitramatically different responses to metal ions includ-ng Cu(II), Zn(II), Cd(II), Ni(II), and Co(II) and whichhus offer the prospect of simultaneous multicompo-

ions are indicated by horizontal lines for the fluorescent indicatorse chelator nitrilotriacetic acid (NTA). Also the relative selectivitymer membrane.

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187FLUORESCENCE BIOSENSING OF TRANSITION METALS

XPERIMENTAL

Variants of human carbonic anhydrase II with aysteine residue inserted in the sequence replacing thesparagine at position 67 (N67C-CA) or the phenylal-nine at position 131 (F131C-CA) were constructed,loned, expressed in E. coli strain BL21(DE3)pACA,solated, and purified essentially as previously de-cribed (25, 26). N67C-CA protein (20 mM) was labeledith a 10-fold molar excess of 7-fluorobenz-2-oxa-1,3-iazole-4-sulfonamide (ABD-F; Molecular Probes Cat.o. F-6053, Eugene, OR)2 dissolved in dimethylform-mide (DMF; final proportion, ,1%) by incubation forh at room temperature in pH 8.0 borate buffer, fol-

owed by gel filtration on Sephadex G-10 to removenreacted reagent. Significant difficulty was encoun-ered in removal of Zn(II) ion from ABD-labeled N67CN67C-ABD) using dipicolinic acid, perhaps due to non-ovalent binding of residual ABD-F to the Zn(II) ion ornterference with the dipicolinic acid binding to thenzyme. Although the apoenzyme could be labeled, itas considered prudent to label the enzyme with metalound. F131C-CA was labeled with 1-(2-maleimidyl-thyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridiniumethanesulfonate (PyMPO maleimide: Molecularrobes Cat. No. M-6026) in DMF (,2.5% of the finaleaction mixture) in borate buffer as above; the degreef conjugation was less than one PyMPO maleimideoiety per CA molecule by spectrophotometry (e412 nm 5

3,000 M21 cm21). Buffers were passed over Chelex-100olumns (Bio-Rad), stored in new or acid-washed poly-thylene plastic bottles, and handled with metal-freeipet tips (Bio-Rad) to minimize metal ion contamina-ion. Zn(II) and Cu(II) were incubated with apoenzymeor at least several hours to assure complete equilibra-ion; equilibration is much faster with the other met-ls. Free metal ion concentrations in the nanomolarange and below are difficult to maintain in the ab-ence of metal ion buffers. Thus, the following buffersere used with their respective metal ions: nitrilotri-cetic acid with Cu(II), Zn(II), and Cd(II) and Bicineith Co(II) and Ni(II). Buffer concentrations were cal-

ulated with a spreadsheet program developed byeith McCall and C. A. Fierke at Duke Universitysing the known pH-dependent stability constants

19). All buffers were maintained at pH 7.0 with 10 mMorpholinepropanesulfonic acid, which has very mod-

st affinity for the metal ions.Steady-state emission spectra and intensities wereeasured on a Spectronics AB-2 spectrophotofluorim-

ter with 8-nm bandpasses and are uncorrected. Fluo-escence intensities were fit to a single binding iso-

2 Abbreviations used: ABD-F, 7-fluorobenz-2-oxa-1,3-diazole-4-ulfonamide; DMF, dimethylformamide; PyMPO maleimides 1-(2-

Valeimidylethyl)-4-(5-(4-methoxyphenyl)-oxazol-2-yl)pyridinium meth-

nesulfonate.

herm using the program Kaleidagraph (Synergyoftware). Fluorescence lifetime data were obtained onn ISS K2 multifrequency phase fluorometer using theltraviolet lines of a Spectra-Physics 2065-7S argon ion

aser (100 mW, all lines, for ABD) or a Kimmon He Cdaser for PyMPO-labeled CA (35 mW at 442 nm) forxcitation, KV-470 and KV-520 bandpass emission fil-ers for the ultraviolet and blue excitation, respec-ively, and dimethylPOPOP in ethanol or Rose Bengaln ethanol as references (27). Frequency-dependenthase and modulation data were analyzed using ISSroprietary software.

ESULTS

Labeling of apo-N67C CA with ABD-F produced aellow–orange conjugate (lmax 5 397 nm) which fluo-esced maximally in the green (Fig. 2). Addition ofn(II) ion to the fluorescent-labeled apoprotein causeddramatic 10-fold increase in fluorescence intensity;

ddition of Cu(II), Cd(II), or Ni(II) caused less dramaticncreases in intensity. Titrating the ABD-N67C withach of the metal ions in buffered solutions demon-trated a concentration dependence of the fluorescencentensity for each metal ion (Fig. 3). The data areell-fit in each case by single binding isotherms withD 5 17 6 4 pM (Cu(II)), 110 6 10 pM (Zn(II)), 9 6 2M (Cd(II)), and 80 6 30 nM (Ni(II)). The affinity ofpo-ABD-N67C for cadmium and nickel ions is thuselatively close to that of the wild-type apoenzyme (9nd 15 nM, respectively) (K. McCall and C. A. Fierke,npublished data). However, the affinity for Cu(II) andn(II) is decreased significantly compared to the wild-ype KDs of 0.1 and 1.0 pM, respectively. The decreasedetal affinity taken together with the large dequench-

ng effect of Zn(II) suggests that the ABD fluorophoreust be near enough to interact with the metal site,hich is unsurprising given that residue 67 is less than0 Å from the metal. However, we note that the sul-onamide moiety of the ABD fluorophore conjugated tohis residue is not properly positioned to bind as aulfonamide anion as is typically the case with arylulfonamide inhibitors (28). This is further borne outy the lack of any excitation or emission shift (Fig. 2)pon binding of the Zn(II), as is seen with dansylamide29), ABD-M (10), or ABD-N (11). The relatively closeroximity of the ABD moiety to the active site suggestshat it may have perturbed the binding of the variousetals; thus, there is no reason to believe the apparent

ffinities derivable from the intensities in Fig. 3 arenaccurate. Finally, we note that the low free metal iononcentrations and the modest fluorescence lifetime ofhe ABD label (see below) are inconsistent with thentensity changes, which are being due to classicalollisional quenching as described by the Stern–
olmer theory.

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The fluorescence lifetimes of N67C-ABD carbonic an-ydrase in the apo form and with metal ions boundere also measured using multifrequency phase flu-rometry. In the absence of metal ions the apo formxhibited a complex decay (Fig. 4) which could be fit (x2

7.0) by three components: a 7.8-ns component withractional intensity of 27%, a 1.3-ns component with6%, and a 0.16-ns component with 17%. Binding of

FIG. 2. Fluorescence excitation (---) and emission sp

IG. 3. Fluorescence intensity (arbitrary units) as a function ofetal ion concentration for apo-N67C-ABD in the presence of Zn21

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wF), Cu (E), Cd (‚), and Ni (Œ). The lines indicate the best fit tohe data.

n(II) to the apoprotein increased the average lifetime,ut not as much as the intensity increased, suggestinghat some static quenching occurs in the apo form. Inarticular, the frequency-dependent phase and modu-ation data were well fit by a three-component decay (x2

1.2), with the main components being 5.7 ns (46%),.8 ns (45%), and 0.36 ns (9%). If the phase angle andodulation are measured at a suitable modulation fre-

uency, significant changes may be observed as a func-ion of analyte (metal ion) concentration if the lifetimesre different enough, as is evidently the case. Thushase shifts and modulations were measured at 101Hz for apo-N67C-ABD as a function of free metal ion

oncentration for Zn(II), Cu(II), and Cd(II) (Figs. 5 and). The phase angle of the apo-N67C-ABD is 30°; addi-ion of Zn(II) increases the lifetime substantially, cre-ting an increase in phase angle of 16° (Fig. 5). Theodulation change is equally dramatic, resulting in a

ecline from 68 to 52% (Fig. 6). In view of the facts thathe ordinary accuracy and precision of phase and mod-lation measurements are roughly a few parts perhousand, it is clear that the Zn(II) concentration cane determined with some precision by this method. Inontrast, the binding of Cu(II) causes a decrease inifetime which results in a decline in phase angle by 8°nd an increase in modulation by 14%. This is at oddsith the slight increase in intensity that accompanies

he binding of copper ion, which we attribute to theum of a decrease of a static quenching process (whichncreases the intensity without changing the lifetime)

ra of N67C-ABD in the holo (—) and apo (– –) forms.

ith a metal-dependent quenching process which de-

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reases the lifetime and intensity. The behavior ofd(II) in this respect mirrors that of Zn(II), but at

ower concentrations. The apparent affinities takenrom the phase angle and modulation data are approx-

IG. 4. Frequency-dependent phase shifts (F, E) and modulationssaturating concentration of Zn(II).

IG. 5. Phase angles at 101 MHz modulation frequency for apo-N67oncentrations.

mately 10 pM for Cu(II), 20 pM for Zn(II), and 3 nM ford(II). Again, these figures are similar to the wild-typeinding affinities; we note that the nonlinear way inhich the phase angles and modulations of the bound

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nd free forms contribute to the observed values (8)akes it difficult to accurately determine the KD solely

rom data of this kind without separate calibration.evertheless, it is apparent that apo-N67C-ABD re-

ponds well to the binding of Cd(II), Cu(II), and Zn(II).

IG. 6. Modulation at 101 MHz modulation frequency for apo-Noncentrations.

FIG. 7. Fluorescence excitation (---, emission at 560 nm) and em

The significantly higher extinction coefficient ofyMPO makes it a brighter fluorophore than ABD.hen conjugated to N67C-CA, it exhibits a large

tokes’ shift with broad emission at 550 nm (Fig. 7),hereas the emission is somewhat to the red when

-ABD measured as a function of Cu21 (F), Cd21 (E), or Zn21 (‚)

ission (—, excitation at 420 nm) spectra of apo-N67C-PyMPO.

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onjugated at F131C-CA (maximum at 565 nm) (re-ults not shown); absorbance and excitation maximaor both labeled variants are at 412 nm, and they cane readily excited by the 442-nm line of the HeCd laserFig. 7). Addition of saturating concentrations of Zn(II)r Cd(II) have a negligible effect on the fluorescencentensity of PyMPO-labeled apo-N67C-CA, but addi-ion of Ni(II), Co(II), or Cu(II) induces decreases inntensity of approximately 25, 75, and 70%, respec-ively. Measurement of intensities of PyMPO-labeledpoN67C-CA as a function of free Ni(II), Co(II), andu(II) yielded the expected binding isotherms for co-alt and nickel (Fig. 8), but not for copper ion (seeelow). The fitted values for nickel and cobalt are 30 6and 16 6 3 nM, which are, respectively, comparable

o and eightfold higher than the wild-type affinities. Fito a single isotherm, the Cu(II) data yielded 0.17 6 0.02M, which is also close to the wild-type value. How-ver, while the cobalt and nickel isotherms showed thesual range of 10 to 90% intensity change over approx-

mately 1.9 log units in metal ion concentration (30),he copper isotherm extended over a broader concen-ration range. The data in Fig. 8 are perhaps bestxplained by binding of a second mole of copper ion inddition to the active site Cu(II); the apparent affini-ies of these two sites are about 0.13 and 30 pM, re-pectively. We have observed binding of Co(II) appar-ntly to an additional site on other variants of CA (12),nd binding of transition metals to polypeptides is wellnown. Furthermore, Cu(II) has been observed by X-ay crystallography to bind to a second site on wild-ype human CA II by Hakansson and his colleagues

IG. 8. Normalized fluorescence intensities of apo-N67C-PyMPOexcitation at 410 nm, emission at 550 nm) as a function of Cu21 (h),o21 (‚), and Ni21 (F) concentrations. The best single binding site fitsre indicated by the solid lines, and the best two-site fit by a dashedine.

31). They found that the second Cu(II) was bound by c

wo histidines (4 and 64) near the active site, whichould account for the apparent quenching of theyMPO by the second Cu(II) and its apparently weakerffinity.We measured the fluorescence decays of the PyMPO-

abeled apo-N67C-CA variant in the absence of metalnd in the presence of saturating concentrations ofi(II), Co(II), and Cu(II). The decays were complex

Fig. 9) and best fit by three components (Table 1). Ass apparent from Fig. 9 and Table 1, the fluorescenceecays in the presence of Ni(II), Co(II), and Cu(II) arearkedly different from that of the apoprotein, as

udged by the frequency-dependent phase shifts andemodulations. The large apparent differences inhase angle and modulation in the range of 90 MHzuggests that PyMPO-labeled CA variants could beuccessfully used for lifetime-based sensing of Cu(II),o(II), and Ni(II). In particular, we note that the phasengle differences at 90.9 MHz between PyMPO-labeled67C-CA in the apo form and with Cu(II), Co(II), andi(II) are 30.3, 19.4, and 11.6°, respectively, with com-ensurate differences in modulation (Figs. 9 and 10).hese differences are more than adequate for lifetime-ased sensing of these metal ions. The very short av-rage lifetimes compared to the approx 15-ns rota-ional correlation time of the protein result in anyifferences in anisotropy between apo and metal-boundorms being negligible (results not shown), and thushese labeled forms of CA are not useful for anisotropy-ased sensing (10).

ISCUSSION

These results make clear the value of fluorescence-ased determinations of free metal ion concentrationssing a biologically derived transducer molecule suchs CA. In particular, the determination of free Cu(II) atubpicomolar levels is at least three orders of magni-ude more sensitive than previous determinations byolecular fluorescence not employing preconcentration

18) and is perhaps comparable to what may bechieved with graphite furnace atomic absorption spec-rophotometry or emission methods or mass spectro-etric techniques. The zinc determinations also repre-

ent comparable improvement over previous molecularuorescence-based measurements. Note that the use ofetal ion buffers to maintain low free metal ion levels

s somewhat artificial, in that it also provides a reser-oir of metal ion which enables a nanomolar concen-ration of fluorescent-labeled apoenzyme to be partiallyaturated by a picomolar concentration of free Zn(II) oru(II). In natural waters we anticipate that conditionsre similar, in that free concentrations of these metalsill be low, with the vast majority of the total metal ionound to indigenous ligands such as dissolved organic
arbon (32). While the factors that determine metal ion

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peciation in natural waters are a large and active areaf research beyond the scope of this paper, it may beaid that this approach may be of value in that itistinctly measures only “free” ion without a separa-ion step, rather than total metal ion, as do most otherechniques. Nevertheless, in a nonbuffered system anxcess of protein would only be fractionally saturatedy a free metal ion concentration, even well above theD. For instance, in a fixed volume 10 nM proteinould only be 10% saturated by 1 nM free Zn, even

hough the KD is 50-fold lower. We anticipate usinghese fluorescent protein transducers in the context ofber optic sensors, where very small amounts of theransducer protein are immobilized on the distal end ofhe optical fiber, and thus the “concentration” of theabeled CA is quite low, and the sensing volume will

IG. 9. Frequency-dependent phase shifts (h, E, {) and modulationf saturating concentrations of Cu ({, }) and Ni (E, F).

TAB

Best 3-component fits to apo-N67C

Metala t1 f1 t2

None 5.00 0.155 2.69Cu21 1.56 0.279 0.72Co21 2.63 0.392 0.85Ni21 3.56 0.207 1.595

a Abbreviations: “metal” refers to the presence of a saturating conanoseconds and fractional intensity of component i; x2 is the sum o
hase and modulation, normalized to the average standard deviationserived lifetimes and fractional intensities were typically 620 ps and 6
ften be large, e.g., the ocean (12). For determinationsn a small, fixed volumes the detection limit for theightly bound metals is thus determined by the lowestoncentration of fluorescent-labeled CA which gives aeliably measurable response. In this regard, theyMPO label is more useful, because of its higherxtinction coefficient, convenient excitability with theeCd laser, and high quantum yield.A key attribute of carbonic anhydrase-based fluo-

escence sensors is their selectivity, as indicated byig. 1, as well as Figs. 3, 5, 6, 8, 10, and 11. Inarticular, carbonic anhydrase is sensitive to Cu andn concentrations in the picomolar range and Cd, Ni,nd Co concentrations in the nanomolar range, evenn the presence of millimolar concentrations of Cand Mg. Even newly developed Zn indicators do not

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xhibit selectivity as good: Mag-Fura-5 responds toree Zn(II) levels in the 20 nM range, but is inter-ered with by Mg in the low millimolar range, whichs present physiologically (14, 33). By comparison,po-N67C-ABD is 1000-fold more sensitive to Zn(II)ith at least 10-fold better rejection of Mg. This levelf selectivity may be crucial in studying, for instance,

FIG. 10. Phase angles at 92 MHz for apo-N67C-PyMPO as

FIG. 11. Modulation at 92 MHz for apo-N67C-PyMPO as a f

n metabolism in the brain (2, 3, 34). Similarly, theetabolism, transport, and toxicity of Cu(II) in hu-ans and other organisms remains a topic of signif-

cant current interest (1, 35). Again, current metal-ofluorescent indicators such as fluorescein-labeledly-His and Gly-Gly (14) do not offer comparable

ensitivity and selectivity.

function of Cu21 (h), Co21 (‚), and Ni21 (F) concentrations.

unction of Cu21 (h), Co21 (‚), and Ni21 (F) concentrations.

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Recently, several workers have developed other bio-ogical or biologically derived molecules for fluores-ence-based sensing of metal ions. Thus, Klemba andegan showed (36) that a purpose-designed proteinith a four helix bundle structure incorporating a zinc-nger metal ion binding motif bound other metals asell. While the affinity (tens of micromolar and up) and

electivity were modest in comparison to carbonic an-ydrase, these workers have shown that polypeptidesan be engineered de novo for biosensor transduction.imilarly, Godwin and Berg developed fluorescent-la-eled zinc finger peptides as a means for determiningn(II) (37). By attaching fluorescent donor and accep-or moieties to a consensus zinc finger peptide theyere able to achieve a modest ratioable response andigh sensitivity, presuming the 5 pM binding constantor zinc is unaffected. The selectivity of the underiva-ized zinc finger for Co(II) (1.1 3 104-fold weaker bind-ng) and Ni(II) (2.8 3 105) with respect to zinc ion isearly as good as that exhibited by carbonic anhydrase1 3 105-fold), and (5 3 104-fold), respectively (37),lthough the stoichiometry of metal ion binding is am-iguous. Finally, Imperiali and her colleagues haveurther exploited the zinc finger motif for binding ofinc and other transition metal ions (38), as well asdapting the Gly-Gly-His peptide motif for this pur-ose (39). In each case they labeled the peptide withansyl or another solvent-sensitive UV-excitable flu-rophore, and observed substantial changes in fluores-ence upon metal ion binding, with good selectivity andensitivity. All these results complement our own inisplaying the power of biologically derived moleculesor sensing purposes.

While the bulk of the selectivity of apocarbonic an-ydrase arises from the unique metal-binding proper-ies of its active site, selectivity can also be achieved byelecting fluorescent-labels which respond differen-ially to the individual metal ions. In particular, thepoN67C-ABD variant exhibits overt changes in life-ime and intensity when Zn(II) binds, a more modesthange in intensity (but not lifetime) when Cu or Cdinds, and smaller changes when Ni is present. Byomparison, the PyMPO-labeled variants are most sen-itive to Cu, Co, and Ni, but show little effect with Znr Cd. In both cases, the binding affinities of labeledariants for the different metals can vary up to ordersf magnitude from the wild type values, in addition toifferences in the fluorescence probe responses to thearious metals. Both ABD and PyMPO were initiallyhosen because of their sensitivity to the polarity ofheir surroundings, in the belief that if the probe werelaced sufficiently close to the metal ion binding site,he presence of the metal would perturb the electro-tatic environment of the fluorophore enough to per-urb its emission. In the case of the covalently bound
BD, the perturbation is evidently more modest than
n the case of similar noncovalent fluorescent aryl sul-onamide inhibitors such as the ethanolamine (ABD-N11)) and b-mercaptoethanol (ABD-M (10)) adducts ofBD-F; nevertheless, it is quite usable. In the case ofyMPO, the presence or absence of the Zn(II) seems toake little difference in its emission.In many natural samples, there will be a preponder-

nce of one metal, but in many others there will beeasurable quantities of several analytes. Under these

onditions, the binding site of the enzyme becomesractionally occupied by more than one kind of metal.he fractional occupancy of the binding site Xi by metalis a function of the metal’s concentration [Mi] andffinity constant Ki:

Xi 5 [Mi]Ki/~1 1 O@Mi#Ki!. [1]

hus, if Cu(II) and Zn(II) are both present at 5 pM withCu 5 0.1 pM and KZn 5 1.0 pM (40), 89.3% of the proteinill have Cu bound, 8.9% will have Zn bound, and 1.8% of

he protein will have no metal bound. Because the affin-ties of wild-type human carbonic anhydrase II are muchreater for Cu(II) and Zn(II) than for nearly all otheretals (Fig. 1), variants with wild-type binding sites are

y far best suited for Cu and Zn sensing. Because thehase and modulation at any frequency are not linearunctions of the concentration, it is necessary know theifetimes of the bound and free forms (Table 1) to calcu-ate the phase and modulation expected for a given mix-ure of metal ions (8). Of course, it is difficult to determinef a mixture is present by simply measuring the phase or

odulation of a labeled variant at a single frequency ase have done. However, it may well be possible to deter-ine the composition of a mixture by determining the

hase and/or modulation at multiple frequencies; by usef variants labeled with different fluorophores such asBD and PyMPO, which exhibit differing responses toarious metals; or using multiple variants with selectiv-ties different than the wild type (K. McCall and C. A.ierke, in preparation). In view of the technical difficultyf preparing metal ion buffers for mixtures of metals atredetermined levels, it may be difficult to test this ap-roach in the manner we have employed heretofore; ef-orts are ongoing to develop approaches to the problem.

CKNOWLEDGMENTS

The authors thank the Office of Naval Research (R.B.T. and.A.F.), the National Institutes of Health (R.B.T. and C.A.F.), and

he National Science Foundation (R.B.T.) for support.

EFERENCES

1. O’Halloran, T. V. (1993) Science 261 713–715.
2. Choi, D. W., and Koh, J. Y. (1998) Annu. Rev. Neurosci. 21,
347–375.

1

1

1

1

1

11

1

1

1

2

2

2

22

2

2

2

2

2

3

3

33

33

33

3

3


3. Cuajungco, M. P., and Lees, G. J. (1997) Neurobiol. Disc. 4,137–169.

4. Tsien, R. Y. (1989) in Annual Review of Neuroscience, Vol. 12,pp. 27, Annual Reviews, Palo Alto, CA.

5. Lindskog, S., Henderson, L. E., Kannan, K. K., Liljas, A., Ny-man, P. O., and Strandberg, B. (1971) in The Enzymes (Boyer,P. D., Ed.), Vol. 5, pp. 587–665, Academic Press, New York.

6. Thompson, R. B., and Jones, E. R. (1993) Anal. Chem. 65, 730–734.

7. Thompson, R. B. (1993) in SPIE Conference on Advances inFluorescence Sensing Technology (Lakowicz, J. R., and Thomp-son, R. B., Eds.), Vol. 1885, pp. 290–299, SPIE, Los Angeles, CA.

8. Thompson, R. B., and Patchan, M. W. (1995) J. Fluoresc. 5,123–130.

9. Elbaum, D., Nair, S. K., Patchan, M. W., Thompson, R. B., andChristianson, D. W. (1996) J. Am. Chem. Soc. 118, 8381–8387.

0. Thompson, R. B., Maliwal, B. P., and Fierke, C. A. (1998) Anal.Chem. 70, 1749–1754.

1. Thompson, R. B., Maliwal, B. P., Feliccia, V., and Fierke, C. A.(1998) in Systems and Technologies for Clinical Diagnostics andDrug Discovery (Cohn, G. E., Ed.), Vol. 3259, pp. 40–47, SPIE,San Jose, CA.

2. Thompson, R. B., Ge, Z., Patchan, M. W., Huang, C.-c., andFierke, C. A. (1996) Biosens. Bioelectr. 11, 557–564.

3. Lindskog, S., and Nyman, P. O. (1964) Biochim. Biophys. Acta85, 462–474.

4. Haugland, R. P. (Ed.) (1996) Handbook of Fluorescent Probesand Research Chemicals, Molecular Probes, Eugene, OR.

5. Simons, T. J. B. (1993) J. Biochem. Biophys. Methods 27, 25–37.6. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol.

Chem. 260, 3440–3450.7. White, C. E., and Argauer, R. J. (1970) Fluorescence Analysis: A

Practical Approach, Dekker, New York.8. Fernandez-Gutierrez, A., and Munoz de la Pena, A. (1985) in

Molecular Luminescence Spectroscopy, Part I, Methods and Ap-plications (Schulman, S. G., Ed.), Vol. 77, pp. 371–546, Wiley-Interscience, New York.

9. Smith, R., and Martell, A. E. (1973) National Institute of Stan-
dards and Technology, U.S. Department of Commerce, Gaithers-burg, MD.
4

0. Ammann, D. (1986) Ion-Selective Microelectrodes: Principles,Design, and Application, Springer-Verlag, Berlin.

1. Sokalski, T., Ceresa, A., Zwickl, T., and Pretsch, E. (1997) J. Am.Chem. Soc. 119, 11347–11348.

2. Thompson, R. B., and Patchan, M. W. (1995) Anal. Biochem. 227,123–128.

3. Coleman, J. E. (1967) J. Biol. Chem. 242, 5212–5219.4. Lindskog, S., and Thorslund, A. (1968) Eur. J. Biochem. 3, 453–

460.5. Nair, S. K., Calderone, T. L., Christianson, D. W., and Fierke,

C. A. (1991) J. Biol. Chem. 266, 17320–17325.6. Kiefer, L. L., Paterno, S. A., and Fierke, C. A. (1995) J. Am.

Chem. Soc. 117, 6831–6837.7. Thompson, R. B., and Gratton, E. (1988) Anal. Chem. 60, 670–

674.8. Nair, S. K., Elbaum, D., and Christianson, D. W. (1996) J. Biol.

Chem. 271, 1003–1007.9. Chen, R. F., and Kernohan, J. (1967) J. Biol. Chem. 242, 5813–

5823.0. Weber, G. (1975) in Advances in Protein Chemistry, Vol. 29, pp.

1–83.1. Hakansson, K., Wehnert, A., and Liljas, A. (1994) Acta Crystal-

logr. D50, 93–100.2. Bruland, K. W. (1989) Limonol. Oceanogr. 34, 269–285.3. Sensi, S. L., Canoniero, L. M. T., Yu, S. P., Ying, H. S., Koh, J.-Y.,

Kerchner, G. A., and Choi, D. W. (1997) J. Neurosci. 17, 9554–9564.

4. Frederickson, C. J. (1989) Int. Rev. Neurobiol. 31, 145–238.5. Prince, R. C., and Gunson, D. E. (1998) Trends Biochem. Sci. 23,

197–198.6. Klemba, M., and Regan, L. (1995) Biochemistry 34, 10094–10100.7. Godwin, H. A., and Berg, J. M. (1996) J. Am. Chem. Soc. 118,

6514–6515.8. Walkup, G. K., and Imperiali, B. (1997) J. Am. Chem. Soc. 119,

3443–3450.9. Torrado, A., Walkup, G. K., and Imperiali, B. (1998) J. Am.

Chem. Soc. 120, 609–610.
0. Hunt, J. A., and Fierke, C. A. (1997) J. Biol. Chem. 272, 20364–
20372.

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Selectivity and Sensitivity of Fluorescence Lifetime-Based Metal Ion Biosensing Using a Carbonic Anhydrase Transducer