Determination of Picomolar Concentrations of Metal Ions Using

Anal. Chem. 1998, 70, 4717-4723

Determination of Picomolar Concentrations of Metal Ions Using Fluorescence Anisotropy: Biosensing with a “Reagentless” Enzyme Transducer Richard B. Thompson,* Badri P. Maliwal, and Vincent L. Feliccia

Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, Maryland 21201 Carol A. Fierke and Keith McCall

Department of Biochemistry, Duke University Medical Center, Box 3711 Durham, North Carolina 27710

Because of their high affinity and selectivity, metalloproteins can be used as transducers in novel sensors, i.e., biosensors, for the determination of trace levels of metal ions in solution. Here, we exploit carbonic anhydrase to determine picomolar to nanomolar concentrations of free transition metal ions by fluorescence anisotropy (polarization) in a reagentless format. Carbonic anhydrase variants engineered with a cysteine replacing a residue chosen near the active site (F131C and H64C) were covalently labeled with derivatives of benzoxadiazole sulfonamide. These labeled variants exhibited changes in anisotropy up to 0.07 upon binding free Cu(II), Co(II), and Zn(II) with apparent Kd’s close to the values observed with wild-type apocarbonic anhydrase. The covalent attachment of the label has significant advantages over noncovalent labels we have described previously. Furthermore, the metal ion-dependent anisotropy changes were predictable using simple theory. The results demonstrate that free transition metal ions can be determined at trace levels in aqueous solution using inexpensive instruments. Determination of metal ions at trace levels (sub-part-per-billion) remains an important task in analytical chemistry, with applications in fields as diverse as environmental monitoring, clinical toxicology, wastewater treatment, animal husbandry, and industrial process monitoring. Powerful methods are available commercially which exhibit high sensitivity, selectivity, reliability, and accuracy. Including atomic absorption and emission spectroscopies, inductively coupled plasma mass spectroscopy (ICPMS), and electrochemical methods. For some applications, these techniques are unsatisfactory because they generally require collection of samples, sample processing including digestion or preconcentration, and instruments ill-suited for use outside the laboratory.1,2 For many applications, a method permitting real time sampling and analysis in the field with inexpensive, portable instruments is desirable. * Corresponding author: (phone) 410 706-7142; (fax) 410 706-7122; (e-mail) [email protected]. (1) Bruland, K. W. Limnol. Oceanogr. 1989, 34, 269-285. (2) Sherrell, R. M.; Boyle, E. A. Deep-Sea Res. 1988, 35, 1319-1334. 10.1021/ac980864r CCC: $15.00 Published on Web 10/15/1998

© 1998 American Chemical Society

Many fluorescence techniques have been developed for metal ion determination, because such techniques are generally quite sensitive and selective, and furthermore, fluorometers for use in the field or even immersed in the ocean are commercially available. Most early work focused on fluorescent indicators: small organic molecules exhibiting changes in fluorescence intensity in the presence of metal ion analytes.3,4 Unfortunately, such intensity-based determinations are difficult to calibrate and are prone to artifact. More recently, Tsien, Haugland, and others have developed the “ratiometric” indicators for metal ions, particularly calcium.5,6 These indicators, such as Fura-2 and Indo-1, exhibit large shifts in emission or excitation wavelength upon binding the analyte, and consequently, the ratio of emission intensities at two selected wavelengths of excitation or emission is a singlevalued function of the analyte concentration. These indicators permit measurement of free calcium inside and outside of cells using fluorescence microscopy, which has revolutionized the study of cell physiology and intracellular signaling.5,6 A significant issue is the difficulty of designing selective ratiometric indicators. In particular, it evidently is difficult to synthesize indicators for zinc (for example) that are sufficiently selective against magnesium or calcium.7 For instance, the widely used calcium indicators Fura-2 and Indo-1 both bind Zn(II) 100-fold more tightly than Ca(II) and only the evidently low concentrations of free Zn(II) in most cells prevent unacceptable interference;6,8 in seawater, the millimolar concentrations of Ca(II) make Fura-2 unusable for Zn(II) without separation. More recently, several workers have developed determinations for various analytes based on changes in fluorescence lifetime of an indicator or indicator system.9-12 This approach shares the (3) Fernandez-Gutierrez, A.; Munoz de la Pena, A. In Molecular Luminescence Spectroscopy, Part I: Methods and Applications; Schulman, S. G., Ed.; WileyInterscience: New York, 1985; Vol. 77, pp 371-546. (4) White, C. E.; Argauer, R. J. Fluorescence Analysis: A Practical Approach; Marcel Dekker: New York, 1970. (5) Tsien, R. Y. In Annual Review of Neuroscience; Annual Reviews, Inc.: Palo Alto, CA, 1989; Vol. 12, p 27. (6) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Molecular Probes, Inc.: Eugene, OR, 1996; p 679. (7) Simons, T. J. B. J. Biochem. Biophys. Methods 1993, 27, 25-37. (8) Jefferson, J. R.; Hunt, J. B.; Ginsburg, A. Anal. Biochem. 1990, 187, 328336.

Analytical Chemistry, Vol. 70, No. 22, November 15, 1998 4717

freedom from artifact of the ratiometric approaches, while also offering a more flexible approach and, in certain cases, a much wider dynamic range.13,14 In particular, Cu(II), Co(II), Zn(II), and Cd(II) may be determined using a lifetime-based approach with a fluorescent-labeled apoenzyme at the tip of an optical fiber, permitting remote, continuous monitoring of the analyte.15 The main issue with lifetime determinations is complex instrumentation, notwithstanding recent advances in low-cost lifetime instrumentation.16,17 Fluorescence anisotropy (closely related to fluorescence polarization18) is a well-known technique frequently used in biophysical studies of macromolecules19,20 and clinically in the form of fluorescence polarization immunoassays.21,22 It remains widely used in the study of macromolecule association and binding reactions and other assays.23,24 The phenomenon of polarization of fluorescence arises when the emission dipole of a fluorescent molecule rotates around another axis at a rate slower than its emissive rate. If, for instance, the emission transition dipole of a fluorophore is more or less rigidly constrained within the framework of a macromolecule with a relatively slow rotational rate, the fluorescence emitted upon polarized excitation will retain some degree of polarization. Fluorescence anisotropy (r) is calculated from the intensities of fluorescence observed through polarizers parallel (I|) and perpendicular (I⊥) to the polarization of the exciting light:

r ) (I| - I⊥)/(I| + 2I⊥)

(1)

A comprehensive theoretical treatment of this phenomenon, including its time dependence, may be found in Steiner.19 For our purposes, we may consider that a fluorophore becoming tightly bound to a macromolecule or experiencing a reduction in its fluorescence lifetime resulting in an apparent increase in its (9) Lippitsch, M. E.; Pusterhofer, J.; Leiner, M. J. P.; Wolfbeis, O. S. Anal. Chim. Acta 1988, 205, 1. (10) Lakowicz, J. R.; Szmacinski, H.; Thompson, R. B. SPIE Conference on Ultrasensitive Laboratory Diagnostics, Los Angeles, CA, 1993; pp 2-17. (11) Carraway, E. R.; Demas, J. N.; DeGraff, B. A.; Bacon, J. R. Anal. Chem. 1991, 63, 337-342. (12) Thompson, R. B. SPIE Conference on Advances in Fluorescence Sensing Technology, Los Angeles, CA, 1993; pp 290-299. (13) Thompson, R. B.; Patchan, M. W. J. Fluoresc. 1995, 5, 123-130. (14) Szmacinski, H.; Lakowicz, J. R. Anal. Chem. 1993, 65, 1668-1674. (15) Thompson, R. B.; Ge, Z.; Patchan, M. W.; Huang, C.-c.; Fierke, C. A. Biosens. Bioelectron. 1996, 11, 557-564. (16) Levy, R.; Guignon, E. F.; Cobane, S.; St. Louis, E.; Fernandez, S. SPIE Conference on Advances in Fluorescence Sensing Technology III, San Jose, CA, 1997. (17) Gruber, W.; O’Leary, P.; Wolfbeis, O. S. Advances in Fluorescence Sensing Technology II, San Jose, CA, 1995; pp 148-158. (18) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983. (19) Steiner, R. F. In Topics in Fluorescence Spectroscopy. Volume 2: Principles; Lakowicz, J. R., Ed.; Plenum Press: New York, 1991; pp 1-52. (20) Weber, G. Adv. Protein Chem. 1953, 8, 415-459. (21) Jolley, M. E.; Stroupe, S. D.; Schwenzer, K. S.; Wang, C. J.; Lu-Steffes, M.; Hill, H. D.; Popelka, S. R.; Holen, J. T.; Kelso, D. M. Clin. Chem. 1981, 27, 1575-1579. (22) Dandliker, W. B.; Kelly, R. J.; Dandliker, J.; Farquhar, J.; Levin, J. Immunochemistry 1973, 10, 219-227. (23) Murakami, A.; Nakaura, M.; Nakatsuji, Y.; Nagahara, S.; Cong, Q. T.; Makino, K. Nucleic Acids Res. 1991, 19, 4097-4102. (24) Davenport, D. In Fluorescence Spectroscopy: An Introduction for Biology and Medicine; Pesce, A. J., Rosen, C.-G., Pasby, T. L., Eds.; Marcel Dekker: New York, 1971; pp 203-240.

4718 Analytical Chemistry, Vol. 70, No. 22, November 15, 1998

emissive rate will exhibit an increased fluorescence anisotropy as predicted from the Perrin equation, where r is the anisotropy, φc is the rotational correlation time, τ is the fluorescence lifetime, and r0 is the anisotropy in the absence of rotation (ordinarily 0.4):

r ) r0/(1 + (τ/φc))

(2)

The anisotropy observed (robs) from a mixture of n fluorophores is the sum of the products of their fractional contributions fn and their anisotropies rn:

robs )

∑f r

n n

(3)

The major use of fluorescence anisotropy for routine chemical analysis heretofore has been for a fluorescence polarization immunoassay, wherein the concentration of a small-molecule hapten (often a drug) is transduced as a change in fluorescence polarization. Polarization immunoassays exploit the fact that a fluorescent-labeled hapten of (for instance) 1000-Da molecular mass with a subnanosecond rotational correlation time and a lifetime of 1-10 ns will exhibit a dramatic increase in anisotropy upon binding to a 150 000-Da immunoglobulin molecule with a principal rotational correlation time of 50 nanoseconds.25 Polarization immunoassays remain popular because they are relatively simple, require simple instrumentation, are accurate and precise, and are relatively free from artifact (see below). Fluorescence anisotropy measurements share many of the advantages of the ratiometric and lifetime-based methods. In particular, when configured in the classic T-format method of Weber,26 anisotropy measurements are explicitly ratiometric. Even when performed by serial measurements with a single detector in the L-format with modern light sources, the anisotropy measurement is very free from artifact; indeed, much “ratiometric” fluorescence microscopy is actually performed by dividing intensities pixel by pixel on images acquired serially using two different excitation wavelengths. These approaches are satisfactory because the (dc) drift of typical lamps is less than 0.5% over the period of the measurement (a few seconds). Fluorescence anisotropy measurements are typically accurate and precise to (0.003 or better.27 Well-known potential error sources include turbid samples because of the high anisotropy of scattered light18 and background fluorescence. Scattered light can usually be eliminated by optical filtration, and background may be corrected for to some degree. Among fluorescence methods and analytical methods in general, anisotropy measurements are therefore very robust. Thus, we sought to modify our approach to transduce the level of the analyte metal ion as a change in fluorescence anisotropy. Together with Christianson and Elbaum, we found that a fluorescent inhibitor whose binding to apocarbonic anhydrase (apo-CA) was largely metal-dependent enabled us to make such a measurement.28 In particular, the fluorescent aryl sulfonamide (25) Yguerabide, J.; Epstein, H. F.; Stryer, L. J. Mol. Biol. 1970, 51, 573. (26) Weber, G. J. Opt. Soc. Am. 1956, 46, 962. (27) Jameson, D. M.; Weber, G.; Spencer, R. D.; Mitchell, G. Rev. Sci. Instrum. 1978, 49, 510-514. (28) Elbaum, D.; Nair, S. K.; Patchan, M. W.; Thompson, R. B.; Christianson, D. W. J. Am. Chem. Soc. 1996, 118, 8381-8387.

inhibitor exhibited very low anisotropy when free in solution due to its lifetime (4 ns) being much greater than its rotational correlation time of ∼100 ps. Because the affinity of the inhibitor is low for the zinc-free apoprotein, little change in anisotropy is seen in the presence of apo-CA. However, upon binding of metal to the apoprotein, the inhibitor affinity is greatly enhanced (KD ) 15 nM). When the inhibitor binds its lifetime hardly changes but its rotational motion is much reduced, resulting in enhanced anisotropy. While the method is sensitive, it does have the drawback that the inhibitor concentration must be carefully controlled for best results; in particular, if the apoprotein concentration is much less than the inhibitor concentration, the measured anisotropy at saturation of the protein will be less than predicted (see below). The dearth of prior reports on metal ion determination by fluorescence anisotropy may be attributed to the facts that few metal ions are luminescent themselves in solution and their complexes with classical metallofluorescent indicators exhibit sufficiently short rotational correlation times in comparison to the lifetime that any change in lifetime is likely to beget a negligibly small change in the measured anisotropy. Similarly, there are very few examples of metal ions inducing large conformational changes in a macromolecule, such that the macromolecule’s rotational motion might change to an extent measurable by fluorescence anisotropy.19 THEORY It is evident from eq 2 that a change in rotational motion, molecular size, or fluorescence lifetime may result in a change in fluorescence anisotropy. Moreover, these effects are largest when the lifetime is similar to the rotational correlation time. We had previously described a method for metal ion determination whereby binding of a particular metal ion to a fluorescent-labeled protein such as apocarbonic anhydrase results in a decrease in the label’s lifetime, usually by Forster energy transfer.15 Most of those data were obtained using fluorescent labels such as CY-3 or CY-5, having rather short lifetimes (15 ns)29 therefore exhibit only slight changes in anisotropy when their lifetimes are reduced by metal binding (eq 2). Thus, we sought fluorescence labels with longer lifetimes that would still exhibit proximity-dependent quenching, or a change in their motion, upon metal binding. We can predict the change in anisotropy expected to be observed upon metal ion binding due to partial quenching of the label using eqs 2 and 3 and assuming that the motion of the label is closely coupled to the overall motion of the protein. If the label lifetime and the protein rotational correlation time were both 15 ns (Chen and Kernohan measured a value of 28.9 ns for the rotational relaxation time F29), one would expect the apoprotein to exhibit an anisotropy of 0.2 (if measured at the maximum of the excitation polarization spectrum where r0 usually equals 0.4). Values of the anisotropy calculated using eqs 2 and 3 as a function of metal ion concentration for cases where binding of the metal reduces the lifetime of the label to varying extents are shown in Figure 1. Note that X-ray data show that, for the wild type at least, HCA II has very similar structures in the holo- and (29) Chen, R. F.; Kernohan, J. J. Biol. Chem. 1967, 242, 5813-5823.

Figure 1. Simulated anisotropy of a macromolecule-bound fluorophore partially quenched by a metal ion. A macromolecule having a rotational correlation time φc ) 15 ns with a tightly bound fluorescent label having a lifetime τ ) 15 ns reversibly binds a metal ion which quenches the label emission by 25 (+), 50 (O), 75 (4), or 90% (9). The fluorescence anisotropy is depicted as a function of the logarithm of the metal ion concentration, expressed as the ratio of the metal ion concentration to its KD.

apoforms30 and, thus, presumably similar rotational rates as well. Evidently, if the quenching is more efficient, the lifetime of the holoform is shorter and the maximum anisotropy is greater. For instance, in the case of 50% quenching the anisotropy increases from 0.2 for the metal-free apoform to 0.30 for the metal-bound holoform. However, the apparent KD of the apoprotein for the metal appears to shift to higher concentrations in the simulation when the quenching is more efficient; for instance, the Kd of the 50% quenched form (e.g., 50% quantum yield) has shifted ∼0.2 log unit, whereas the 90% quenched (10% quantum yield) has shifted ∼1.1 log units. This is because in this simulation the quantum yield decreases commensurately with the decline in lifetime, and thus the bound form does not contribute a significant fraction of the emission until it represents the vast majority of emitters. For instance, when the protein is half-saturated with metal ([M2+ ] ) Kd), which quenches the emission by 90%, only 11% of the fluorescence observed is contributed by the protein with bound metal. Thus, on this basis alone one could expect an apparent shift in the equilibrium constant; this effect may be corrected for by measuring the lifetimes or quantum yield. However, due to the accuracy with which anisotropy can be measured, even modest degrees of quenching provide satisfactory anisotropy changes. An important advantage of fluorescent labeling the protein covalently as compared with using a diffusible fluorescent inhibitor is that the affinity of the inhibitor for the holoprotein is no longer a matter for concern. For example, the fluorescent aryl sulfonamide ABD-M exhibits a substantial increase in anisotropy upon binding to holocarbonic anhydrase,37 but its affinity for the holoprotein is rather modest (KD ) 0.3 uM), whereas the apoenzyme’s affinity for Zn(II) is rather high (KD ) 10 pM). Consequently, at a zinc concentration of 1 nM, an equivalent total concentration of apoprotein would become saturated with zinc; (30) Hakansson, K.; Carlsson, M.; Svensson, L. A.; Liljas, A. J. Mol. Biol. 1992, 227, 1192-1204.

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Table 1 varianta

M2+b

F131C-ABD-T Co Cu H64C-ABD Zn

τ1c

f1d

τ2c

f2d

τ3c

16.87 14.09 8.07 10.18 9.73

0.66 0.31 0.56 0.40 0.30

5.82 3.20 2.85 2.44 1.64

0.22 0.49 0.31 0.42 0.36

1.08 0.89 0.61 0.42 0.36

f3d

〈τ〉e

χ2f

0.12 12.57 0.6 0.20 6.15 0.8 0.14 5.44 0.7 0.19 1.4 0.34 1.2

a Variant of labeled apocarbonic anhydrase measured. b Metal ion added. c τi, lifetime (in ns) of the ith component. d fi, fractional intensity of the ith component. e 〈τ〉, the average lifetime (in ns). f χ2, reduced χ2, the ordinary criterion for goodness of fit.

Figure 2. Human carbonic anhydrase II. Residues replaced with cysteine (F131C and H64C) and the active site Zn(II) (large ball) are highlighted.

however, only a tiny fraction of the protein would subsequently have ABD-M bound to it at a concentration more than 2 orders of magnitude below its binding constant. EXPERIMENTAL SECTION Variants of human carbonic anhydrase II (H64C, F131C) (Figure 2) were cloned, expressed, and isolated from Escherichia coli strain BL21(DE3)pACA as previously described.31,32 Variant H64C was labeled in pH 8 borate buffer for 4 h at room temperature using ABD-F (7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide; Molecular Probes Catalog No. D-6053, Molecular Probes, Eugene, OR); excess reagent was removed by repeated centrifugation in a Centriprep filtration device (Amicon) or passage over a Sephadex G-25 column. ABD-(T) (7-(5-maleimidyl)pentylaminobenz-2-oxa-1,3-diazole-4-sulfonamide) was customsynthesized by Molecular Probes and conjugated to apocarbonic anhydrase variant F131C as described above. In all cases, the average number of fluorophores conjugated per protein molecule was 1.0 or less, as determined by spectrophotometry using an extinction coefficient at 430 nm of 8000 M-1 cm-1 for the fluorescent labels and 49 000 M-1 cm-1 at 280 nm for the protein. Zn(II) was removed from some variants as previously described using dipicolinate at pH 6.2;33 in the case of H64C-ABD, dipicolinate was apparently less effective in catalyzing release of Zn(II). In this case, care was required to fully remove unreacted ABD-F, which may act as a sulfonamide ligand for the active site Zn, hindering its removal. Alternatively, the Zn may be replaced with the less tightly bound Ni(II) prior to derivatization with ABD-F and the Ni(II) removed by dialysis at low pH in the presence of EDTA. Buffers were rendered metal-free by passage over a Chelex-100 column (Bio-Rad). Steady-state spectra and anisotropies were measured with a Spectronics AB-2 fluorometer; lifetimes were measured on an ISS K2 phase fluorometer using the ultraviolet multiline output of a small-frame Ar ion laser (Spectra Physics 2065) and dimethylPOPOP as a reference compound or (31) Nair, S. K.; Calderone, T. L.; Christianson, D. W.; Fierke, C. A. J. Biol. Chem. 1991, 266, 17320-17325. (32) Kiefer, L. L.; Paterno, S. A.; Fierke, C. A. J. Am. Chem. Soc. 1995, 117, 6831-6837. (33) Hunt, J. B.; Rhee, M. J.; Storm, C. B. Anal. Biochem. 1977, 79, 614-617.

4720 Analytical Chemistry, Vol. 70, No. 22, November 15, 1998

the 442-nm line of a Kimmon HeCd laser with Rose Bengal in ethanol as a standard, both as previously described.34 Differential polarized-phase fluorometry was performed as described previously, using the HeCd or a frequency-doubled Spectra-Physics Tsunami picosecond laser at 390 nm.35 Zn(II) levels were buffered at the indicated levels by the use of NTA at pH 7.0 and 30 °C, and Cu(II) levels were similarly buffered at pH 7.0 and 25 C.36 We note that H64C, especially when fluorescent-labeled in the apoform, is significantly less stable than other variants we have used, and thus it is important not to expose it to extremes of temperature or concentrate it. RESULTS Since carbonic anhydrase has a ∼15 ns rotational correlation time (based on a rotational relaxation time F of 28.9 ns29 determined using a Perrin-Weber plot), inspection of the Perrin equation suggests that label lifetimes comparable to the relevant rotational correlation time should result in maximal changes in anisotropy. Thus, we sought a label with a lifetime in the 15-ns range which was likely to be partially quenched upon binding to the metal. In fact, a form of the sulfonamidobenzoxadiazole label we had used previously37 modified to react selectively with sulfhydryl residues was satisfactory, exhibiting a complex decay with an average lifetime of 12.8 ns (see Table 1) when coupled to position F131C on the protein (Figure 3). Differential polarizedphase fluorometry (a sensitive probe of rotational motion)35 of the apo-F131C-ABD(T) conjugate was performed in order to determine whether the ABD(T) label closely followed the rotational motion of the whole protein or exhibited significant segmental motion independent of the protein. The data (Figure 4) were well fit by a single rotational correlation time of 14.7 ns (χ2 ) 0.8), with no significant segmental motion. Addition of Co(II) to the apoprotein resulted in quenching of the ABD(T) fluorescence intensity to the extent of 70%, which saturated in the micromolar range (Figure 5). This behavior is consistent with binding of Co(II) to the active site of the enzyme, resulting in partial quenching; by comparison, collisional quenching would not be significant at Co(II) concentrations below micromolar according to the Stern-Volmer relation, nor would it result in saturating behavior.18 The lifetime of Co(II)-saturated apo-F131C-ABD(T) (34) Thompson, R. B.; Gratton, E. Anal. Chem. 1988, 60, 670-674. (35) Mantulin, W. W.; Weber, G. J. Chem. Phys. 1977, 66, 4092-4099. (36) Smith, R.; Martell, A. E. National Institute of Standards and Technology, U.S. Department of Commerce, Gaithersburg, MD, 1973. (37) Thompson, R. B.; Maliwal, B. P.; Fierke, C. A. Anal. Chem. 1998, 70, 17491754.

Figure 3. Frequency-dependent phase shifts (O, b, 4) and demodulations (0, 9, ]) for apocarbonic anhydrase variant F131CABD(T) in the absence of metal (O, 0), and in the presence of saturating amounts of Cu(II) (], 4) and Co(II) (b, 9). The best threecomponent fits to the data sets are indicated by the lines, with the derived values collected in Table 1.

Figure 4. Differential polarized-phase fluorometry of apoF131CABD(T) in the absence of metal: differential phase angles (9) and modulation ratios (O) are depicted as a function of frequency, with the lines indicating the best fit to these data.

was measured and well fit by three components; the decline in average lifetime (52%, Table 1) was somewhat less than the decline in intensity (70%; Figure 5), suggesting a modest level of static quenching is present. According to the simulated values in Figure 1, saturation with the analyte resulting in quenching of between 50 and 75% should result in an increase of anisotropy from 0.20 to between 0.27 and 0.31. In the case of apo-F131C-ABD(T), saturation with Co(II) results in an increase in anisotropy from 0.175 to 0.245, a comparable increase (Figure 5). While the lifetime and rotational correlation times do not exactly match those used in the simulation, nevertheless the agreement is remarkably close and suggests that even simple simulations such as those depicted in Figure 1 are useful in designing biosensor transducers. Having previously noted that F131C-ABD(T) exhibits Cudependent decreases in its fluorescence intensity like other carbonic anhydrase variants with fluorescent labels close to the active site, the corresponding fluorescence lifetimes and anisotropies were also measured as a function of free Cu(II) concentration;

Figure 5. Fluorescence intensity (b) and anisotropy (0) of ABD(T)-labeled apo-F131C carbonic anhydrase II as a function of free Co(II) concentration. Excitation was at 442 nm and emission at 550 nm, and the free Co(II) concentrations were maintained at the stated levels by buffering with millimolar concentrations of Bicine.

Figure 6. Fluorescence anisotropy (0) and intensity (b) of ABD(T)-labeled apo-F131C carbonic anhydrase II as a function of free Cu(II) concentration. The free copper concentration is maintained at the stated level by buffering with NTA and the pH at 7.0 by 10 mM MOPS. Excitation at 426 nm; emission at 550 nm.

the results are depicted in Figures 3 and 6, respectively. It is apparent that as Cu(II) concentration is increased, the fluorescence lifetime (Table 1) and intensity both decline and the anisotropy increases (Figure 6), grossly as predicted in Figure 1. In particular, the intensity declines ∼75% and the average lifetime declines 60%, and the anisotropy increases from 0.17 to 0.245. No change was observed when the apoprotein was treated with Zn(II) (results not shown), and the decline in lifetime is too large to explain as resulting from trivial collisional quenching, due to the very low concentrations of free Cu(II) present. The anisotropy changes in Figure 5 are quite usable, and the apparent KD of the apoprotein as determined by anisotropy (∼2 pM) is ∼0.5 log unit higher than that previously observed (0.4 pM) (K.M. C.A.F., unpublished results) in accord with the simulation in Figure 1. We note that care was taken to fully equilibrate the apoenzyme samples in the (very low) concentration of free copper ion present. Unlike Co(II) and Cu(II), Zn(II) is not a notably effective quencher, and thus we sought other means to transduce its binding in the active site. We suspected that the presence of zinc in the active site might perturb the emission of a solvent-sensitive probe if the label were sufficiently close to the active site. Thus Analytical Chemistry, Vol. 70, No. 22, November 15, 1998

4721

Figure 7. Intensity (b) and anisotropy (O) of ABD-F-labeled apoH64C carbonic anhydrase as a function of free Zn(II) concentration. Excitation at 390 nm, emission at 500 nm, free Zn(II) buffered with NTA, pH maintained at 7.0 with 15 mM MOPS, and temperature at 30 °C.

we labeled the H64C variant (Figure 2) with the moderately solvent-sensitive probe ABD-F, and the protein exhibited zincdependent changes in its intensity and anisotropy (Figure 7). While ABD is an aromatic sulfonamide which might in principle act as an inhibitor in a manner akin to other aryl sulfonamides such as dansylamide, acetazolamide, or azosulfamide, no fluorescence spectral evidence was seen of the deprotonation of the sulfonamide of covalently attached ABD, as is seen with other aryl sulfonamides.38 Indeed, modeling studies with carbonic anhydrase II crystal structures indicated that an ABD moiety immobilized at position H64C was incapable of interacting with the Zn(II) in the active site without substantial disruption of the protein structure.39 Nevertheless, the ABD-H64C exhibits changes in fluorescence intensity as a function of Zn(II) concentration, in this case approximately doubling in intensity as the Zn(II) concentration goes up (Figure 7). In this case, the change in apparent lifetime of the ABD-H64C upon binding of Zn(II) is modest (Table 1). The small difference in lifetime between apoand holoforms suggests that binding of Zn(II) to the active site potentiates some static quenching process, resulting in an increased quantum yield with little change in lifetime. The anisotropy increases significantly as well, increasing ∼30% going from free to bound (Figure 7). Such a change is reliably and accurately measurable, being ∼20-fold larger (0.060 vs 0.003) than the typical precision of the method. Ordinarily, a modest change in lifetime such as this should result in a negligible change in the anisotropy, which is manifestly not the case. The results in Figure 7 are best explained by a change in the segmental motion of the ABD fluorophore upon binding of the Zn(II). Inasmuch as histidine-64 in wild-type carbonic anhydrase undergoes substantial motion during the catalytic cycle of the wild-type protein,40,41 segmental motion in this part of the polypeptide is unsurprising. We measured the mobility of the ABD label on the protein in the (38) Einarsson, R.; Zeppezauer, M. Acta Chem. Scand. 1970, 24, 1098-1102. (39) Nair, S. K.; Elbaum, D.; Christianson, D. W. J. Biol. Chem. 1996, 271, 10031007. (40) Nair, S. K.; Christianson, D. W. J. Am. Chem. Soc. 1991, 113, 9455-9458. (41) Krebs, J. F.; Fierke, C. A.; Alexander, R. S.; Christianson, D. W. Biochemistry 1991, 30, 9153-9160.

4722 Analytical Chemistry, Vol. 70, No. 22, November 15, 1998

Figure 8. Differential polarized-phase fluorometry of ABD-F labeled apo-H64C carbonic anhydrase II in the presence and absence of Zn(II). Differential phase angles (0, 9) and modulation ratios (O, b) are depicted for the apo- (b, 9) and holo-proteins (O, 0) as a function of frequency, together with the best fits to the data (lines).

presence and absence of zinc by differential polarized phase fluorometry; the results are depicted in Figure 8. While the data are somewhat noisy, the difference in mobility of the probe in the presence and absence of zinc is unmistakable. The apoenzyme exhibited rotational correlation times of 15.4 ( 0.7 (0.234 ( 0.002 fractional contribution to the anisotropy), 0.4 (0.101 ( 0.002), and 0.005 ns (0.070) (χ2 ) 0.8), whereas with Zn(II) bound the data were best fit by correlation times of 15.9 ( 0.7 (fractional contribution to the anisotropy, 0.293 ( 0.001), 2.4 (0.075 ( 0.002), and 0.005 ns (0.056) (χ2 ) 0.5). Noteworthy are the 6-fold increase in the second correlation time and the decline in the contribution of the third correlation time component. Inasmuch as the motion of the probe is likely to be highly hindered and most apparent at higher frequencies than our instrument can achieve, it is clear that the mobility is substantially reduced upon Zn(II) binding to this variant, providing the observed response. DISCUSSION What is the significance of a reagentless system for determining metal ions by fluorescence anisotropy? While it is clear that fluorescence anisotropy-based determinations employing separate, diffusible small molecules have demonstrated a greater response,37 reagentless approaches offer significant advantages. First, because the fluorophore is tethered to the apoprotein, there is no uncertainty about the relative levels of apoprotein and fluorophore. This is potentially of interest in fluorescence anisotropy microscopy of biological specimens42,43 because the fluorophore (but not the apoprotein) may diffuse into or out of cellular compartments or organelles, complicating interpretation. Second, developing sensors using a reagentless approach is likely to be easier, since the fluorescent-labeled apoprotein might be covalently attached to the surface of an optical fiber or entrapped in a hydrogel, whereas the diffusible fluorophore can only be entrapped with the apoprotein within a semipermeable membrane, if one can be made. While we do not demonstrate it here, the work of Betts et al.44 demonstrates that a fiber-optic sensor based on fluorescence (42) Dix, J. A.; Verkman, A. S. Biophys. J. 1990, 57, 231-240. (43) Fushimi, K.; Dix, J. A.; Verkman, A. S. Biophys. J. 1990, 57, 241-254.

anisotropy is feasible. As mentioned above, in a reagentless system there is no difficulty imposed by differences in affinity of the apoprotein for the metal and the holoprotein for the fluorophore. This is important because while variants of CA have been constructed that bind Zn(II) with affinities varying over 11 orders of magnitude in concentration,45 it is unlikely that inhibitor affinities can be varied over as large a range.46 Thus, apart from second-order effects of the fluorophore label on the metal ion affinities, the affinity of the protein may be varied independently of the nature of the covalent label. By comparison, some variants with altered affinity45 and/or kinetics47 exhibit significantly reduced affinity for sulfonamide inhibitors, limiting the scope (at the current state of the art) of anisotropy-based determination using these diffusible fluorophores. It is important to note that the variants following fluorescent derivatization retain metal ion affinities comparable to the wildtype apoprotein. For instance, apo-F131C-ABD(T) exhibits affinities for Cu and Co of 0.3 pM and 100 nM, respectively, compared with wild-type affinities of 0.4 pM and 50 nM, respectively. Similarly, apo-H64C-ABD exhibits a KD for Zn(II) of 80 pM (Figure 7) vs 4 pM for the wild type. While some perturbation has occurred, clearly if the wild-type binding site is intact, the affinity and selectivity will be largely retained. However, it should be borne in mind that while apo-F131C-ABD(T) displays only modest changes in its fluorescence properties upon the binding of Zn or Cd, these ions nevertheless bind and thus can interfere with the determination of Cu or Co by this method, if they are sufficiently plentiful. This issue will be dealt with in a future paper. While it is not trivial to construct fluorescence anisotropy-based assays for metal ions, it nevertheless is a more flexible approach than those developed for fluorescence lifetime-based assays and sensors. In particular, one may devise a lifetime-based assay by exploiting any of several phenomena which alter the apparent fluorescence lifetime of a suitable label. These phenomena are mainly quenching mechanisms and include energy transfer,10,12 electron transfer, and quenching by paramagnetic ions and free radicals. In light of eq 2 and Figure 1, any of these phenomena may also be exploited for anisotropy-based sensing. In addition to lifetime changes, changes in the apparent rotational diffusion may also be exploited for anisotropy-based sensing, even though there may be little change in lifetime, as is the case for ABDH64C. One can conceive of many metal-dependent conformational changes (such as those induced in calmodulin by the binding of calcium ions19) which could be utilized for this purpose. The recent development of long-lived fluorescent labels for immunoassays based on anisotropy48 will expedite the development of anisotropy-based assays for other analytes and ligands, which (44) Betts, T. A.; Bright, F. V.; Catena, G. C.; Huang, J.; Litwiler, K. S.; Paterniti, D. P. In Laser Techniques in Luminescence Spectroscopy; Vo-Dinh, T., Eastwood, D., Eds.; ASTM STP-1006; American Society for Testing and Materials: Philadelphia, 1990; pp 88-95. (45) Ippolito, J. A.; Baird, T. T.; McGee, S. A.; Christianson, D. W.; Fierke, C. A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 5017-5021. (46) Nair, S. K.; Krebs, J. F.; Christianson, D. W.; Fierke, C. A. Biochemistry 1995, 34, 3981-3989. (47) Huang, C.-c.; Lesburg, C. A.; Kiefer, L. L.; Fierke, C. A.; Christianson, D. W. Biochemistry 1996, 35, 3439-3446. (48) Terpetschnig, E.; Szmacinski, H.; Lakowicz, J. R. Anal. Biochem. 1995, 227, 140-147.

create conformational changes or alter the aggregation state in macromolecules. Anisotropy-based metal ion determinations using carbonic anhydrase II offer unique advantages. In particular, the selectivity of metal ion binding by apocarbonic anhydrase II is greater than current fluorescent indicators for metal ions6,7 (K.M. and C.A.F., unpublished results) and as least as good as biological and biomimetic molecules designed for the purpose.49,50 Furthermore, the absolute affinity for ions such as Cu(II) and Zn(II), as well as the kinetics of their equilibration and their relative affinity (K.M. and C.A.F., in preparation), can all be modified by mutagenesis of the protein, either by a structure-based design process or a random, combinatorial one. Finally, the carbonic anhydrase macromolecule “scaffold” permits innocuous site-directed mutagenesis of the protein to optimize transducer response.51 The large polypeptide structure has sufficient reserve stability that its structure may usually be modified by site-directed mutagenesis without fatally compromising its stability; by comparison, mutagenesis of smaller macromolecules typically has a greater impact on their stability. Notwithstanding these advantages, it is not trivial to devise anisotropy-based assays. This is apparent upon consideration of eq 2. In particular, the lifetime of the fluorescent label (if its rotational motion closely matches that of the macromolecule) must be approximately the same as its rotational correlation time, as depicted in the simulation. If the lifetime is significantly shorter, the anisotropy is high and will only increase slightly upon quenching. Similarly, if the lifetime is severalfold longer than the correlation time, the initial anisotropy is very low and will not increase dramatically upon quenching. If, as in the example of ABD-labeled H64C or other fortuitous cases, metal binding results in a conformational change or an apparent increase in quantum yield, the anisotropy change can be much greater. At best, such behavior can be difficult to predict. While the fluorophore ABD when attached to position 64 does not appear to interact with the zinc in the holoenzyme, modification of the probe to permit greater flexibility might permit such interaction with the metal ion, leading to dramatic changes in all spectral parameters (R.B.T. et al., in preparation). Trivially, it may be difficult to select a fluorophore that is sensitive to the presence of the metal that also has the desired fluorescence properties, especially a suitably long lifetime. Nevertheless, the vast and growing palette of fluorophores and their potential influence by metal ions makes it likely that a suitable anisotropy response can be achieved in many cases. ACKNOWLEDGMENT The authors thank Dr. David Christianson for many fruitful discussions and the Office of Naval Research, the National Institutes of Health, and the National Science Foundation for support. Received for review August 4, 1998. Accepted August 25, 1998. AC980864R (49) Klemba, M.; Regan, L. Biochemistry 1995, 34, 10094-10100. (50) Walkup, G. K.; Imperiali, B. J. Am. Chem. Soc. 1997, 119, 3443-3450. (51) Thompson, R. B.; Ge, Z.; Patchan, M. W.; Fierke, C. A. J. Biomed. Opt. 1996, 1, 131-137.

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