Fluorescence-based biosensing of zinc using carbonic anhydrase

  • Carol A. Fierke
  • Richard B. Thompson


Measurement of free zinc levels and imaging of zinc fluxes remains technically difficult due to low levels and the presence of interfering cations such as Mg and Ca. We have developed a series of fluorescent zinc indicators based on the superb sensitivity and selectivity of a protein, human apo-carbonic anhydrase II, for Zn(II). These indicators transduce the level of free zinc as changes in intensity, wavelength ratio, lifetime, and/or anisotropy; the latter three approaches permit quantitative imaging of zinc levels in the microscope. A unique attribute of sensors incorporating biological macromolecules as transducers is their capability for modification by site-directed mutagenesis. Thus we have produced variants of carbonic anhydrase with improved affinity for zinc, altered selectivity, and enhanced binding kinetics, all of which are difficult to modify in small molecule indicators.

Key words

biosensor carbonic anhydrase fluorescence hippocampus wavelength ratiometric zinc 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Alberts IL, Nadassy K. 1998 Analysis of zinc binding sites in protein crystal structures. Protein Sci 7, 1700–1716.PubMedCrossRefGoogle Scholar
  2. Belli SL, Zirino A. 1993 Behavior and calibration of the copper(II) ion-selective electrode in high chloride media and marine waters. Anal Chem 65, 2583–2589.CrossRefGoogle Scholar
  3. Benters A, Flogel U, Schafer T, Leibfritz D, Hechtenberg S, Beyers-mann D. 1997 Study of the interactions of cadmium and zinc ions with cellular calcium homeostasis using 19F-NMR spectroscopy. Biochem J 322, 793–799.PubMedGoogle Scholar
  4. Chen RF, Kernohan J. 1967 Combination of bovine carbonic anhydrase with a fluorescent sulfonamide. J Biol Chem 242, 5813–5823.PubMedGoogle Scholar
  5. Christianson DW. 1991 Structural biology of zinc. Adv Prot Chem 42, 281–355.CrossRefGoogle Scholar
  6. Christianson DW, Alexander RS. 1989 Carboxylate histidine zinc interactions in protein — structure and function. J Am Chem Soc 111, 6412–6419.CrossRefGoogle Scholar
  7. Christianson DW, Fierke CA. 1996 Carbonic anhydrase — Evolution of the zinc binding site by nature and by design. Acc Chem Res 29, 331–339.CrossRefGoogle Scholar
  8. Clark HA, Hoyer M, Philbert MA, Kopelman R. 1999 Optical nanosensors for chemical analysis inside single living cells. I. Fabrication, characterization, and methods for intracellular delivery of PEBBLE sensors. Anal Chem 71, 4831–4836.PubMedCrossRefGoogle Scholar
  9. Cox JD, Hunt JA, Compher KM, Fierke CA, Christianson DW. 2000 Structural influence of hydrophobic core residues on metal binding and specificity in carbonic anhydrase II. Biochemistry 39, 13687–13694.PubMedCrossRefGoogle Scholar
  10. Denny MF, Atchison WD. 1994 Methylmercury-induced elevations in intrasynaptosomal zinc concentrations: an 19F-NMR study. J Neurochem 63, 383–386.PubMedCrossRefGoogle Scholar
  11. DiTusa CA, McCall KA, Christensen T, Mahapatro M, Fierke CA, Toone EJ. 2001 Thermodynamics of metal ion binding. Il. Metal ion binding by carbonic anhydrase variants. Biochemistry, 40, 5345–5351.PubMedCrossRefGoogle Scholar
  12. Dix JA, Verkman AS. 1990 Mapping of fluorescence anisotropy in living cells by ratio imaging: Application to cytoplasmic viscosity. Biophvs J 57, 231–240.CrossRefGoogle Scholar
  13. Eigen M, Hammes GG. 1963 Elementary steps in enzyme reactions as studied by relaxation spectrometry. Adv Enzymol Relat Areas Mol Biol 25, 1–38.PubMedGoogle Scholar
  14. Elbaum D, Nair SK, Patchan MW, Thompson RB, Christianson DW. 1996 Structure-based design of a sulfonamide probe for fluorescence anisotropy detection of zinc with a carbonic anhydrase-based biosensor. J Am Chem Soc 118, 8381–8387.CrossRefGoogle Scholar
  15. Eriksson AE, Jones TA. 1988 Refined structure of human carbonic anhydrase II at 2.0 A resolution. Proteins 4, 274–282.PubMedCrossRefGoogle Scholar
  16. Fernandez-Gutierrez A, Munoz de la Pena A. 1985 Determinations of inorganic substances by luminescence methods. In: Schulman S.G. ed. Molecular Luminescence Spectroscopy, Part l: Methods and Applications; New York: Wiley-Interscience, 371–546.Google Scholar
  17. Frederickson CJ, Suh SW, Silva D, Frederickson CJ, Thompson RB. 2000 Importance of zinc in the central nervous system: the zinc-containing neuron. J Nutr (Suppl.) 130, 1471S - 1483S.Google Scholar
  18. Fushimi K, Dix JA, Verkman AS. 1990 Cell membrane fluidity in the intact kidney proximal tubule measured by orientation-independent fluorescence anisotropy imaging. Biophvs J 57, 241–254.CrossRefGoogle Scholar
  19. Glusker JP. 1991 Structural aspects of metal liganding. Adv Prot Chem 42, 1–76.CrossRefGoogle Scholar
  20. Grynkiewicz G, Poenie M, Tsien RY. 1985 A new generation of calcium indicators with greatly improved fluorescence properties. J Biol Chem 260, 3440–3450.PubMedGoogle Scholar
  21. Hakansson K, Carlsson M, Svensson LA, Liljas A. 1992 Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes. J Mol Biol 227, 1192–1204.PubMedCrossRefGoogle Scholar
  22. Hakansson K, Wehnert A, Liljas A. 1994 X-ray analysis of metal-substituted human carbonic anhydrase II derivatives. Acta Crys D50, 93–100.Google Scholar
  23. Haugland RP. 1996 Handbook of Fluorescent Probes and Research Chemicals. Oregon: Molecular Probes, Inc., Eugene.Google Scholar
  24. Henkens RW, Sturtevant JM. 1968 The kinetics of the binding of Zn(II) by apocarbonic anhydrase. J Am Chem Soc 90, 2669–2676.CrossRefGoogle Scholar
  25. Hirano T, Kikuchi K, Urano Y, Higuchi T, Nagano T. 2000 Highly zinc-selective fluorescent sensor molecules suitable for biological applications. J Am Chem Soc 122, 12399–12400.CrossRefGoogle Scholar
  26. Huang C-C, Lesburg CA, Kiefer LL, Fierke CA, Christianson DW. 1996 Reversal of the hydrogen bond to zinc ligand histidine-119 dramatically diminishes catalysis and enhances metal equilibration kinetics in carbonic anhydrase II. Biochemistry 35, 3439–3446.PubMedCrossRefGoogle Scholar
  27. Hunt JA, Ahmed M, Fierke CA. 1999 Metal binding specificity in carbonic anhydrase is influenced by conserved hydrophobic amino acids. Biochemistry 38, 9054–9060.PubMedCrossRefGoogle Scholar
  28. Hunt JA, Fierke CA. 1997 Selection of carbonic anhydrase variants displayed on phage: aromatic residues in zinc binding site enhance metal affinity and equilibration kinetics. J Biol Chem 272, 20364–20372.PubMedCrossRefGoogle Scholar
  29. Ippolito JA, Baird TT, McGee SA, Christianson DW, Fierke CA. 1995a Structure-assisted redesign of a protein-zinc binding site with femtomolar affinity. Proc Natl Acad Sci USA 92, 50175021.Google Scholar
  30. Ippolito JA, Christianson DW. 1993 Structure of a His3Cys zinc binding site in human carbonic anhydrase II. Biochemistry 32, 9901–9905.PubMedCrossRefGoogle Scholar
  31. Ippolito JA, Christianson DW. 1994 Structural consequences of redesigning a protein-zinc binding site. Biochemistry 33, 1524115249.Google Scholar
  32. Ippolito JA, Nair SK, Fierke CA, Christianson DW. 1995b Structure of His94Asp carbonic anhydrase II in a new crystalline form reveals a partially occupied zinc binding site. Prot Engin 8, 975–980.CrossRefGoogle Scholar
  33. Iverson TM, Alber BE, Kisker C, Ferry JG, Rees DC. 2000 A closer look at gamma-class carbonic anhydrases: high resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila. Biochemistry 39, 9222–9231.CrossRefGoogle Scholar
  34. Jensen KK, Martini L, Schwartz TW. 2001 Enhanced fluorescence resonance energy transfer between spectral variants of green fluorescent protein through zinc-site engineering. Biochemistry 40, 938–945.PubMedCrossRefGoogle Scholar
  35. Kiefer LL, Fierke CA. 1994 Functional characterization of human carbonic anhydrase II variants with altered zinc binding sites. Biochemistry 33, 15233–15240.PubMedCrossRefGoogle Scholar
  36. Kiefer LL, Ippolito JA, Fierke CA, Christianson DW. 1993a Redesigning the zinc binding site of human carbonic anhydrase II: Structure of a His2Asp-Zn2+ metal coordination polyhedron. J Am Chem Soc 115, 12581–12582.CrossRefGoogle Scholar
  37. Kiefer LL, Krebs JF, Fierke CA. 19936 Engineering a cysteine residue into the zinc binding site of carbonic anhydrase II. Biochemistry 32, 9896–9900.Google Scholar
  38. Kiefer LL, Paterno SA, Fierke CA. 1995 Hydrogen bond network in the metal binding site of carbonic anhydrase enhances zinc affinity and catalytic efficiency. JAm Chem Soc 117, 6831–6837.CrossRefGoogle Scholar
  39. Kimber MS, Pai EF. 2000 The active site architecture of Pisum sativum beta-carbonic anhydrase is a mirror image of that of alpha-carbonic anhydrases. EMBO J 19, 1407–1418.PubMedCrossRefGoogle Scholar
  40. Kuhn MA, Hoyland B, Carter S, Zhang C, Haugland RE. 1995 Fluorescent ion indicators for detecting heavy metals. SPIE Conference on Adv Fluor Sens Tech ll (San Jose, California), Vol. 2388, 238–244.Google Scholar
  41. Lesburg CA, Christianson DW. 1995 X-ray crystallographic studies of engineered hydrogen bond networks in a protein-zinc binding site. J Am Chem Soc 117, 6838–6844.CrossRefGoogle Scholar
  42. Lesburg CA, Huang C-C, Christianson DW, Fierke CA. 1997 Histidine to carboxamide ligand substitutions in the zinc binding site of carbonic anhydrase II alter metal coordination geometry but retain catalytic activity. Biochemistry 36, 15780–15791.PubMedCrossRefGoogle Scholar
  43. Levy R, Guignon EF,Cobane S, St. Louis E, Fernandez S. 1997 Compact, rugged, and inexpensive frequency domain fluorometer. SPIE Conference on Advances in Fluorescence Sensing Technology III,San Jose, CA vol. 2980, 81–89.Google Scholar
  44. Lindskog S, Henderson LE, Kannan KK, Liljas A, Nyman PO, Strandberg B. 1971 Carbonic anhydrase. In: Boyer PD, ed. The Enzymes. New York: Academic Press: 587–665.Google Scholar
  45. Lindskog S, Nyman PO. 1964 Metal-binding properties of human erythrocyte carbonic anhydrases. Biochim Biophys Acta 85, 462474.Google Scholar
  46. Lippitsch ME, Pusterhofer J, Leiner MJP, Wollbeis OS. 1988 Fiber-optic oxygen sensor with the fluorescence decay time as the information carrier. Anal Chim Acta 205, 1–6.CrossRefGoogle Scholar
  47. Maren TH. 1977 Use of inhibitors in physiological studies of carbonic anhydrase. Am J Physiol 232, F291 - F297.PubMedGoogle Scholar
  48. McCall KA, Fierke CA. 2000 Colorimetric and fluorimetric assays to quantitate micromolar concentrations of transition metals. Anal Biochemistry 284, 307–315.CrossRefGoogle Scholar
  49. Mitsuhashi S, Mizushima T, Yamashita E, Yamamoto M, Kumasaka T, Moriyama H, Ueki T, Miyachi S, Tsukihara T. 2000 X-ray structure of beta carbonic anhydrase from the red alga, Porphyridium purpureum, reveals a novel catalytic site for CO2 hydration. J Biol Chem 275, 5521–5526.PubMedCrossRefGoogle Scholar
  50. Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY. 1997 Fluorescent indicators for Cat+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887.PubMedCrossRefGoogle Scholar
  51. Pearce LL, Gandley RE, Han W, Wasserloos K, Stitt M, Kanai AJ, McLaughlin MK, Pitt BR, Levitan ES. 2000 Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein. Proc Natl Acad Sci USA 97, 477–482.PubMedCrossRefGoogle Scholar
  52. Pearson RG. 1966 Acids and bases. Science 151, 172–177.PubMedCrossRefGoogle Scholar
  53. Rae TD, Schmidt PJ, Pufahl RA, Culotta VC, O’Halloran TV. 1999 Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase. Science 284, 805–808.PubMedCrossRefGoogle Scholar
  54. Roe RR, Pang YP. 1999 Zinc’s exclusive tetrahedral coordination governed by its electronic structure. J Mol Model 5, 134–140.CrossRefGoogle Scholar
  55. Rulisek L, Vondrasek J. 1998 Coordination geometries of selected transition metal ions (Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+) in metalloproteins. J lnorg Biochem 71, 115–127.CrossRefGoogle Scholar
  56. Simons TJB. 1993 Measurement of free zinc ion concentration with the fluorescent probe mag-fura-2 (furaptra). J Biochem Biophys Meth 27, 25–37.PubMedCrossRefGoogle Scholar
  57. Szmacinski H, Lakowicz JR. 1993 Optical measurements of pH using fluorescence lifetimes and phase-modulation fluorometry. Anal Chem 65, 1668–1674.PubMedCrossRefGoogle Scholar
  58. Thompson RB. 1991 Fluorescence-based fiber optic sensors. In: Lakowicz JR, ed. Topics in Fluorescence Spectroscopy. Vol. 2: Principles. New York: Plenum Press: 345–365.Google Scholar
  59. Thompson RB. 1993 Fiber optic ion sensors based on phase fluorescence lifetime measurements. SPIE Conference on Advances in Fluorescence Sensing Technology, Los Angeles, CA, vol. 1885, 290–299.Google Scholar
  60. Thompson RB, Frisoli JK, Lakowicz JR. 1992 Phase fluorometry using a continuously modulated laser diode. Anal Chem 64, 2075–2078.CrossRefGoogle Scholar
  61. Thompson RB, Ge Z, Patchan MW, Fierke CA. 1996a Performance enhancement of fluorescence energy transfer-based biosensors by site-directed mutagenesis of the transducer. J Biomed Optics 1, 131–137.CrossRefGoogle Scholar
  62. Thompson RB, Ge Z, Patchan MW, Huang C-C, Fierke CA. 1996b Fiber optic biosensor for Co(II) and Cu(II) based on fluorescence energy transfer with an enzyme transducer. Biosensors Bioelectron 11, 557–564.CrossRefGoogle Scholar
  63. Thompson RB, Jones ER. 1993 Enzyme-based fiber optic zinc biosensor. Anal Chem 65, 730–734.CrossRefGoogle Scholar
  64. Thompson RB, Whetsell WO Jr., Maliwal BP, Fierke CA, Frederickson CJ. 2000a Fluorescence microscopy of stimulated Zn(II) release from organotypic cultures of mammalian hippocampus using a carbonic anhydrase-based biosensor system. J Neurosci Meth 96, 35–45.CrossRefGoogle Scholar
  65. Thompson RB, Maliwal BP, Feliccia VL, Fierke CA, McCall K. I 998a Determination of picomolar concentrations of metal ions using fluorescence anisotropy: biosensing with a `reagentless’ enzyme transducer. Anal Chem 70, 4717–4723.Google Scholar
  66. Thompson RB, Maliwal BP, Fierke CA. 1998b Expanded dynamic range of free zinc ion determination by fluorescence anisotropy. Anal Chem 70, 1749–1754.PubMedCrossRefGoogle Scholar
  67. Thompson RB, Maliwal BP, Fierke CA. 1999 Selectivity and sensitivity of fluorescence lifetime-based metal ion biosensing using a carbonic anhydrase transducer. Anal Biochem 267, 185–195.PubMedCrossRefGoogle Scholar
  68. Thompson RB, Maliwal BP, Zeng HH. 2000b Zinc biosensing with multiphoton excitation using carbonic anhydrase and improved fluorophores. J Biomed Optics 5, 17–22.CrossRefGoogle Scholar
  69. Thompson RB, Patchan MW. 1995a Fluorescence lifetime-based biosensing of zinc: origin of the broad dynamic range. J Fluoresc 5, 123–130.CrossRefGoogle Scholar
  70. Thompson RB, Patchan MW. 1995b Lifetime-based fluorescence energy transfer biosensing of zinc. Anal Biochem 227, 123–128PubMedCrossRefGoogle Scholar
  71. Thompson RB, Walt DR. 1994 Emerging strategies for molecular biosensors. Naval Res Rev 46, 19–29.Google Scholar
  72. Thompson RB, Zeng HH, Loetz M, Fierke C. 2000 Issues in enzyme-based metal ion biosensing in complex media. In-vitro Diagnostic Instrumentation (San Jose, CA), vol. 3913, 120–127CrossRefGoogle Scholar
  73. Weber G. 1956 Photoelectric method for the measurement of polar-ization of fluorescence of solutions. J Opt Soc Am 46, 962CrossRefGoogle Scholar
  74. White CE, Argauer RJ. 1970 Fluorescence Analysis: A Practical Approach. New York: Marcel Dekker, Inc.Google Scholar
  75. Yamashita MM, Wesson L. 1990 Where metal ions bind in proteins. Proc Natl Acad Sci USA 87, 5648–5652.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2001

Authors and Affiliations

  • Carol A. Fierke
    • 1
  • Richard B. Thompson
    • 2
  1. 1.Departments of Chemistry and BiochemistryUniversity of MichiganAnn ArborUSA
  2. 2.Department of Biochemistry and Molecular BiologyUniversity of Maryland School of MedicineBaltimoreUSA

Personalised recommendations