Determining the Fluorescence Spectrum of a Protein

Roger H. Pain1

1 Jozef Stefan Institute, Ljubljana, null
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 7.7
DOI:  10.1002/0471140864.ps0707s38
Online Posting Date:  January, 2005
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Abstract

Fluorescence spectra of proteins are determined chiefly by the polarity of the environment of the tryptophan and tyrosine residues and by their specific interactions. A thorough consideration of fluorescence spectrometers and their calibration is provided along with important information regarding spectrometer cells, buffers and clarification of samples. Protocols are provided for recording fluorescence spectra and for measuring fluorescence quenching to probe the accessibility of tryptophan residues to small molecules (to yield information about the structural environment of the tryptophan). The technique involves quantifying the decrease in protein fluorescence intensity in the presence of increasing concentrations of quencher, followed by analysis of the data to give details of the interaction of the quencher with the tryptophan residue. Finally, a Support Protocol gives details on how to interpret fluorescence spectra.

     
 
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Table of Contents

  • Unit Introduction
  • Strategic Planning
  • Basic Protocol 1: Recording a Fluorescence Emission Spectrum
  • Basic Protocol 2: Determination of Fluorescence Quenching
  • Support Protocol: Basic Theory and Interpretation of Fluorescence Spectra
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Recording a Fluorescence Emission Spectrum

 Materials
  • Calibrated fluorescence spectrometer, buffer solution, and cleaned cuvettes (see Strategic Planning)
  • Clarified protein solution of known concentration (see Strategic Planning for clarification methods and unit 7.2 for concentration determination) with absorbance usually in the range of 0.05 to 0.1 at the wavelength to be used for excitation

Basic Protocol 2: Determination of Fluorescence Quenching

 Materials
  • Protein solution in buffer, A280 = 0.05 to 0.1
  • Quenchers: these are most conveniently made up as concentrated stock solutions; examples of frequently used quenchers include:
    • 5 M NaI or 2.5 M KI solution containing 1 mM Na2S2O3 to prevent formation of reactive I3–
    • 5 M CsCl (optical grade, Aldrich)
    • 8 M acrylamide (Electran grade from BDH; 295 = 0.236 liter/mol/cm)
    • 2.5 M succinimide (recrystallized from ethanol with activated charcoal treatment; 295 = 0.03 liter/mol/cm)
  • Additional reagents and equipment for recording a fluorescence spectrum (see Basic Protocol 1)
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Figures

  •  FigureFigure 7.7.1 Rayleigh and Raman bands in fluorescent spectra, as seen in scans for solvent baseline and hen egg white lysozyme (EWL) solutions (solid lines). Circles represent the spectrum of EWL with baseline subtracted. Parameters: EWL A280 = 0.05; ex = 280 nm; excitation and emission bandwidths, 2.5 nm; scan rate, 100 nm/min; five scans accumulated. Spectra were measured using a Perkin Elmer LS50B fluorescence spectrometer.
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  •  FigureFigure 7.7.2 Temperature dependence of fluorescence in a polar environment. (A) Tyrosine (6 µM) at 303 nm (ex = 274 nm). (B) Tryptophan (1 µM) at 355 nm (ex = 278 nm). Both chromophores are in a polar environment, 0.01 M potassium phosphate, pH 7.0. The dependence is much smaller in a nonpolar environment, such as the interior of a protein. Reprinted from Schmid (1991) with permission of Oxford University Press.
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  •  FigureFigure 7.7.3 Fluorescence spectrum of a “tyrosine only” protein, ribonuclease (RNase). Parameters: RNase A275 = 0.05; ex = 275 nm; one scan, smoothed; other settings as for Figure 7.7.1. Baseline was measured with the same settings and subtracted. Measurements made using a Perkin-Elmer LS50B.
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  •  FigureFigure 7.7.4 The effect of scanning parameters on the authenticity of spectra. The Raman band from Figure 7.7.1 is shown for a single scan at 1500 nm/min (dotted line); ten scans at 1500 nm/min (dashed line); and ten scans at 100 nm/min (solid line). The times required for scanning the complete baselines for these three measurements were 0.087 min, 0.87 min, and 13 min, respectively. Measurements made using a Perkin-Elmer LS50B.
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  •  FigureFigure 7.7.5 The inner filter effect. A cuvette (10 × 10–mm) is represented in plan view, with the collimated incident beam from the monochromator having intensity I0. As a result of absorption by the protein solution, the intensity of the beam through the cuvette will decrease steadily, emerging with intensity I. The values are illustrated for a solution having an absorbance at the excitation wavelength of 0.1. The optics of the fluorescence detector are focused so that only fluorescence originating from the volume depicted by the heavily shaded square is “seen” by the photomultiplier. Thus the observed normalized fluorescence intensity will be less than that expected from the protein at infinite dilution. The fluorescence passes through the protein solution on its way to the detector and will be further decreased in intensity if the solution absorbs at the wavelengths of the emitted radiation.
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  •  FigureFigure 7.7.6 Fluorescence quenching by different quenchers–analysis of data. (A) Fluorescence intensities from 3-phosphoglycerate kinase (PGK), expressed as a fraction of the unquenched fluorescence, are plotted according to the Stern-Volmer equation (see Basic Protocol 2, step ) at 316 nm (filled symbols) and 350 nm (open symbols). Quenchers used were succinimide (circles; treated with activated charcoal and recrystallized with ethanol; 2.5 M stock solution) and potassium iodide (squares; 2.5 M stock solution with 1 mM Na2S2O3). Fluorescence intensities were adjusted for dilution and for quencher absorbance using a molar extinction coefficient 295 = 0.03 liter/mol/cm for succinimide. The higher level of quenching at 350 nm reflects the fact that the more exposed of the two tryptophan residues has a more red-shifted fluorescence maximum. (B) Modified Stern-Volmer plot for the quenching of PGK fluorescence by succinimide. The data in panel A for quenching at 350 nm is plotted according to the modified Stern-Volmer equation (see Basic Protocol 2, step annotation). The intercept on the y-axis is 2.36 ± 0.2. The results in panels A and B are characteristic of a protein where part of the fluorescence is emitted by residue(s) that are inaccessible to a given quencher, in this case, iodide and succinimide. The tryptophan(s) are less accessible to iodide than to succinimide, with implications for the polarity of their environment. Measurements made using a Perkin-Elmer MPF3-L. Reprinted from Varley et al. (1983) with permission of Elsevier Science.
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  •  FigureFigure 7.7.7 The processes of absorption and emission of radiation by a chromophore. The electronic structure of a chromophore is in the ground state (S0) under normal conditions. Absorption of radiation, a rapid process, leads to excitation to the vibrational states of an excited state (S1). Under normal thermal conditions, the excitation energy is rapidly degraded to the lower one or two vibrational levels. Thus, although excitation takes place into a range of levels in the excited electronic state, emission, in the form of fluorescence, takes place from the lowest vibrational level to a range of vibrational levels in the ground electronic state. The lengths of the vertical lines representing absorption and emission illustrate the relative energies of the radiation in each process and indicate why fluorescence almost always occurs at longer wavelengths than absorption. The vertical broken line represents the dissipation of absorbed energy by means other than radiation.
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  •  FigureFigure 7.7.8 Quenching of the tyrosine fluorescence of -lactoglobulin. Spectra were recorded in the absence (a, c) and presence (b, d) of 9.48 M urea, and excited at 275 nm (solid lines, tyrosine and tryptophan excited) and 297 nm (broken lines, tyrosine not excited). Parameters: protein concentration 16.5 µM in 0.025 M phosphate, 0.068 M NaCl, pH 6.2; ex = 8 nm and em = 9 nm;. Perkin-Elmer MPF 2A spectrofluorometer. Reprinted from Creamer (1995) with permission from the American Chemical Society.
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  •  FigureFigure 7.7.9 Quenching of fluorescence of tumor necrosis factor (TNF) on denaturation. Fluorescence emission spectra are shown for native TNF (solid line), guanidine-unfolded TNF (dotted line), and acid-denatured TNF (dashed line). Parameters: TNF concentration, 30 µg/ml; ex = 280 nm; bandwidths ranging from 16 to 24 nm. Measurements made using Perkin-Elmer MPF3 spectrofluorimeter. Reprinted from Hlodan and Pain (1994) with permission of Elsevier Science.
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  •  FigureFigure 7.7.10 Assignment of fluorescence to tryptophan W333 in 3-phosphoglycerate kinase (PGK). In the presence of 1.12 M succinimide (shown to quench the exposed tryptophan fluorescence; see Fig. 7.7.6), fluorescence is further quenched by acrylamide, which is able to gain access to the buried tryptophan. Fluorescence was measured at 316 nm (filled circles) and at 350 nm (open circles) and plotted according to the Stern-Volmer equation (see Basic Protocol 2, step ). Fluorescence intensities were corrected for dilution and for quencher absorbance using 295 = 0.236 for acrylamide. The linear plot indicates the quenching of a single residue. Measurements made using a Perkin-Elmer MPF3-L. Reprinted from Varley et al. (1991) with permission of Elsevier Science.
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  •  FigureFigure 7.7.11 The binding of retinol to -lactoglobulin that has been denatured by exposure to high pressure. The sample containing 270 µM -lactoglobulin was pressurized to 400 MPa for 15 min. After release of pressure, retinol in ethanol was added and fluorescence measured. Parameters: final protein concentration 8.5 µM in 20 mM phosphate buffer; ex = 330 nm; em = 470 nm. Circles, native -lactoglobulin; triangles, pressurized -lactoglobulin. Reprinted from Ikeuchi et al. (2001) with permission from the American Chemical Society.
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  •  FigureFigure 7.7.12 Depolarization of fluorescence indicates rotation of the chromophore. Monochromatic radiation from the source (S) has all but the vertically polarized electric vector removed by the polarizer (P). This is absorbed only by those molecules in which the transition dipole of the chromophore is aligned vertically. In the case where these molecules do not rotate appreciably before they fluoresce (“no rotation”; indicated by shading), their emitted radiation will be polarized parallel to the incident radiation. The intensity of radiation falling on the detector (D) will be zero when the analyzer (A) is oriented perpendicular to the polarizer. In the case where the molecules rotate significantly before fluorescence takes place, some of the excited chromophores will emit radiation with a horizontal polarization (“rotation”) and some with a vertical polarization. Finite intensities will be measured with both parallel and perpendicular orientations of the analyzer. The fluorescence from the remainder of the excited molecules will not be detected. The heavy arrows on the left of the diagram illustrate the case where there is rotation.
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Literature Cited

Literature Cited
    Agashe, V.R., Shashtry, M.C.R., and Udgaonkar, J.B. 1995. Initial hydrophobic collapse in the folding of barstar. Nature 377:754-757.
    Campbell, I.D. and Dwek, R.A. 1984. Biological Spectroscopy. Benjamin/Cummings, Menlo Park, Calif.
    Cardamone, M. and Puri, K. 1992. Spectrofluorimetric assessment of the surface hydrophobicity of proteins. Biochem. J. 282:589-593.
    Christensen, H. and Pain, R.H. 1994. "The contribution of the molten globule model". In Mechanisms of Protein Folding (R.H. Pain, ed.) pp. 55-79. Oxford University Press, Oxford.
    Creamer, L.K. 1995. Effect of sodium dodecyl sulfate and palmitic acid on the equilibrium unfolding of bovine -lactoglobulin. Biochemistry 34:7170-7176.
    Eftink, M.R. and Ghiron, C. 1981. Fluorescence quenching studies with proteins. Anal. Biochem. 114:199-227.
    Freifelder, D. 1982. Physical Biochemistry: Applications to Biochemistry and Molecular Biology. Freeman, New York.
    Gruebele, M. 1999. The fast protein folding problem. Annu. Rev. Phys. Chem. 50:485-516.
    Hlodan, R. and Pain, R.H. 1994. Tumour necrosis factor is in equilibrium with a trimeric molten globule at low pH. FEBS Lett. 343:256-260.
    Ikeuchi, Y., Nakagawa, K., Endo, T., Suzuki, A., Hayashi, T., and Ito, T. 2001. Pressure-induced denaturation of monomer -lactoglobulin is partially irreversible: Comparison of monomer form (highly acidic pH) with dimer form (neutral pH). J. Agric. Food Chem. 49:4052-4059.
    Kuwajima, K. and Arai, M. 2000. The molten globule state: The physical picture and biological significance. In Mechanisms of Protein Folding (R.H. Pain, ed.) pp. 138-174. Oxford University Press, Oxford.
    Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy. Plenum Press, New York.
    Losso, J.N., Kummer, A., Li-Chan, E., and Nakai, S. 1993. Development of a particle concentration fluorescence immunoassay for the quantitative determination of IgG in bovine milk. J. Agric. Food Chem. 41:682-686.
    Schmid, F.X. 1989. Spectral methods of characterizing protein conformation and conformational changes. In Protein Structure (T.E. Creighton, ed.) pp. 251-285. IRL Press, Oxford.
    Varley, P.G., Dryden, D.T., and Pain, R.H. 1991. Resolution of the fluorescence of the buried tryptophan in yeast 3-phosphoglycerate kinase using succinimide. Biochim. Biophys. Acta 1077:19-24.
 Key Reference
    Lakowicz, J.R. 1983. See above.

An essential text, providing a thorough, but clear and readable description of the practice and interpretation of fluorescence spectroscopy, with particular reference to proteins.

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