Principles of Fluorescence Spectroscopy

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382 FLUORESCENCE ANISOTROPY B. Now assume that the quantum yield of DNS increases twofold upon binding to BSA. What is K d ? C. How would you determine whether the quantum yield of DNS changes upon binding to BSA? D. Predict the time-resolved anisotropies for each solution at 20EC. Assume P 0 = 0.3913 for DNS and that Table 10.6. Polarization Values of DNS [BSA] [DNS] P the rotational correlation times of DNS and BSA are well approximated by that predicted for an anhydrous sphere. The molecular weight of BSA is near 66,000 g/mol. The viscosity of water at 25EC is 0.894 cP (g cm –1 sec –1 ). The density of BSA can be taken as 1.2 g/cm 3 . 0 1 x 10 –7 M 0.0149 2 x 10 –5 M 1 x 10 –7 M 0.2727 >>Kd 1 x 10 –7 M 0.3913
11 In the preceding chapter we described the measurement and interpretation of steady-state fluorescence anisotropies. These values are measured using continuous illumination, and represent an average of the anisotropy decay by the intensity decay. The measurement of steady-state anisotropies is simple. However, interpretation of the steady-state anisotropies usually depends on an assumed form for the anisotropy decay, which is not observed in the experiment. Additional information is available from measurements of the time-dependent anisotropy, that is, the values of r(t) following pulsed excitation. The form of the anisotropy decay depends on the size, shape, and flexibility of the labeled molecule. The measured decays can be compared with the decays calculated from various molecular models. Anisotropies decays can be measured using the time-domain (TD) or the frequency-domain (FD) method. It is important to understand the factors which affect the anisotropy decays. For a spherical molecule the anisotropy is expected to decay with a single rotational correlation time (θ). Perhaps the most frequent interpretation of the correlation time is in terms of the overall rotational correlation time of a protein. The measured values of θ can be compared with the values predicted for a hydrated sphere of equivalent molecular weight (eq. 10.46). However, anisotropy decays are usually multi-exponential, which can be the result of numerous factors. Multi-exponential anisotropy decays are expected for non-spherical fluorophores or proteins. The correlation times in the anisotropy decay are determined by the rates of rotation about the various molecular axes. By examination of the correlation time it is sometimes possible to estimate the shapes of proteins. In addition to shape, anisotropy decays are affected by the segmental flexibility of the macromolecule. For instance, tryptophan anisotropy decays of proteins frequently display correlation times that are too short to be due to rotational diffusion of the whole protein. These compo- Time-Dependent Anisotropy Decays nents are usually due to independent motions of the tryptophan residue within the protein or on the surface of the protein. The rates and amplitudes of tryptophan side-chain motions have been used to study the nanosecond dynamics of proteins. Anisotropy decays can also be affected by resonance energy transfer between the same type of fluorophore, that is, depolarization due to homotransfer. Anisotropy decays of membrane-bound probes have been particularly informative. Membrane-bound probes often display unusual behavior in which the anisotropies do not decay to zero. This occurs because some probes do not rotate freely in membranes, at least not within the ns decay times of most fluorophores. The extent of rotation is often limited by the anisotropic environment of a membrane. The nonzero anisotropies at long times can be interpreted in terms of the order parameters of the membrane. In this chapter we present examples of simple and complex anisotropy decays to illustrate the information available from these measurements. In the following chapter we describe more advanced concepts in anisotropy decay analysis. 11.1. TIME-DOMAIN AND FREQUENCY-DOMAIN ANISOTROPY DECAYS Suppose a fluorophore is excited with a pulse of vertically polarized light, and that it rotates with a single correlation time. The anisotropy decay is determined by measuring the decays of the vertically (||) and horizontally (⊥) polarized emission. If the absorption and emission transition moments are colinear, the time-zero anisotropy is 0.4. In this case the initial intensity of the parallel component is threefold larger than that of the perpendicular component (Figure 11.1, left). If the fundamental anisotropy is greater than zero (r 0 > 0), vertically polarized excitation pulse results in an initial population of fluorophores which is enriched in the parallel orientation. 1 The decay of the differ- 383
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Principles of Fluorescence Spectros
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Joseph R. Lakowicz Center for Fluor
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Preface The first edition of Princi
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x GLOSSARY OF ACRONYMS DNS dansyl o
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xii GLOSSARY OF ACRONYMS TRES time-
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xiv GLOSSARY OF MATHEMATICAL TERMS
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xvi CONTENTS 2.9.4. Conversion betw
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xviii CONTENTS 6. Solvent and Envir
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xx CONTENTS 10.4.4. Alignment of Po
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xxii CONTENTS 14.7.1. Simulations o
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xxiv CONTENTS 19.12. New Approaches
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xxvi CONTENTS 25.3. Optical Propert
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2 INTRODUCTION TO FLUORESCENCE Figu
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6 INTRODUCTION TO FLUORESCENCE inst
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10 INTRODUCTION TO FLUORESCENCE Fig
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12 INTRODUCTION TO FLUORESCENCE ns,
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14 INTRODUCTION TO FLUORESCENCE 1.7
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24 INTRODUCTION TO FLUORESCENCE due
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28 INSTRUMENTATION FOR FLUORESCENCE
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3 Fluorescence probes represent the
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PRINCIPLES OF FLUORESCENCE SPECTROS
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98 TIME-DOMAIN LIFETIME MEASUREMENT
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100 TIME-DOMAIN LIFETIME MEASUREMEN
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5 In the preceding chapter we descr
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6 Solvent polarity and the local en
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238 DYNAMICS OF SOLVENT AND SPECTRA
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278 QUENCHING OF FLUORESCENCE 8.1.
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280 QUENCHING OF FLUORESCENCE Figur
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282 QUENCHING OF FLUORESCENCE ly ev
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290 QUENCHING OF FLUORESCENCE relat
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298 QUENCHING OF FLUORESCENCE σ ar
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320 QUENCHING OF FLUORESCENCE 47. R
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322 QUENCHING OF FLUORESCENCE 113.
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328 QUENCHING OF FLUORESCENCE P8.3.
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Page 351 and 352: 332 MECHANISMS AND DYNAMICS OF FLUO
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444 ENERGY TRANSFER Figure 13.1. Fl
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446 ENERGY TRANSFER wavelength is e
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448 ENERGY TRANSFER Table 13.1. Cal
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450 ENERGY TRANSFER Figure 13.6. Ab
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452 ENERGY TRANSFER safe for many p
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454 ENERGY TRANSFER Figure 13.12. P
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456 ENERGY TRANSFER Figure 13.16. E
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458 ENERGY TRANSFER Figure 13.21. R
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460 ENERGY TRANSFER Figure 13.24. R
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462 ENERGY TRANSFER tiple acceptors
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464 ENERGY TRANSFER Figure 13.29. A
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466 ENERGY TRANSFER Figure 13.33. F
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468 ENERGY TRANSFER Table 13.3. Rep
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470 ENERGY TRANSFER 55. Clegg RM. 1
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472 ENERGY TRANSFER Tong AK, Jockus
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474 ENERGY TRANSFER Figure 13.39. O
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14 In the previous chapter we descr
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15 In the previous two chapters on
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16 The biochemical applications of
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578 TIME-RESOLVED PROTEIN FLUORESCE
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608 MULTIPHOTON EXCITATION AND MICR
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19 Fluorescence sensing of chemical
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676 NOVEL FLUOROPHORES Figure 20.1.
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678 NOVEL FLUOROPHORES Figure 20.7.
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680 NOVEL FLUOROPHORES Figure 20.12
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698 NOVEL FLUOROPHORES 33. Acherman
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700 NOVEL FLUOROPHORES 103. Demas J
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702 NOVEL FLUOROPHORES 176. Montalt
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21 During the past 20 years there h
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22 Fluorescence microscopy is one o
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758 SINGLE-MOLECULE DETECTION Figur
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766 SINGLE-MOLECULE DETECTION small
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770 SINGLE-MOLECULE DETECTION Table
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786 SINGLE-MOLECULE DETECTION face
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788 SINGLE-MOLECULE DETECTION TCSPC
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790 SINGLE-MOLECULE DETECTION 53. H
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792 SINGLE-MOLECULE DETECTION Ke PC
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794 SINGLE-MOLECULE DETECTION Polar
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24 In the previous chapter we descr
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862 RADIATIVE-DECAY ENGINEERING: SU
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Appendix I Corrected Emission Spect
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Appendix II Fluorescence Lifetime S
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890 APPENDIX III P ADDITIONAL READI
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892 APPENDIX III P ADDITIONAL READI
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894 ANSWERS TO PROBLEMS A1.4. A. Th
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896 ANSWERS TO PROBLEMS Energy tran
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898 ANSWERS TO PROBLEMS Figure 4.65
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900 ANSWERS TO PROBLEMS expected to
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904 ANSWERS TO PROBLEMS where f is
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906 ANSWERS TO PROBLEMS [BSA] Obser
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908 ANSWERS TO PROBLEMS emission. T
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910 ANSWERS TO PROBLEMS dx rA A (
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912 ANSWERS TO PROBLEMS B. A covale
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914 ANSWERS TO PROBLEMS The α i va
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920 ANSWERS TO PROBLEMS CHAPTER 24
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Index A Absorption spectroscopy, 12
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