fluorescence polarization.doc

Fluorescence SpectroscopyReturn to the Chemistry Home Page FluorescenceSchematic state energy level diagramS is singlet and T is triplet. The S0 state is the ground state and the subscript numbers identify individual states.TimescaleS0 Sn absorptionSn S1 internal conversion (10-11 - 10-14 sec)S1 S0 + hn fluorescence (10-7 - 10-9 sec)S1 Tn intersystem crossing (10-8 sec)S1 S0 internal conversion (10-5 - 10-7 sec)T1 S0 + hn phosphorescence (10 - 10-3 sec)T1 S0 internal conversion (10 - 10-3 sec)Characteristics of Excited States1. Energy 2. Lifetime 3. Quantum Yield 4. PolarizationPhosphorescence occurs at longer wavelength than does fluorescenceOften, the emission band is red-shifted relative to the absorption band: Stokes shiftExcited states decay exponentially with timeI = I0e-t/tI0 is the initial intensity at time zero,I is the intensity at some later time t, and t is the lifetime of the excited state. Also, kF = 1/ t, where kF is the rate constant for fluorescence. Quantum Yield = FFF = number of fluorescence quanta emitted divided by number of quanta absorbed to a singlet excited stateFF = ratio of photons emitted to photons absorbedQuantum yield is the ratio of photons emitted to photons absorbed by the system: FF = kF / kF + kISC + knr + kq + krPolarizationMolecule of interest is randomly oriented in a rigid matrix (organic solvent at low temperature or room temperature polymer). Plane polarized light is used as the excitation source.The degree of polarization is defined as follows:where I| and I are the intensities of the observed parallel and perpendicular components, respectively. a is the angle between the emission and absorption transition moments. If a is 0 than P = +1/2, and if a is 90 than P = -1/3. Figure 26, Becker, pg. 84Polarization of fluorescence of phenol in propylene glycol at -70C shows that the transition moments of the corresponding absorption bands are mutually perpendicular. Phosphorescence is usually slow (seconds) therefore quenching by impurities including oxygen usually makes phosphorescence difficult to observe. Low temperature glasses and rigorous exclusion of oxygen is usually necessary to observe phosphorescence. Since this condition is not biological, fluorescence is the primary emission process of biological relevance. Experimental MeasurementsSteady-state measurements: F, ITime-Resolved measurements: tEmission spectra are obtained when the excitation monochrometer M1 is fixed and the emission monochrometer M2 is scanned.If M2 is fixed and M1 is scanned the result is an excitation spectrum. Excitation and absorption spectra should be identical.Relative quantum yields are determined by using a standard such as quinine sulfate in 1 N H2SO4 (fF = 0.70), or fluorescein in 0.1 N NaOH (fF = 0.93). The areas under the emission band of the standard relative to the sample are compared. It is of course important that the absorption at lex are matched.Excited-state decay rates can be measured by exciting the sample with a short pulse of light and monitoring the emission as a function of time. Figure 8-14, Cantor & Schimmel, pg. 442.Fluorescence decay of a pure sample showing a single exponential decay. The dark line shows the excitation pulse. Time correlated single photon counting was used to obtain this data. This technique counts the number of emitted photons hitting a detector at times, t, following excitationOne critical difference between steady-state and kinetic measurements of fluorescence is that the value of tF is not a function of concentration of the sample while the value of FF is concentration dependent. Only at low concentration is the value of FF linearly dependent on concentration. The reason is the so-called inner filter effect.The inner filter effect:At low concentration the emission of light is uniform from the front to the back of sample cuvette. At high concentration more light is emitted from the front than the back. Since emitted light only from the middle of the cuvette is detected the concentration must be low to assure accurate FF measurements.Fluorescence characteristics of chromophores found in proteins and nucleic acids. Generally, quantum yields are low and lifetimes are short. AbsorptionFluorescenceSensitivitySubstanceConditionlmax(nm)emax 10-3lmax(nm)fFtF(nsec)emaxfF 10-2TryptophanH2O, pH 72805.63480.202.611TyrosineH2O, pH 72741.430PhenylalanineH2O, pH 72570.22820.046.40.08AdenineH2O, pH 726013.43212.6 10-40.020.032GuanineH2O, pH 72758.13292.6 10-40.020.024CytosineH2O, pH 72676.13130.8 10-40.020.005UracilH2O, pH 72609.53080.4 10-40.020.004NADHH2O, pH 73406.24700.0190.401.2Figure 8-15, Cantor & Schimmel, pg. 444Fluorescence emission spectra of human serum albumin (solid line), tryptophan alone (dashed line), and an 18:1 molar ratio of tyrosine to tryptophan (gray line): Excitation at 245 nm.The 18:1 sample approximates the relative occurrence of these amino acids in the protein. Note that the spectrum of the protein closely resembles that of pure tryptophan because tyrosine sensitivity is low and its emission is most likely quenched by tryptophan (via energy-transfer mechanism).Commonly, fluorescent probe molecules are used to characterize protein and nucleic acids. Sensitivity is higher. lmax is also different from biomolecule so selective excitation is possible. Fluorescence generally is much more sensitive to the environment of the chromophore than is light absorption. Therefore, fluorescence is an effective technique for following the binding of ligands or conformational changes. The sensitivity of fluorescence is a consequence of the relatively long time a molecule stays in an excited singlet state before deexcitation. Absorption, or CD, is a process that is over in 10-15 sec. On this time scale, the molecule and its environment are effectively static. In contrast, during the 10-9 to 10-8 sec that a singlet remains excited, all kinds of processes can occur, including protonation or deprotonation reactions, solvent-cage relaxation, local conformational changes, and any processes coupled to translational or rotational motion.A number of fluorescent molecules have a very convenient propertyin aqueous solution their fluorescence is very strongly quenched, but in a nonpolar or a rigid environment (like in a protein or nucleic acid) a striking enhancement is observed.Figure 8-17, Cantor & Schimmel, pg. 447.In addition protein protects the probe from quenchers such as oxygen.F0/F = fo/f = kF + kIC + kISC + kq(Q)/(kF + kIC + kISC) = 1 + kqt0(Q), where F = fluorescence in the presence of quencher, F0 = fluorescence in the absence of quencher. Therefore a plot of F0/F versus concentration of Q will yield a value for kq. Quenching of tryptophan fluorescence by collision with oxygen:Tryptophan: kq = 12 109 M-1 sec-1 (diffusion controlled)Carbonic anhydrase: kq = 2.6 109 M-1 sec-1Singlet-Singlet Energy TransferIf the emission band of a molecule (D) coincides with the absorption band of another (A) two processes occur: emission of D is quenched, emission of A is sensitized. Figure 8-18, Cantor & Schimmel, pg. 449Frster theory can be used to determine distance between chromophoresThe rate of energy transfer = kT = (1/tD)(R0/R)-6. tD = lifetime of D in the absence of A.R0 = characteristic transfer distance = 9.7 103 (J k2 n-4 fD)1/6 cm, where J = eA(n)fD(n)n-4 dvJ is a measure of the spectral overlap between donor emission and acceptor absorption (shaded region in figure). FD is the normalized fluorescence of the donor; n is the refractive index of the medium between donor and acceptor; fD is the quantum yield of donor in the absence of acceptor; and k2 is a complex geometric factor that depends on the orientation of donor and acceptor. If both donor and acceptor are free to tumble rapidly on the time scale of fluorescence emission, k2 approaches a limiting value of 2/3. If the efficiency of energy transfer is expressed as E = kT/(kT + 1/tD) thanE = R06/(R06 + R6).Figure 8-20, Cantor & Schimmel, pg. 455For dansyl-(L-proline)n-a-naphthyl for n = 1 to 12. For this pair R0 = 50.Energy transfer plays a large role in determining the emission spectrum of normal proteins.The fluorescence of tyrosine is overlapped by the absorption of tryptophan. The R0 for this donor-acceptor pair is about 9 . This is short enough, given the average size of globular proteins, such that most tyrosines in proteins are quenched by singlet-singlet energy transfer by tryptophan. The result is that observed emission comes from primarily tryptophan (see above).Fluorescence Anisotropy/PolarizationFigure 16-14 and legend, pg. 529, MarchellSteady-state experiment continuous irradiation with polarized light of molecules in solution.Polarization = I| and I are time-independent steady state values for fluorescent intensity polarized parallel and perpendicular. Po is the maximum P which occurs when the rotational motion is very slow compared to the singlet excited state lifetime.trot = rotational correlation time = the characteristic lifetime of rotational diffusion. For large proteins trot is large.If trot tF and the polarization, P, reaches its maximum value, Po.The slope of the plot of versus will be which yields a value for V since tF is known. This in turn yields a value for trot and an estimate for the radius of the molecule (note: these equations are valid only for spherical molecules).Perrin plot for human macroglobulin (MW = 900,000 Da) and two fragments (MW = 180,000 and 50,000 Da) yields values for trot.The protein is first covalently labeled with dansyl chloride which has strong fluorescence when bound to proteins and a tF = 12 ns.900 kDa = trot = 80 ns180 kDa = trot = 69 ns50 kDa = trot = 58 nsIf the 50 kDa and 900 kDa were both rigid molecules one would expect a 260% reduction in trot, but only get 25% reduction. Therefore, the 900 kDa protein must be highly flexible.Time-resolved Fluorescence Depolarization (Anisotropy).A short pulse of vertically polarized light is directed at the sample;the light is absorbed, promoting the molecule to an excited singlet state;following vibrational relaxation, light is emitted (fluorescence) at lower energy;if the molecule rotates during the time interval between absorption and emission, there will be a decrease in the polarization with time at a rate that reflects the rate at which the molecule rotates diffusionally.Fluorescence Anisotropy = at times, t, after the light pulse is turned off.The overall fluorescent intensity I|(t) + 2I(t) will decrease exponentially in time according to the lifetime, tF, of the excited singlet state.The A(t) will decrease with a time constant, trot, which represents the time it takes for a molecule to rotate diffusionally, i.e., A(t) = Aoexp(-t/trot).A dansyl-labeled protein can be used to determine directly the trot in a time-resolved experiment.Figure 16-15, pg. 532, Marshell(a) dansyl-labeled protein in membrane(b) free dansyl-labeled proteinA(t) is bi-exponential, i.e., two trots.In (a) trotA = 3 ns, trotB = 700 ns.In (b) trotA = 3 ns, trotB = 45 ns.The fast component is due to local flexibility at the site of attachment of the label to the protein, and the slow component corresponds to rotational diffusion of the whole protein molecule.Clearly, in a membrane the protein is highly immobilized.Rotational Correlation Times, trot, of Proteins, Determined Either from Experimentally Measured Fluorescence Depolarization Decay Rates or from Theoretical Models of the Protein as a Sphere in a Continuous Medium.ProteinMolecularWeighttrot (fromfluorescencedepolarization)trot (from )trot(expt) / trot(calc)Apomyoglobin17,0008.3 nsec4.4 nsec1.9b-lactoglobulin18,400Trypsin25,000Chymotrypsin25,000Carbonic anhydrase30,00011.28.01.4b-lactoglobulin (dimer)36,000Apoperoxidase40,000Serum albumin66,00041.717.42.4Fluorescence depolarization studies of an antibody to which a fluorescent hapten was bound provided evidence for internal flexibility in the immunoglobulin molecule.Antibodies were grown specific to a dansyl-hapten.Depolarization studies of the hapten-antibody complex reveals two trots ( 33 and 168 ns).The fast component may be due to flexibility in the hinge region that joins the fragments and the slow component is most likely due to rotation of the whole complex.Fluorescence Polarization (FP)Note 1.4Share | PrinciplesFluorescence polarization measurements provide information on molecular orientation and mobility and processes that modulate them, including receptorligand interactions, proteinDNA interactions, proteolysis, membrane fluidity and muscle contraction (Figure 1).Because polarization is a general property of fluorescent molecules (with certain exceptions such as lanthanide chelates), polarization-based readouts are somewhat less dye dependent and less susceptible to environmental interferences such as pH changes than assays based on fluorescence intensity measurements. Experimentally, the degree of polarization is determined from measurements of fluorescence intensities parallel and perpendicular with respect to the plane of linearly polarized excitation light, and is expressed in terms of fluorescence polarization (P) or anisotropy (r):Note that both P and r are ratio quantities with no nominal dependence on dye concentration. Because of the ratio formulation, fluorescence intensity variations due to the presence of colored sample additives tend to cancel and produce relatively minor inteferences. P has physically possible values ranging from 0.33 to 0.5. In practice, these limiting values are rarely attained. Measured values of P in bioanalytical applications typically range from 0.01 to 0.3 or 10 to 300 mP (mP = P/1000). This measurement range is not as narrow as it might appear to be because very precise measurements (P 0.002 or 2 mP) are readily obtainable with modern instrumentation.Figure 1. Physical basis of fluorescence polarization assays. Dye molecules with their absorption transition vectors (arrows) aligned parallel to the electric vector of linearly polarized light (along the vertical page axis) are selectively excited. For dyes attached to small, rapidly rotating molecules, the initially photoselected orientational distribution becomes randomized prior to emission, resulting in low fluorescence polarization. Conversely, binding of the low molecular weight tracer to a large, slowly rotating molecule results in high fluorescence polarization. Fluorescence polarization therefore provides a direct readout of the extent of tracer binding to proteins, nucleic acids and other biopolymers. Dependence of Fluorescence Polarization on Molecular MobilityInterpretation of the dependence of fluorescence polarization on molecular mobility is usually based on a model derived in 1926 from the physical theory of Brownian motion by Perrin.where Po is the fundamental polarization of the dye (for fluorescein, rhodamine and BODIPY dyes, Po is close to the theoretical maximum of 0.5), is the excited-state lifetime of the dye and is the rotational correlation time of the dye or dye conjugate. These relationships can be expressed in terms of fluorescence anisotropy in an equivalent and mathematically simpler manner. For a hydrodynamic sphere, can be estimated as follows:where = solvent viscosity, T = temperature, R = gas constant and V = molecular volume of the fluorescent dye or dye conjugate. In turn, V can be estimated from the molecular weight of the dye or dye conjugate with appropriate adjustments for hydration. Simulations of these relationships are shown in Figure 2., leading to the following general conclusions: Fluorescence polarization increases as molecular weight increases. Fluorescence polarization increases as solvent viscosity increases. Fluorescence polarization decreases as the excited state lifetime of the dye () increases.Note that these simulations assume that the dye is rigidly attached to a spherical carrier. When conventional parameter estimates for proteins in aqueous solutions are used, is found to increase by about 1 ns per 2400 dalton increase of molecular weight.Figure 2. Simulation of the relationship between molecular weight (MW) and fluorescence polarization (P). Simulations are shown for dyes with various fluorescence lifetimes (): 1 ns (cyanine dyes) in purple, 4 ns (fluorescein and Alexa Fluor 488 dyes) in red, 6 ns (some BODIPY dyes) in green and 20 ns (dansyl dyes) in blue. At MW = 1000, P = 0.167 for = 1 ns, P = 0.056 for = 4 ns, P = 0.039 for = 6 ns and P = 0.012 for = 20 ns. Simulations assume Po (the fundamental polarization) = 0.5 and rig