Fluorescence polarization spectrum

Weber G. (1960) Fluorescence Polarization Spectrum and Electronic Energy Transfer in Tyrosine, Tryptophan and Related Compounds, Biochem. J. 75, 335-345. 

The fluorescence polarization spectrum of Rhodamin B and the SoFresponding absorption spectrum are given in the Figure 4.17B. The transition in bands 1 and 5 have the same polarization direction, bands 3 pud 4 are polarized almost perpendicular to 1 and the polarization of 2 gjt at some intermediate angle. These reflect the relative orientation of phe transition moments in the respective bands. 

Figure 4.17 A. Fluorescence polarization spectrum. A general case of a molecule with two absorption bands a and b with transition moment vectors at right angles to each other. (1) Absorption spectrum (2) Polarization of flourescence excitation spectrum. 

B. Fluorescence polarization spectrum and absorption spectrum of Rhodamin B. 

Denaturation. The stability of LADH and its denaturation has been studied under a variety of conditions including acid pH, and different concentrations of urea and guanidine hydrochloride. At pH 5, LADH loses its activity while still in the dimeric state, and at lower pH dissociation occurs, as can be seen in the drastic change in the fluorescence polarization spectrum.1410 The spectral data obtained are consistent with unfolding of the tertiary structure, some of which occurs before subunit dissociation. 

Acid Denaturation. LADH loses activity and zinc at pH 5 while still in the dimeric state 177,182). At lower pH dissociation occurs into subunits 182-184) and there are drastic changes in the protein fluorescence spectrum 185-187) and the fluorescence polarization spectrum 182). Different time dependences for the changes of the tyrosine and tryptophan difference fluorescence peaks are observed 187), which is consistent with a slower quenching of the buried Trp-314 (Section II,C,3,c) compared to the more exposed tyrosines. This interpretation implies that a partial unfolding of the tertiary structure occurs prior to the dissociation into subunits at acid pH. 

The equilibrium and rate constants for NADH binding to the three isozymes EE, ES, and SS of the horse enzyme have been determined 305). Differences in binding to the two types of chains were found both for the binding strength and the pH dependence. Changes in the absorption spectrum 306,307), the fluorescence polarization spectrum (305), the optical rotatory dispersion spectrum 309), and the effect of DzO on the fluorescence spectrum 310) have been studied for the binary enzyme coenzyme complexes compared to the free molecules. 

The degree of absorption polarization and the ratio of the extinction coefficient and exhibits a sharp increase starting at about 29,000 cm . Tliis indicates that a weak transition, namely the L j-transition, is buried below the much stronger L -band at wavelengths above 29,000 cm". A comparison of the absorption polarization spectrum, the fluorescence polarization spectrum and the circular dichroism spectrum shows that the 0-0-vibrational band of the L transition is located at about 29,000 cm [7]. This example demonstrates the usefulness of the liquid crystal method to localize weak hidden optical transitions. 

Figure 9.29 Two-photon fluorescence excitation spectrum of 1,4-difluorobenzene. The upper and lower traces are obtained with plane and circularly polarized radiation, respectively, but the differences are not considered here. (Reproduced, with permission, Ifom Robey, M. J. and Schlag, E. W., Chem. Phys., 30, 9, 1978)...

To qualify the environment into which the colorant molecule is embedded, the actual fluorescence spectrum is compared with the one under standard conditions. If the fluorescence emission spectrum is shifted to longer wavelengths (bathochromic shift), it can be concluded that the molecular enviromnent is of a more polar nature or is polarized by the excited fluorophore. Conversely, a fluorescence shift to shorter wavelengths (hypsochromic shift) indicates a transfer of the fluorophore from a polar... 

Additional evidence for conformational changes in the transporter has come from measurement of the intrinsic fluorescence of the protein tryptophan residues, of which there are six, in the presence of substrates and inhibitors of transport. The fluorescence emission spectrum of the transporter has a maximum at about 336 nm, indicating the presence of tryptophan residues in both non-polar environments (which would emit maximally at about 330 nm) and in polar environments (which would emit at 340-350 nm) [154], The extent of quenching by the hydrophilic quencher KI indicates that more than 75% of the fluorescence is not available for quenching, and so probably stems from tryptophan residues buried within the hydrophobic interior of the protein or lipid bilayer [155]. Fluorescence is quenched... 

Fluorescent probes are divided in two categories, i.e., intrinsic and extrinsic probes. Tryptophan is the most widely used intrinsic probe. The absorption spectrum, centered at 280 nm, displays two overlapping absorbance transitions. In contrast, the fluorescence emission spectrum is broad and is characterized by a large Stokes shift, which varies with the polarity of the environment. The fluorescence emission peak is at about 350 nm in water but the peak shifts to about 315 nm in nonpolar media, such as within the hydrophobic core of folded proteins. Vitamin A, located in milk fat globules, may be used as an intrinsic probe to follow, for example, the changes of triglyceride physical state as a function of temperature [20]. Extrinsic probes are used to characterize molecular events when intrinsic fluorophores are absent or are so numerous that the interpretation of the data becomes ambiguous. Extrinsic probes may also be used to obtain additional or complementary information from a specific macromolecular domain or from an oil water interface. 

It is possible, however, that the electrochromic response of some styrylpyridi-nium probes, for example, RH421 (see Fig. 2), is enhanced by a reorientation of the dye molecule as a whole within the membrane. There is a steep gradient in polarity on going from the aqueous environment across the lipid headgroup region and into the hydrocarbon interior of a lipid membrane. Therefore, any small reorientation of a probe within the membrane is likely to lead to a change in its local polarity and hence a solvatochromic shift of its fluorescence excitation spectrum. Such a... 

Fig. B5.2.1. Corrected excitation spectrum (broken line) and excitation polarization spectrum of indole in propylene glycol at -58 °C. The fluorescence is observed through a cut-off filter (Corning 7-39 filter) (reproduced with permission from Valeur and Weber3 ).

Since ANS dissolved in a polar medium does not fluoresce, one cannot record its fluorescence excitation spectrum. For tryptophan, ethidium bromide, and riboflavin, one can see that for each molecule, the absorption spectrum looks like the fluorescence... 

Model compound and difference spectral studies have been carried out on tyrosine and related compounds by Wetlaufer (1956), Laskowski (1957), Edelhoch (1958), Chervenka (1959), Bigelow and Geschwind (1960), Yanari and Bovey (1960), and Foss (1961) (see also Section VI,Z)). Studies of the fluorescence of tyrosine—activation spectrum, fluorescence emission spectrum, and quantum yield—have been reported by Teale and Weber (1957). The pH-dependence of the fluorescence of tyrosine and related compounds was studied by White (1959) fluorescence-polarization studies from the same laboratory were reported by Weber (1960a) for simple compounds, and for proteins (Weber, 1960b). Teale (1960) has carried out extensive studies on the fluorescence characteristics of a score of proteins. 

The fluorescence polarization excitation spectrum has been measured for thymine in aqueous solution. " The depolarization at the red edge is attributed to the hidden n, ir transition. Ionization of the lowest excited singlet and triplet states have been determined by the effect of pH on the absorption, fluorescence, and phosphorescence spectra of purines and pyrimidines. " Spectral, polarization, and quantum yield studies of cytidylyl-(3, 5 )-adenosine have also been published. Intermediates in the room-temperature flash photolysis of adenine and some of its derivatives have been identified hydrated electron, radical cations and anions, and neutral radicals resulting from their reactions have been assigned. Photoionization occurs via the triplet state. FMN encapsulated in surfactant-entrapped water pools interacts with polar head groups, entrapped water molecules, and outer apolar solvent. ... 

In the bulk, the low concentration of ground-state pairs excludes their observation by absorption. The formation of the excited-state complex, termed exciplex, is a collisional process electronic excitation of either the acceptor or the donor leads to the formation of a locally excited state (for instance, in hydrocarbon molecules, it is a nn state). During the lifetime of this state, a collision with the other partner (which is in the ground state) leads to the formation of the exciplex. This mechanism is compatible with the fact that the absorption and fluorescence excitation spectra of the system are identical with those obtained by superimposing the spectra of the individual components. At the same time, the fluorescence emission spectrum changes drastically—a broad band, red shifted with respect to the bare molecule s emission spectrum, appears. It is usually devoid of vibrational structure, and is shifted to longer wavelengths as the solvent polarity increases [1],... 

Fig. 26.8. Fluorescence-excitation spectrum from an individual RC-LHl complex from Rps. palustris as a function of the polarization of the excitation light, (a) Top Stack of 312 individual spectra recorded consecutively. Between two successive spectra the polarization of the incident radiation has been rotated by 6.4°. The horizontal axis corresponds to the photon energy, the vertical axis to the scan number or equivalently to the polarization angle and the intensity is given by the gray scale. The excitation intensity was 10 W/cm. Bottom Spectrum that corresponds to the average of the 312 consecutively recorded spectra, (b) Top Fluorescence intensity of the three bands marked by the arrows in the lower part as a function of the polarization of the incident radiation (dots) together with cos -type functions fitted to the data black). Bottom Two fluorescence-excitation spectra from the stack that correspond to mutually orthogonal polarization of the excitation light. The spectra where chosen such that the horizontal polarization yielded maximnm intensity for the narrow feature at the low-energy side. Adapted from [62]...

Figure 5.18. Absorption (A) and fluorescence spectrum (P) of azulene in ethanol at 93 K. FP and AP(P) denote the polarization spectrum of fluorescence and the excitation-polarization spectrum of fluorescence, respectively (by permission from Ddrr, 1966).

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