Fluorescence emission maximum

Fluorescence emission maximum Fluorescence quantum yield Molar absorption coefficient (e) at peak wavelengths (as monomer)... 

The bioluminescence spectrum of P. stipticus and the fluorescence and chemiluminescence spectra of PM are shown in Fig. 9.7. The fluorescence emission maximum of PM-2 (525 nm) is very close to the bioluminescence emission maximum (530 nm), but the chemiluminescence emission maximum in the presence of a cationic surfactant CTAB (480 nm) differs significantly. However, upon replacing the CTAB with the zwitter-ionic surfactant SB3-12 (3-dodecyldimethylammonio-propanesulfonate), the chemiluminescence spectrum splits into two peaks, 493 nm and 530 nm, of which the latter peak coincides with the emission maximum of the bioluminescence. When PM-1 is heated at 90°C for 3 hr in water containing 10% methanol, about 50% of PM-1 is converted to a new compound that can be isolated by HPLC the chemiluminescence spectrum of this compound in the presence of SB3-12 (curve 5, Fig. 9.7) is practically identical with the bioluminescence spectrum. 

Element or compound determined Fluorescent emission maximum... 

Presently, the only commercially available dyes that are applied because of then-ability to form fluorescent aggregates are trimethine cyanines JC-1 and JC-9 (Fig. 11) [25], the first one being studied much more extensively than the second one. The dye JC-1 is known to form red-fluorescent (emission maximum at 590 nm) J-aggregates in mitochondria possessing strong intramitochondrial negative potential, while upon depolarization of the mitochondrial membrane, the dye monomer green emission (maximum at 527 nm) is observed [25]. JC-9 demonstrates similar properties [25]. Such properties permit the application of these dyes for, e.g., detection of apoptotic electrical depolarization of mitochondria [25]. 

Plastocyanin from parsley, a copper protein of the chloroplast involved in electron transport during photosynthesis, has been reported to have a fluorescence emission maximum at 315 nm on excitation at 275 nm at pH 7 6 (2°8) gjncc the protein does not contain tryptophan, but does have three tyrosines, and since the maximum wavelength shifts back to 304 nm on lowering the pH to below 2, the fluorescence was attributed to the emission of the phenolate anion in a low-polarity environment. From this, one would have to assume that all three tyrosines are ionized. A closer examination of the reported emission spectrum, however, indicates that two emission bands seem to be present. If a difference emission spectrum is estimated (spectrum at neutral pH minus that at pH 2 in Figure 5 of Ref. 207), a tyrosinate-like emission should be obtained. 

For single-tryptophan proteins there is some correlation between blue-shifted fluorescence emission maximum and phosphorescence lifetime (Table 3.2). Another correlation is that three of the proteins which exhibit phosphorescence, azurin, protease (subtilisin Carlsberg), and ribonuclease Tlt are reported to show resolved fluorescence emission at 77 K. Both blue-shifted emission spectra and resolved spectra are characteristic of indole in a hydrocarbon-like matrix. 

Table 3.2. Correlation between Fluorescence Emission Maximum and Phosphorescence Lifetime in Single-Tryptophan Proteins 1...

The binding of 8-anilino-l-naphthalenesulfonate (ANS) to ciliary dynein ATPase resulted in a marked increase in the dye s fluorescence intensity, accompanied by a blue shift in the observed fluorescence emission maximum" (Fig. 6). While dynein has 37 3 dye binding... 

The fluorescence emission maximum, quantum yield, and lifetime of a fluorophore are very sensitive to its immediate environment. A blue shift in the emission maximum and an increase in the fluorescence quantum yield or lifetime is generally observed when a fluorophore is transferred form a polar solvent to a nonpolar one or when it binds to a hydro-phobic protein site. Furthermore, fluorescence quenching or enhancement may result from interactions of the fluorophore with various structural elements in its vicinity. 

The tryptophan fluorescence emission maximum of nearly all model compounds in H20 was located at 350 nm, indicating that the indole ring was completely exposed to the aqueous environment. 

In contrast to the variability of the fluorescence emission maximum observed with tryptophan, the tyrosine fluorescence emission maximum is always located at 303 nm and is not environment dependent. 

Fluorescence quantum yield and emission maximum determinations as a function of peptide concentration may also permit the detection of peptide self-aggregation at concentrations below 10-4 M, because the peptide fluorophore is likely to be located in a different environment in the peptide aggregate. For example, the concentration-dependent changes in the tryptophan fluorescence emission maximum of mellitin were monitored to determine the equilibrium dissociation constant and thermodynamic parameters of the monomer-tetramer self-association reaction of this peptide. 25 Similarly, measurement of the changes in the tryptophan fluorescence intensity of gramicidin A as a function of its concentration permitted the determination of an average monomer-dimer equilibrium con-stant. 26 ... 

The intrinsic UV fluorescence of proteins is dominated by the tryptophan indole rings. The absorption maximum is 280-290 nm with the fluorescence maximum ranging from 315-355 nm, depending on the local environment of the indole side-chains. Quantum yields range from 0.04 to 0.50 0.10 is a common value. As the local environment polarity or dielectric constant increases, the fluorescence maximum shifts up to 355 nm, such as for an indole ring in water or buffer. Trp moieties in highly hydro-phobic environments fluoresce at 315-320 nm. Thus the fluorescence emission maximum (and the quantum yield) provide indirect information as to the local environment of the Trp fluors. 

The pentacyclic thioxanthylium salt 624 absorbs at 582 nm (emax 105 000 M-1 cm-1) with a fluorescence emission maximum at 600 nm. The dye displays photodynamic activity in vitro against chemosensitive hamster ovary cell lines (Equation 207) <2006BMC8635>. 

The fluorescence emission maximum of CGTase is located at 338 nm, and its spectrum bandwidth is 55 nm (Figure 7.12a). Thus, both embedded and surface tryptophan residues contribute to protein fluorescence. Although CGTase contains many tyrosine residues, the absence of a shoulder or a peak at 303 nm (Figure 7.12b), when excitation is performed at 273 nm, suggests that tyrosine residues do not contribute to CGTase emission. 

Quantum yield and fluorescence emission maximum are sensitive to the surrounding environment. This can be explained as follows. Fluorophore molecules and amino acids of the binding sites (in the case of an extrinsic fluorophore such as TNS, fluorescein, etc.) or the amino acids of their microenvironment (case of Trp residues) are associated by their dipoles. Upon excitation, only the fluorophore absorbs the energy. Thus, the dipole of the excited fluorophore has an orientation different from that of the fluorophore in the ground state. Therefore, the fluorophore dipole-solvent dipole interaction in the ground state is different from that in the excited state (Figure 7.18). 

ELEMENT OR COMPOUND DETERMINED FLUORESCENT EMISSION maximum (nm) EXAMPLE OF APPLICATION... 

Progress curves at a single wavelength are monitored. The time courses are the average of several independent kinetic runs. Usually these studies are performed under pseudo-first-order conditions that allow the experimenter to analyze the kinetic time courses as a sum of exponential functions. The single wavelength can be chosen from the time-resolved spectra, and is always selected close to the absorbance maximum (or fluorescence emission maximum) of reactant, intermediate, or product. 

In general, binding of the peptide to a protein will result in a shift in the tryptophan fluorescence emission maximum from approximately 355 nm (free peptide) to shorter wavelength as the tryptophan enters a more hydrophobic environment, and an overall intensification of the fluorescence emission intensity. The extent of the wavelength shift gives some information about the environment of the tryptophan in the complex. More importantly, the fluorescence enhancement on binding of the peptide may be used to determine the dissociation constant (K ) at the low protein/peptide concentrations required to study high affinity interactions. 

Although in some cases an energy-transfer step may be involved, the specific emitter in a bioluminescent reaction is generally an intermediate or product whose excited state is populated during the reaction. Free flavin in aqueous solution has a fluorescence emission maximum at 525 nm, while bacterial luminescence both in vivo and in vitro emits at a maximum around 495 nm. These results and their implications are reported and discussed elsewhere (Balny and Hastings, 1975). 

Figure 32-6 shows typical fluorescence emission maximum wavelengths for the cutaneous porphyrias. The plasma in VP contains porphyrin covalently bound to protein with a fluorescence emission maximum at 624 to 628 nm. In other porphyrias, porphyrin is noncovalently bound to albumin and hemopexin. A normal fluorescence emission scan in plasma from a patient with clinically active skin lesions excludes all cutaneous porphyrias as their cause. The scan may become normal in PCT and HCP as skin lesions heal, and the diagnosis may be missed unless individual porphyrins are also measured in urine and feces from such patients. In addition, the scan may be abnormal during an acute attack of AIP or HCP in the absence of skin lesions and is always abnormal in clinically manifest VP, whether skin lesions are present or not (Table 32-5). 

Pyrene is extremely sensitive to the polarity of the microenvironment surrounding it. As the polarity of the microenvironment increases, the emission intensity of the first vibronic band (/,) increases, while the emission intensity of the third vibronic band (I3) decreases. Thus, ///. is related to the dipolarity of the microenvironment surrounding the pyrene molecules for example /,//3 shifts from 0.58 in cyclohexane to 1.87 in water.72 73 PRODAN and DCM are solvatochromic fluorophores whose fluorescence band position is extremely sensitive to the polarity of the surrounding microenvironment. For example, the fluorescence emission maximum of PRODAN shifts from 401 nm in cyclohexane to 531 nm in water.74-75... 

Table 12.1 Effect of Solvent Polarity on the Fluorescence Emission Maximum (X /nm) of Some Angelicin Derivatives...

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