Environment-sensitive fluorophores

Vazquez ME, Blanco JB, Imperiali B (2005) Photophysics and biological applications of the environment-sensitive fluorophore 6-N, N-Dimethylamino-2, 3-naphthalimide. J Am Chem Soc 127 1300-1306... 

Z. M. Shakhsher and W. R. Seitz, Optical detection of cationic surfactants based on ion pairing with an environment-sensitive fluorophore, Anal. Chem. 62, 1758-1762(1990). 

Exogenous Environment-Sensitive Fluorophores and Glucose Binding Proteins... 

The fluorescence of FlAsH and ReAsH are, like the parent fluorophores fluorescein and resorufin, relatively insensitive to the local protein environment that the tetracysteine is surrounded by. Such insensitivity is useful in quantitative studies such as protein localization or trafficking in cells when the protein monitored may experience different local environments. However, environment-sensitive fluorophores are very useful probes of changes in... 

Fluorophores capable of CHEF and other types of environment-sensitive (or solvatochromic) fluorophores have been utilized in sensors for kinase activity. Chelation-sensitive fluorophores manifest altered fluorescent properties upon chelation of various metal ions, while environment-sensitive fluorophores exhibit altered excitation and emission properties with changing environment, such as solvent polarity (Figure 1.1). 

Figure 1.1 Environment-sensitive fluorescence, (a) Fluorescence emission spectrum of the environment-sensitive fluorophore, 4-DMN, in solvents of various polarities and (b) structure of the Sox fluorophore demonstrating chelation-enhanced fluorescence.

The mutant proteins were expressed in E. coli, released from the periplasmic space by osmotic shock, and purified in a single step by perfusion chromatography using an anion-exchange column. After the purity of the proteins were verified, they were then labeled with several environment-sensitive fluorophores (Figure 2), such as 6-aciyloyl-2-dimethylaminonaphthalene(acrylodan),... 

Fluorescence spectra and quantum yields are generally more dependent on the environment than absorption spectra and extinction coefficients. For example, coupling a single fluorescein label to a protein reduces fluorescein s quantum yields 60% but only decreases its molar extinction coefficient by 10%. Interactions either between two adjacent fluorophores or between a fluorophore and other species in the surrounding environment can produce environment-sensitive fluorescence. 

More so than absorption, fluorescence is sensitive to the chromophore s environment. Many fluorophores can show very substantial shifts in both of emission and fluorescence lifetime in response to changes in solvent or other environmental factors. When the change results from a variation in solvent, this is termed solvatochromism (the term is applicable to both absorption and emission). This can often be put to good use, in which A ax of a fluorophore can be used to probe whether the local environment is, for example, relatively hydrophobic or hydrophilic. Look back at Table 3.1 to see several examples of solvent scales based upon solvatochromism. Examples of two typical compounds that show large solva-tochromic effects are shown. 

The simplest fluorescence measurement is that of intensity of emission, and most on-line detectors are restricted to this capability. Fluorescence, however, has been used to measure a number of molecular properties. Shifts in the fluorescence spectrum may indicate changes in the hydrophobicity of the fluorophore environment. The lifetime of a fluorescent state is often related to the mobility of the fluorophore. If a polarized light source is used, the emitted light may retain some degree of polarization. If the molecular rotation is far faster than the lifetime of the excited state, all polarization will be lost. If rotation is slow, however, some polarization may be retained. The polarization can be related to the rate of macromolecular tumbling, which, in turn, is related to the molecular size. Time-resolved and polarized fluorescence detectors require special excitation systems and highly sensitive detection systems and have not been commonly adapted for on-line use. 

Many fluorophores are sensitive to changes in the hydropho-bicity of the immediate environment. Therefore, bringing these fluorophores into a different environment may also produce a change in FRET, when a second fluorophore is affected by the emission change of the first. Fluorophores like Nile Red with changes of up to 100 nm when transferred from water to an aprotic organic solvent are principally suitable for such an approach [71], Molecular rotors have the characteristic of having a quantum yield that depends on the viscosity. Such dyes are formed by an electron donor unit and an electron acceptor unit that can rotate relative to each other upon photoexcitation with a behavior that depends on the viscosity of the environment. These dyes have been included in FRET probes for viscosity studies [53],... 

Chen AK, Cheng ZL, Behlke MA, Tsourkas A (2008) Assessing the sensitivity of commercially available fluorophores to the intracellular environment. Anal Chem 80 7437-7444... 

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