Biochemical Fluorophores

FluCTophores are divided into two general classes, intrinsic and extrinsic. Intrinsic fluorophc are those which occur naturally. Extrinsic fluorophores are those which are added to a sanqple that does not dis 4ay the desired spectral propeities. In proteins, the dominant fluorophore is the indcAt group of tryptophan (Bgure 1.17). Ind(4e absorbs 

FigMVd 1.17. Abempdm and ensssionspectEaf lMomolectdes.TiyK nyplopheneii ion from piDteiiis.Af ft Spectra trinsic nenbnne 

Rgure 1.18. Ruoco(4)0its for covalent labefing (DNS 0 and F2TC) and fluorescent nucleotide analogs (c-ATP and /iR-beozo AMP). 

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Equation [1.5] can be derived from Eqs. [ 1.1 ]-[l. 3]. Many biochemical fluorophores do not behave predictably as unsubstituted aromatic compounds. Ifence, there is often poor agreement between die value of x calculated from Eq. [1.5] and that calculated from the absorption and emission spectra (Eq. [1.4]). These discr ancies occurfor a variety of unknown and known reasons, such as a fraction of the fluorophores being located next to quenching groups, which sometimes occurs for tryptophan residues in proteins. 

In the present review, we describe the effects of different silver nanostructures that were prepared by various methods in our laboratories on the emission intensity of fluorophores with various quantum yields and on biochemical fluorophores. The silver nanostructures consist of subwavelength size nanoparticles of silver deposited on inert substrates. These particles display a surface plasmon absorption, which in the small... 

Tokunaga M, Kitamura K, Saito K, Iwane A H and Yanagida T 1997 Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy Biochem. Biophys. Res. Commun. 235 47-53... 

Fluorescence analysis has been extended to many nonfluorescent species by the development of a wide range of derivatizing agents that form a fluorescent product. This approach has been especially useful with biochemical molecules, many of which are not natural fluorophores. 

Jackson, P., The use of polyacrylamide-gel electrophoresis for the high-reso-lution separation of reducing saccharides labeled with the fluorophore 8-ami-nonaphthalene-l,3,6-trisulfonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device, Biochem. ]., 270, 705, 1990. 

The lifetime of the excited state of fluorophores may be altered by physical and biochemical properties of its environment. Fluorescence lifetime imaging microscopy (FLIM) is thus a powerful analytical tool for the quantitative mapping of fluorescent molecules that reports, for instance, on local ion concentration, pH, and viscosity, the fluorescence lifetime of a donor fluorophore, Forster resonance energy transfer can be also imaged by FLIM. This provides a robust method for mapping protein-protein interactions and for probing the complexity of molecular interaction networks. 

Noise can be also introduced by biochemical heterogeneity of the specimen. This can be a major cause of uncertainty in biological imaging. The high (three-dimensional) spatial resolution of fluorescence microscopy results in low numbers of fluorophores in the detection volume. In a typical biological sample, the number of fluorophores in the detection volume can be as low as 2-3 fluorophores for a confocal microscope equipped with a high NA objective at a fluorescent dye concentration of 100 nM. This introduces another source of noise for imaging applications, chemical or molecular noise, related to the inherent randomness of diffusion and the interaction of molecules. 

Fluorescence or Forster resonance energy transfer (FRET) is widely accepted as being one of the most useful methods to observe biochemical events in vitro and in living cells. Generally, there are two forms of FRET sensors those based on a pair of genetically encoded fluorophores, usually employing fluorescent proteins from jellyfish or corals, or those based on small molecules that make use of small organic fluorophores. 

Stepping outside of the subject of biochemical protein dynamics there is also a healthy literature on the use of sensing using fluorophores. Spectral and lifetime characteristics of fluorophores are dependent on their environment, for example, pH, O2, and Ca2+, these features are a useful tool, particularly in the study of the basic biology of the cell (see for instance Chapter 4). 

Hennig A, Florea M, Roth D, Enderle T, Nau WM (2007) Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore. Anal Biochem 360 255-265... 

Malicka J, Gryczynski I, Gryczynski Z, Lakowicz JR (2003) Effects of fluorophore-to-silver distance on the emission of cyanine-dye-labeled oligonucleotides. Anal Biochem 315 57-66... 

Figure 3.34 — Manifolds for implementation of a sensor containing a packed non-regenerable reagent and a regenerable fluorophore. (A) Flow-through sensor system 1 eluent vessel 2 pump 3 injection valve 4 TCPO reactor 5 CL cell 6 light-tight box with PMT 7 amplifier 8 recorder. (B) Design of the packed two-layer sensor 1 inlet capillary 2 inlet cap with frit 3 quartz tube 4 TCPO layer 5 frit 6 luminophore layer 7 outlet cap with frit 8 outlet capillary. (C) Manifold for implementation of the previous cell in biochemical applications (Reproduced from [240] and [241] with permission of the American Chemical Society and Elsevier Science Publishers, respectively).

Many of the fully aromatic 6-6-5 tricyclics show good fluorescence properties, and in some instances this has been utilized in biochemical applications. The reader is directed to the literature cited in Section 7.22.12.1 (benzo-separated purines) for examples of how the sensitivity of the fluorescence properties of tricyclic fluorophores to environmental conditions can be utilized to analyze the binding parameters associated with biomacromolecules. Of particular interest in this regard is a method known as fluorescence-polarization titration (83B2347). 

Most biochemical assays used to determine the potency of inhibitors against (1) aminopeptidases and (2) endopeptidases with minor contributions to the substrate binding efficiency by the S site, are based on the measurement of FI as readout. AMC and RhllO are the most common fluorophores in these cases. For studies of enzyme kinetics, the application of AMC is sufficient. If an assay is intended to determine the inhibitory potencies of small chemical compounds, it is recommended to use red-shifted dyes like RhllO to minimize interference of compound fluorescence with the fluorescence originating from the assay dye. 

Lozinsky, E., Martin, V.V., Berezina, T.A., Shames, A., Weis, A.L., and Likhtenshtein, G. I. (1999) Dual fluorophore-nitroxide probes for analysis of vitamin C in biological liquids, J. Biochem. Biophys. Meth. 38,29-42. 

Y8. Yin, D., Biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores. Free Radicals Biol. Med. 21, 871-888 (1996). 

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