Extrinsic Fluorophores

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. 

The fast, sensitive, reliable, and reproducible detection of (bio)molecules including quantification as well as biomolecule localization, the measurement of their interplay with one another or with other species, and the assessment of biomolecule function in bioassays as well as in vitro and in vivo plays an ever increasing role in the life sciences. The vast majority of applications exploit extrinsic fluorophores like organic dyes, fluorescent proteins, and also increasingly QDs, as the number of bright intrinsic fluorophores emitting in the visible and NIR is limited. In the near future, the use of fluorophore-doped nanoparticles is also expected to constantly increase, with their applicability in vivo being closely linked to the intensively discussed issue of size-related nanotoxicity [88]. 

Extrinsic fluors are produced via a chemical reaction where the added reagent either enhances emission of a weak emitter through association or the analyte is derivatized with a fluor tag. 8-Hydroxyquinoline (HQS) is an example of an extrinsic complexing reagent (Reaction 11.1) where the native ligand is a marginal fluorophore but forms intense emitting metal chelates. This approach affords sensitive detection of... 

Fluoroimmunoassays comprise a subclass of extrinsic labehng methods where various selective antigen (Ag)- antibody (Ab) immunoassay fluorescent labeling schemes yield a emission signal. One common scheme involves an enzyme-linked immunosorbent assay (ELISA) depicted in Figure 11.2 where the free Ab is tagged with a fluorophore. Numerous analytes can be detected via these types of selective lock-and-key methods. ... 

INTRINSIC AND EXTRINSIC FLUORESCENCE. Intrinsic fluorescence refers to the fluorescence of the macromolecule itself, and in the case of proteins this typically involves emission from tyrosinyl and tryptopha-nyl residues, with the latter dominating if excitation is carried out at 280 nm. The distance for tyrosine-to-tryp-tophan resonance energy transfer is approximately 14 A, suggesting that this mode of tyrosine fluorescence quenching should occur efficiently in most proteins. Moreover, tyrosine fluorescence is quenched whenever nearby bases (such as carboxylate anions) accept the phenolic proton of tyrosine during the excited state lifetime. To examine tryptophan fluorescence only, one typically excites at 295 nm, where tyrosine weakly absorbs. [Note While the phenolate ion of tyrosine absorbs around 293 nm, its high pXa of 10-11 in proteins typically renders its concentration too low to be of practical concern.] The tryptophan emission is maximal at 340-350 nm, depending on the local environment around this intrinsic fluorophore. 

One can employ linearly polarized light to excite selectively those fluorophores that are in a particular orientation. The difference between excitation and emitted light polarization changes whenever fluorophores rotate during the period of time between excitation and emission. The magnitude of depolarization can be measured, and one can therefore deduce the fluorophore s rotational relaxation kinetics. Extrinsic fluorescence probes are especially useful here, because the proper choice of their fluorescence lifetime will greatly improve the measurement of rotational relaxation rates. One can also determine the freedom of motion of the probe relative to the rotational diffusion properties of the macromolecule to which it is attached. When held rigidly by the macromolecule, the depolarization of a probe s fluorescence is dominated by the the motion of the macromolecule. 

Fluorescence spectroscopy S Conformational change with ligand binding induces change in fluorescence properties of intrinsic or extrinsic fluorophore... 

Analogously, the fluorescence quantum yield of an extrinsic fluorescent probe contained in a peptide can be measured by comparison with an appropriate standard. If the fluorescent peptide exists in a conformational equilibrium, the fluorophore may be located in a number of different environments and may have a distinct quantum yield (ip,) in each environment. In this case the determined fluorescence quantum yield represents a population-weighted average of the individual [Pg.700]

A large number of fluorescence decay measurements have been performed with proteins.127 Studies on the fluorescence decay of tyrosine and tryptophan and their derivatives, and on biologically active peptides containing intrinsic or extrinsic fluorophores have also been carried out and a few illustrative examples will be reviewed here. 

A suitable fiuorescent probe is an organic molecule, which must change its characteristic parameters with changes in its microenvironment and the parameter must be measurable when the probe is added to the system [54]. The fluorescent probes are categorized as either extrinsic, intrinsic, or covalently bound probes. The intrinsic probes allow a system to be observed without any chemical perturbation. This occurs when the system to be characterized has an in-built fluorescent chromophore unit like tryptophan, tyrosine and phenyl alanine in protein. In some cases the fluorophore is covalently... 

For chromophores that are part of small molecules, or that are located flexibly on large molecules, the depolarization is complete—i.e., P = 0. A protein of Mr = 25 kDa, however, has a rotational diffusion coefficient such that only limited rotation occurs before emission of fluorescence and only partial depolarization occurs, measured as 1 > P > 0. The depolarization can therefore provide access to the rotational diffusion coefficient and hence the asymmetry and/or degree of expansion of the protein molecule, its state of association, and its major conformational changes. This holds provided that the chromo-phore is firmly bound within the protein and not able to rotate independently. Chromophores can be either intrinsic—e.g., tryptophan—or extrinsic covalently bound fluorophores—e.g., the dansyl (5-dimethylamino-1-naphthalenesulfonyl) group. More detailed information can be obtained from time-resolved measurements of depolarization, in which the kinetics of rotation, rather than the average degree of rotation, are measured. For further details, see Lakowicz (1983) and Campbell and Dwek (1984). 

Small-molecule extrinsic fluorophores are generally used in structural and folding studies of RNA molecules (Fig. 8.5). There are a number of factors that will influence the choice of donor—acceptor fluorophores... 

In order to measure quantum yields of an extrinsic fluorophore bound to a protein and which emits at longer wavelengths than in the UV, standards such as 3,3 -diethylthiacarbocyanine iodide (DTC) in methanol (Op = 0.048) and rhodamine 101... 

Fluorophores, small molecules that can be part of a molecule (intrinsic fluorophores) or added to it (extrinsic fluorophores), can be found in different cells, and so they can be... 

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). 

In static quenching, an intrinsic or extrinsic fluorophore is used to examine the interaction between two macromolecules. 

The fluorophore should be bound to one of the two proteins only (case of extrinsic fluorophores) or should be part of it (case of intrinsic fluorophores). Also, the binding parameters of the fluorophore-macromolecule complex can be determined by following the fluorescence modification of the fluorophore observables. 

Whether fluorophores are intrinsic or extrinsic to the macromolecule (protein, peptide, or DNA), depolarization is the result of two motions, fluorophore local motions and macromolecule global rotation. 

The three proteins show intrinsic fluorescence due to the presence of Trp residues. Thus, although binding occurs between LCA and LTF or STF, we cannot use fluorescence of Trp residues of LCA to follow this interaction, since there will bean overlapping with the fluorescence of Trp residues of LTF or STF. Therefore, it is necessary to use an extrinsic probe, which is bound to one protein only. A covalently bound fluorophore such as fluorescein is very suitable to perform binding experiments, since there will be no real binding between the fluorophore and the added protein, c Figure 13.6 shows the fluorescence intensity at 515 nm of fluorescein bound to LCA in the presence of increasing concentrations of LTF or STF. 

Fluorescence measurements on proteins require both an appropriate fluorescence technique and the presence of a suitable fluorophore. The techniques used for the application of fluorescence to proteins are described later in this article. In this section, we briefly consider three classes of fluorophores that are used widely to study proteins native fluorophores including fluorescent amino acids, extrinsic fluorescent labels, and auto-fluorescent proteins. Each has advantages for probing proteins and has distinct drawbacks No perfect fluorophore exists for studying proteins. 

Among nonisotopic techniques, fluorescence (both intrinsic and extrinsic) offers a convenient mode of detection, and the sensitivity of some fluorescent labels is comparable to that of radiolabeled iodine. Recent innovations include the use of polarized light for excitation, such that the degree of polarization of the emission as well as its intensity can provide information about the concentration and size-related behavior (e.g., rotational diffusion) of the fluorescent-labeled molecule. One disadvantage of steady-state fluorescence techniques is that many analytical samples either autofluoresce or quench the fluorescence of the substance of interest. A recent development that circumvents this problem utilizes long-lived fluorophores such as the lanthanide metal ions as labels. Detection is time resolved and data are collected after the decay of spurious or otherwise unwanted fluorescence, i.e., after 100-200 psec. 

Table 4.4 Summary of the main extrinsic fluorophores that may be combined with proteins or nucleic acids. Main absorption and fluorescence characteristics are given. See Fig. 4.15 and Table 4.1 for structure abbreviations and other abbreviations used in the table.

Figure 4.15 Structures of some useful extrinsic fluorophores that may be chemically combined with a biological macromolecule of interest for structure and/or function investigations.

One extreme view of chemical introduction of an extrinsic fluorescent probe is found in the case ofthe alanine derivative of the fluorophore 6-dimethylamino-2-acylnaphthalene (DAN) (Figure 4.23). This derivative fluorophore, given the trivial name Aladan, is incorporated into a polypeptide by solid-phase synthetic chemistry (although a molecular biology technique known as nonsense suppression is now available for the introduction of unnatural amino-acid residues into recombinant proteins). The fluorescent emission maximum (Tnax) of Aladan shifts dramatically on different solvent exposures, from 409 nm in heptane to 542 nm in water, yet at the same time remains only mildly changed by variations in pH or salt concentration. This compares to a maximum environment-mediated shift of around 40 nm for intrinsic tryptophan fluorescence. In addition, there is little spectral overlap between extrinsic Aladan fluorescence and intrinsic fluorescence from tryptophan or tyrosine. 

The selection of extrinsic fluorescent probe is driven by the consideration of which biological macromolecule or lipid is to be labelled, the requirement for compatibility between the intended fluorescent probe (in terms of solubility in water, pH sensitivity and so on) and the properties of the molecule to be labelled. Also, choice of the fluorescent probe should be consistent with experimental objectives. For instance, FRET experiments require that extrinsic donor and acceptor fluorophores should be properly matched for their capacity to participate in the FRET effect (see Section 4.5.4). 

Aladan substitution of internal core amino-acid residues provides an approach to characterise the physical characteristics of protein cores. Steady-state fluorescence alone can provide initial insight to the immediate environment of Aladan in the protein core. However, time-resolved fluorescence spectroscopy can be used to understand variations in protein core composition and structure as a function of time through the characterisation of Aladan fluorescence intensity and /max changes that are caused by small fluctuations in the relative permittivity, e, of the protein interior with time (fs-ps timescale). Such spectroscopy is possible since fluorescence lifetimes, Tr, are typically in the ns range (see Section 4.5). Also, time-resolved fluorescence spectroscopy can be performed with non-covalently linked extrinsic fluorophores such as ethidium bromide (EtBr). This fluorophore intercalates between the bases of DNA or RNA double helix and in so doing acquires a substantial increase in (j) and hence fluorescence intensity at /max (595 nm). Should there be a disruption or collapse in double-helical structure, then intercalation fails and fluorescent intensity drops... 

The fluorescence intensity decrease of any fluorescent probes whether free in solution, bound to a protein or to a membrane such as extrinsic fluorophores or being parts of a protein such as intrinsic fluorophores can take place with one, two or several fluorescence lifetimes. 

Fluorophnes can be part of a molecule and m this case we call them intrinsic fluorophores. or they can be added to a molecule and th will be Icnown as extrinsic fluorophores. We are going here to describe the properties of ibe most known fluorophores. 

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