Extrinsic fluorescence

Currently, there are a wide variety of highly fluorescent extrinsic dyes that may be grouped in two classes ... 

The conformation of bovine myelin basic protein (MBP) in AOT/isooctane/water reversed micellar systems was studied by Waks et al. 67). This MBP is an extrinsic water soluble protein which attains an extended conformation in aqueous solution 68 but is more density packed at the membrane surface. The solubilization of MBP in the AOT reversed micelles depends on the water/AOT-ratio w0 68). The maximum of solubilization was observed at a w0-value as low as 5.56. The same value was obtained for another major protein component of myelin, the Folch-Pi proteolipid 69). According to fluorescence emission spectra of MBP, accessibility of the single tryptophane residue seems to be decreased in AOT reversed micelles. From CD-spectra one can conclude that there is a higher conformational rigidity in reversed micelles and a more ordered aqueous environment. 

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. 

Fluorescence spectroscopy is also particularly well-suited to clarify many aspects of polymer/surfactant interactions on a molecular scale. The technique provides information on the mean aggregation numbers of the complexes formed and measures of the polarity and internal fluidity of these structures. Such interactions may be monitored by fluorescence not only with extrinsic probes or labelled polymers, but also by using fluorescent surfactants. Schild and Tirrell [154] have reported the use of sodium 2-(V-dodecylamino) naphthalene-6-sulfonate (SDN6S) to study the interactions between ionic surfactants and poly(V-isopropylacrylamide). 

The potential of using extrinsic (fluorescent) probes for monitoring the initial stages of oxidation was explored for the photo-oxidation of a UV-cured aliphatic polyurethane-acrylate-based adhesive [68]. The two probes investigated were p-dimethylamino salicylic acid (p-DASA) and 2, 7 -difluorescein (Oregon Green... 

Figure 24 (a) and (b) Fluorescence spectra from extrinsic probes in cured adhesive under... 

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]. 

Fluorescent probes can be divided into three classes (i) intrinsic probes-, (ii) extrinsic covalently bound probes and (iii) extrinsic associating probes. Intrinsic probes are ideal but there are only a few examples (e.g. tryptophan in proteins). The advantage of covalently bound probes over the extrinsic associating probes is that the location of the former is known. There are various examples of probes covalently... 

A criticism often aimed at the use of extrinsic fluorescent probes is the possible local perturbation induced by the probe itself on the microenvironment to be probed. There are indeed several cases of systems perturbed by fluorescent probes. However, it should be emphasized that many examples of results consistent with those obtained by other techniques can be found in the literature (transition temperature in lipid bilayer, flexibility of polymer chains, etc.). To minimize the perturbation, attention must be paid to the size and shape of the probe with respect to the probed region. 

The indole chromophore of tryptophan is the most important tool in studies of intrinsic protein fluorescence. The position of the maximum in the tryptophan fluorescence spectra recorded for proteins varies widely, from 308 nm for azurin to 350-353 nm for peptides lacking an ordered structure and for denatured proteins. (1) This is because of an important property of the fluorescence spectra of tryptophan residues, namely, their high sensitivity to interactions with the environment. Among extrinsic fluorescence probes, aminonaphthalene sulfonates are the most similar to tryptophan in this respect, which accounts for their wide application in protein research.(5)... 

Quenching of fluorescence of tryptophan residues, coenzyme fluoro-phores, or extrinsic probes buried in the interior of proteins by colli-sional quencher molecules diffusing through the protein matrix/7,25 27)... 

The fluorescence quantum yield of native DNA ( =4x 10 5)(,0>l,is much too small and its fluorescence lifetimes (ti 10 ps, t2 s 65 ps)(,2) are far too short to be useful for studying its rotational Brownian dynamics, so one must employ an extrinsic probe. Most commonly used is ethidium dye. Upon... 

If the soluble protein that specifically adsorbs to the fiber can be extrinsi-cally labeled, the background problem can be avoided. Of course, in vivo proteins cannot be labeled. However, it is conceivable that a protein labeled with a bulky extrinsic group (e.g., fluorescent dextrans) could be confined by a molecular sieve membrane (e.g., a dialysis membrane) within a closed volume surrounding the specifically derivatized optical fiber. When exposed to the (unlabeled) protein in the biological fluid under investigation, the membrane-clad fiber would allow some unlabeled protein to permeate in and... 

Both the physics and the chemistry of proximity to a surface can alter the excited-state lifetime and rotational motion of a fluorescent molecule. An extrinsic label attached to BSA has been found to reduce its fluorescence lifetime upon BSA adsorption to fused silica.(95) The decrease is too large to arise from the physical near-field proximity effects discussed in Section 7.3 ... 

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. ... 

In some manufacturing process analysis applications the analyte requires sample preparation (dilution, derivatization, etc.) to afford a suitable analytical method. Derivatization, emission enhancement, and other extrinsic fluorescent approaches described previously are examples of such methods. On-line methods, in particular those requiring chemical reaction, are often reserved for unique cases where other PAT techniques (e.g., UV-vis, NIR, etc.) are insufficient (e.g., very low concentrations) and real-time process control is imperative. That is, there are several complexities to address with these types of on-line solutions to realize a robust process analysis method such as post reaction cleanup, filtering of reaction byproducts, etc. Nevertheless, real-time sample preparation is achieved via an on-line sample conditioning system. These systems can also address harsh process stream conditions (flow, pressure, temperature, etc.) that are either not appropriate for the desired measurement accuracy or precision or the mechanical limitations of the inline insertion probe or flow cell. This section summarizes some of the common LIF monitoring applications across various sectors. 

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. 

Extrinsic fluorescence is used whenever the natural fluorescence of a macromolecule is inadequate for accurate fluorescence measurement. In this case, one can attach a fluorescent reporter group by using the reactive isocyanate or isothiocyanate derivatives of fluorescein or rhodamine, two intensely fluorescent molecules. One can covalently also label a protein s a- and e-amino groups with dansyl chloride (/.e., A,A-dimethylaminonaphtha-lenesulfonyl chloride). Another useful reagent is 8-ani-lino-l-naphthalenesulfonic acid (abbreviated ANS). This compound is bound noncovalently by hydrophobic interactions in aqueous solutions, ANS is only very fluorescent, but upon binding within an apolar environment, the quantum yield of ANS becomes about 100 times greater. 

FLUORESCENCE MEASUREMENTS OF LIGAND BINDING. In principle, ligand binding may either enhance or quench the intrinsic or extrinsic fluorescence of its macromolecular receptor or it may change the polarization of the fluorescence emission (see below). 

Figure 6. Enhanced ANS fluorescence attending binding of this extrinsic environmentally sensitive probe to sites on sea urchin sperm tail dynein.

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... 

The determination of fluorescence parameters of peptides requires the presence of either natural fluorescent amino acid residues (intrinsic fluorescence) or of extrinsic fluorescent probes covalently attached to the peptide at appropriate sites. The use of extrinsic fluorescent probes is mandatory in cases where the conformational or rotational behavior of a peptide is examined in the presence of proteins that contain intrinsic fluorescent amino acids. 

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