Extrinsic fluorescent probes

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... 

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)... 

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. 

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. 

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]

Extrinsic fluorescent probes can provide additional useful information about the integ-... 

In other media like micelles, cyclodextrin, binary solvent mixtures, and proteins (47-55), lifetime distributions are routinely used to model the decay kinetics. In all of these cases the distribution is a result of the (intrinsic or extrinsic) fluorescent probe distributing simultaneously in an ensemble of different local environments. For example, in the case of the cyclodextrin work from our laboratory (53-55), the observed lifetime distribution is a result of an ensemble of 1 1 inclusion complexes forming and coexisting. These complexes are such that the fluorescent probe is located simultaneously in an array of environments (polarities, etc.) in, near, and within the cyclodextrin cavity, which manifest themselves in a distribution of excited-state lifetimes (53-55). In the present study our experimental results argue for a unimodal lifetime distribution for PRODAN in pure CF3H. The question then becomes, how can a lifetime distribution be manifest in a pure solvent ... 

Taniguchi, K Tosa, H Suzuki, K Kamo, Y. (1988). Microenvironment of two different extrinsic fluorescence probes in Na+,K+-ATPase changes out of phase during sequential appearance of reaction intermediates. J. Biol. Chem. 263,12943-12947. 

Comprehensive reviews (Kl, Ul) of the active sites of cholinesterase both postulated the presence not only of an esteratic site for butyrylcholinesterase but also of an anionic site. Additionally, in the region of the anionic site, there are two hydrophobic areas, one directly surrounding the anionic group and the second located at some distance from it (Kl). The presence of hydrophobic areas has been established (B32, C3, H29, H45, MIO) by the use of fluorescent probes with spectral responses which reflect the environment of the probe. Such probes can be used to monitor changes in the conformations of enzymes and can be designed to be active-site-directed, competitive inhibitors (H30). Aspects of the spectroscopy of intrinsic and extrinsic fluorescent probes have been reported (C3). 

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). 

I. Tasaki, Energy Transduction in the Nerve Membrane and Studies of Excitation Processes with Extrinsic Fluorescent Probes, Ann. NY Acad. Sci. Ill, 247-267 (1974). 

The application of fluorescence measurements to a large variety of chemical, biochemical, biological and physical problems is extensive. The majority of fluorescence applications involve the use of extrinsic fluorescence probes. These are chromophoric molecules that are attached to or adsorbed onto another molecule and their fluorescence is measured. These molecules probe the properties of the substances to which they are attached. The catalogue and manual published by Molecular Probes (9) is an excellent collection of a large number of different examples of the use of fluorescence, especially as they relate to bio-... 

In addition to the complicated response of the fluorophore to various stimuli, one more aspect should be home in mind. Only a small number of systems contain intrinsic fluorophores and are inherently fluorescent. Such systems (e.g., tryptophan-containing proteins) can be studied directly and reliable information on the positions, mobility, and accessibility of tryptophan residues for different molecules can be relatively easily obtained. In a majority of cases, a successful fluorescence study requires the addition of a low content of an extrinsic fluorescent probe, which modifies not only optical but also other properties of the studied system. An extrinsic probe feels only the effect of its immediate microenvironment, which has undoubtedly been altered by its insertion. Even though the change in the system is negligible at a macroscopic level, most fluorescence methods report the behavior of the tiny perturbed part of the system. Therefore, the extent and nature of possible perturbation of the system must also be investigated to enable description of the behavior of the unperturbed system. 

As nonconjugated and nonaromatic polymers are nonfluorescent, studies of their phase behavior using fluorescence spectroscopy require the use of extrinsic fluorescent labels [38-40]. These can be either selectively dissolved into the polymer phases [41] or, most commonly, attached covalently to the polymer chains [42-47]. A criticism that is often made of using extrinsic fluorescent probes is the possible local perturbation induced by the probe itself on the nanoenvironment to be probed. In order to minimize such perturbation, the size and shape of the probe should be chosen so as to cause the minimum possible perturbation on the probed region. 

A variety of extrinsic fluorophores can be attached to proteins to serve as fluorescence probes. These can be selected to maximize sensitivity and to avoid contamination (i.e., by moving to longer absorption and emission wavelengths) from other absorbing components. With both intrinsic and extrinsic fluorescence probes, the method focuses only on these probes sites, which might be as few as a single site on a protein. 

Ooi, A. Yano, R Okagaki, T. Thermal stability of carp G-actin monitored hy loss of polymerization activity using an extrinsic fluorescent probe. Fish. Sci. 2008, 74,193-199. 

The use of an extrinsic fluorescence probe bound to a protein is not recommended for the study of the folding process since the presence of the probe may modify the pathway of folding. 

Antibodies were labeled with an extrinsic fluorescent probe for the generation of a fluorescent signal during biosensor detection. The fluorescent probe used was tretramethyl-rhodamine-5-isothiocyanate (TRITC) purchased from Molecular Probes, Inc., Eugene, Oregon and stored in a desiccator at less than 0 °C prior to use. 

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