Extrinsic probes

Extrinsic probes must be used when the system under study has no useffil intrinsic reporter groups or a reaction produces no fluorescence change from intrinsic reporter groups. In such cases one may use an appropriate fluorescent group which is added to the system to report the reaction. Extrinsic reporter groups come in many forms such as non ovalentIy and covalently bound fluorescent labels. Many of the latter can be covalently attached to the protein of interest at a specific site. 

Chemical modification reagents for labelling reactive amino acid side chains (predominantly cysteine or lysine) have been available for many years and used to probe for residues dose to or in the active site of enzymes. Although fluorescent derivative,s of these reagents have been used less frequently, they occasionally reward persistent experimentation by offering more detailed 

The presence of fluorescent groups such as pyrene, fluorescein, eosin etc. may also give characteristics to the protein or hgand that were previously not present. For example, such groups on ligands generally decrease water solubility and sometimes cause the compound to adsorb to the walls of plastic containers and cuvettes. 

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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). 

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

Expression system VFPs intrinsic probes others primary or secondary extrinsic probes Target aa Target kDa Target aa Covalent Fluorogenic Arbitrary... 

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... 

A wide range of techniques exist, which each of them provide their own advantages. It is relevant to be observant towards how much protein is needed for the investigation and which intrinsic or extrinsic probe that is being monitored. 

The goal of these studies is to measure the association constants of the LTF-LCA and STF-LCA complexes. LCA is given as a fluorescein-LCA complex (from Sigma). The extinction coefficient of the complex is 132.996 mM-1 cm-1 at 495 nm. Two molecules of fluorescein are bound to one molecule of LCA. Binding of the STF or LTF to LCA-fluorescein complex induces a variation in the fluorescence intensity of the extrinsic probe. LTF and STF concentrations can be determined spectrophotometrically at 280 nm with an absorption E1 % = 14.3 and 14.0, respectively. 

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. 

The fluorescence lifetime and polarization of an extrinsic probe bound tightly to a spherical protein have been measured at different oxygen concentrations. The following data are obtained ... 

The usual experimental technique is to observe the fluorescence of a probe molecule incorporated into the system under investigation and make the assumption that the motion of the probe reflects that of its environment. Probe molecules may be divided into two cathodes intrinsic probes (such as fluorescent amino acid residues naturally occurring in a protein under investigation) and extrinsic probes either bound chemically to the macromolecule or simply dispersed in the stem. The criteria that need to be applied when selecting suitable candidates for probe molecules have been discussed by Stryer Ideally the probe should... 

Fluorescence In general orientational order ((S2) Generally, extrinsic probe... 

The discussion below indicates the variety of options for studying monolayers and related systems, which underlies the relative popularity of fluorescence techniques. At the same time the eaveat must be repeated that extrinsic probes have to be introduced in the monolayer. This insertion does affect the structure and the question remains by how much the perturbed layer differs from the original one. The argument that the disturbance is ineonsequential if the mole fraction of the probe is low does not hold because by fluoreseence measurements typically only the modified part is seen. 

There are only a few examples of the use of extrinsic probes to investigate accessibility of quenchers to protein-complexed excited states. Polycyclic aromatic hydrocarbons were used by Geacintov et al. to probe bovine serum albumin (BSA) [205]. The accessibility of oxygen to the probe molecules was... 

The second point is a fundamental property of the probe approach this issue is being addressed by designing probes that resemble as much as possible the native DNA base pairs. One can also make the argument that the use of an extrinsic probe such as an intercalator, which binds to DNA by insertion of a planar portion of itself into the base stack, is afunctional probe in the sense that the ability of unusual DNA structures/differentially flexible DNA sequences to bind to any molecule is a measurable parameter tied to the DNA function of being read, packaged, and unpackaged by external molecules. The principal photophysical methods to study DNA sequence-directed structure and dynamics are discussed in the following sections. 

The decrease of the fluorescence intensity of the Trp residues in presence of calcofluor white is the result of an energy transfer Forster type from the Trp residues to the extrinsic probe. The efficiency of this energy transfer depends on three parameters, the distance R between the donor (Trp residues) and the acceptor (calcofluor white), the spectral overlap between the fluorescence spectrum of the donor and the absorption spectrum of the acceptor and the orientation factor k which gives an indication on how the dipoles of acceptor in the fundamental state and donor in the excited state are aligned. 

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