Fluorophores extrinsic probes

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 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. 

In the static quenching, one uses intrinsic or extrinsic fluorophore to probe the interaction between two macromolecules. The fluorophore should be bound to one of the two proteins only (case of the extrinsic fluorophores) or should be part of it (case of the intrinsic fluorophores) if we want to perform such experiments. Also, binding parameters of the fluorophore-macromolecule complex can be determined by following fluorescence modification of the fluorophore observables. 

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. 

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. 

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 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... 

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. 

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). 

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. 

In instances where nature has not provided an appropriate fluorophore, one can often add an extrinsic label. The earliest probes include dansyl chloride [1] and ANS (Fig. 3). Dansyl chloride can be covalently attached to macromolecules by reaction with amino groups. ANS often binds spontaneously but non-covalently to proteins and membranes, probably by hydrophobic and electrostatic interactions. The emission of both molecules is sensitive to the polarity of the surrounding environment. ANS is nearly non-fluorescent in water, but fluoresces strongly upon association with serum albumin, immunoglobulins and other proteins. A wide variety of covalent and non-covalent probes are available [2,3]. 

Thousands of fluorescent probes are known, uid it is not practical to describe them all. This ch rto contains an overview of the various types of fluorO ores, dieir spec-tnd propoties, and their applications. Fluoro tores can be Ixoadly divided into two main classes, intrinsic and extrin-ric. Intrinsic fluorophores are those which occur naturally. These include the aromatic amino acids, NADH, flavins, and derivatives of pyridoxal and chlorophyll. Extrinsic fluorophores are added to the sample to provide fluorescence when none exists or to change the spectral properties of die sample. Extrinsic fluorophores include dansyl chloride, fluorescein, rhodamine. and numerous odier substances. 

There is presently interest in the use of fluorescence from tissues, either from the intrinsic fluorophores or fiom extrinsically added probes. Much of the fluorescence... 

Solvent polarity and the local environment have profound effects on the emission spectra of polar fluorophores. These effects are the origin of the Stokes shift, which is one of the earliest observations in fluorescence. Emission spectra are easily measured, and as a result, there are num ous publications on emission spectra of fluoropho-res in different solvents and when bound to proteins, membranes, and nucleic acids. One common use of solvent effects is to determine the polarity of the probe binding site on the macromolecule. This is accomplished by comparison of the emission Spectra and/or quantum yields of the fluorophore when it is bound to the macromolecule and when it is dissolved in solvents of different polarity. However, there are many additional instances where solvent effects are used. Suppose a fluorescent ligand binds to a protein. Binding is usually accompanied by a spectral shift due to the different environment for the bound ligand. Alternatively, the ligand may induce a spectral shift in the intrinsic or extrinsic protein fluorescence. Additionally, fluorophores often display spectral shifts when they bind to membranes. 

An anisotropy decay more typical of proteins is shown by phospholipase A2. This enzyme catalyzes the hydrolysis of phospholipids and is active mostly when located at a lipid-water interface. Phospholipase Ai has a single tryptophan resichie (trp-3), which serves as the intrinsic probe. The anisotropy decay is clearly more complex than a single exponential. At long times, the correlation time is 6.5 ns, consistent with overall rotational diffusion. However, in comparison with LADH, there is a dramatic decrease in anisotropy at short times (Figure 11.14), The correlation time of the fast component is less than 50 ps, and this motion accounts for one-(hird of the total anisotropy. Independent tryptophan motions have been observed in a large number of proteins " and have been predicted by molecular dynamics calculations. Fast components in the anisotropy decay are also observed for labeled pro teins. Hence, segmental motions of intrinsic and extrinsic fluorophores appear to be a common feature of proteins. 

The required accuracy for < ) depends, of course, on the purpose of the measurement.) Protein motions with < ) values greater than this will probably require that a longer-lived extrinsic fluorophore (e.g. pyrene) be attached to the molecule. Motions on the microsecond to second timescales can be monitored using phosphorescent probes (see Section 4.2). 

Fluorescent probes are relatively small molecules that are used to label biomolecules such as proteins, antibodies and nucleic acids. They contain functional groups and specific physical and chemical characteristics that confer suitability for their use as detection moieties. To date, thousands of fluorescent probes are known each with varying spectral properties. Fluorophores may be intrinsic or extrinsic in nature. Intrinsic fluorophores are naturally occurring whereas extrinsic fluorophores are added to generate a fluorescence signal to facilitate measurement of a specific target molecule. Fluorescent labels have provided excellent sensitivity for a range of assay systems that can be applied to the determination of almost any analyte. 

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. 

---