Fluorescent equilibrium probes

Fluorescent Equilibrium Probes. Himel and co-workers (23, 24, 25) have synthesized active-site-directed fluorescent equihbrium probes which are competitive inhibitors of the active site of cholinesterase enzymes. The fluorescence intensity of the probe-enzyme complex is decreased by any foreign molecule (insecticide) which competes with the equilibrium fluorescent probe for the active site of the enzyme or which changes the equilibrium dynamics by exo area reaction with the enzyme. This highly specific and sensitive spectroscopic method is being developed as an analytical method for insecticides (26). 

If one wants to understand why such changes occur, one can look at a few of the basic equilibrium properties of such complexes. Figure 1 illustrates the trends which occur when a sample is titrated with copper, monitoring three different parameters. The black dots indicate the relative amount of bound copper as indicated by free copper ions sensed with an ion-selective electrode (Xc of left ordinate). The triangles represent the change of the absorbance of the solution at 465 nm (right ordinate). The curve with the open circles is the relative quenching of the fulvic acid fluorescence (Q of left ordinate). We see that we are able to probe several different types of sites with different types of probes for this multidentate system. 

It should be emphasized that best design (each application corresponding to a particular design), proper choice, and correct use of fluorescent probes require a thorough knowledge of the basic phenomena involved in ion recognition medium effect on complexation equilibrium, fundamental photophysical processes, and possible changes from other causes than complexation. 

The fluorescent probe 2,6-TNS and other similar aminonaphthalene derivatives (1,8-ANS, DNS) were considered to be indicators of the polarity of protein molecules, and they were assumed to be bound only to hydrophobic sites on the protein surface. The detection of considerable spectral shifts with red-edge excitation has shown that the reason for the observed short-wavelength location of the spectra of these probes when complexed to proteins is not the hydrophobicity of their environment (or, at least, not only this) but the absence of dipole-relaxational equilibrium on the nanosecond time scale. Therefore, liquid solvents with different polarities cannot be considered to simulate the environment of fluorescent probes in proteins. 

The interaction of ACh with the Torpedo nAChR. The data shown compare the equilibrium binding parameters obtained from fluorescence studies using covalently attached fluorescent probes and those obtained from radiolabelled [ H]ACh binding studies or functional measurements of cation flux. These data support a model in which the Torpedo nAChR carries sites of different affinities for ACh. We have previously suggested that occupancy of the lower affinity sites leads to channel activation whereas the higher affinity sites may play a role in desensitization processes... 

Schneider et al. [63] investigated the photochemistry of the spiro-oxazine merocyanines pumping and probing at 570 nm in acetonitrile. The found that the solution bleached within the <5-psec pulse duration. The bleached state recovered with at least a biexponential behavior, and from their fluorescence decay measurements, three exponentials were required to fit the decay. They attribute these findings to the possibility of three merocyanine isomers that are in equilibrium. Their compounds feature geminal ethyl groups on the indoline moieties and this may influence the system as compared to NOSIl. 

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 two-state transition is usually identified by all spectroscopic probes changing simultaneously as the equilibrium changes. The far ultraviolet circular dichroism signals, which are a measure of secondary structure, should change in parallel with the near ultraviolet, which are a measure of tertiary structure. Fluorescence and near ultraviolet absorbance spectra also probe tertiary structure and should change in parallel with each other and the circular dichroism spectra. Ideally, there should be isosbestic or isodichroic points where spectra converge. 

Evidence from protein fluorescence indicates that the equilibrium conformation of the central regions of the polypeptide backbone, which are amenable to probing by emission from the tryptophan residues, is not changed overall to any major extent in the Pr -< Pfr transformation. The conformational reorganization of the protein, which is induced by the Z - E isomerization (which in turn is presumed to represent the primary photoreaction of the overall Pr -> Pfr transformation), appears to be confined mostly to the domain housing the bilatriene chromophore. 

Dynamic processes at thermodynamic equilibrium that occur within a time range from sub-microseconds to seconds can be probed without the imposition of a transient disturbance by optical intensity fluctuation spectroscopy. As such, dynamic light scattering (DLS) [155] measures the fluctuation of quasielastic scattering intensity and fluorescence correlation spectroscopy (FCS) [156-158] measures concentration fluctuations of specific fluorescent molecules... 

Fluorescence Of Monolayers Containing Pyrene-Labeled Probes. A fluorescence probe method was also used as a complementary technique to study the thermodynamics of SA film formation. Mixed monolayers containing the fluorescence probe pyrene hexadecanoic acid, Py-C16, in host fatty acids of different lengths were prepared by adsoiption from solutions containing mostly the host fatty acid and a small fraction of Py-C16 (approximately 1 to 5 mol %). All monolayers were prepared under equilibrium adsoiption conditions. For fluorescence measurements only A1 substrate was used because when glass is used an impurity fluorescence from glass interferes with the pyrene fluorescence. 

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