Fluorescence results

Fig. 46. Scheme of optical transitions, explaining the dual fluorescence resulting from proton transfer in excited electronic state. 

The first chemists to prepare a monocyclic pyrylium salt were von Kostanecki and Rossbach who in 189 6 described the fluorescence in dilute aqueous solution of the reaction product obtained from l,3,5-triphenjdpontane-l,5-dione (benzylidene-diacetophenone) and sulfuric acid. However, they failed to isolate the compound which caused the fluorescence and did not suspect that it was a pyrylium salt. It was only in 1916-1917 that Dilthey recognized that this fluorescence resulted from 2,4,6-triphenylpyrylium (3). 

Solution of this coupled set of differential equations allows the concentrations of each of the anthracene electronic states to be determined as a function of time. In a previous publication, Nelson et al 1 used this approach to investigate the relative importance of electron transfer from the singlet and triplet states of anthracene. In this contribution, we will use these simulations to predict profiles of the anthracene ground state as a function of time so that the simulation results may be compared with the steady-state fluorescence results presented above. 

The fluorescence resulting from an idcnm.il treatment ot social standard solutions of phenylalanine (0.1-1.0 mmol 1 ) is used to calculate the test concentration. 

Cowgill pointed out that there are essentially two distinct quenching processes of tyrosine fluorescence resulting from association with the peptide bond.(3) Tyrosines affected by these mechanisms are classified in Table 1.3 as... 

Figure 6.3. Substrate-labeled fluorescent immunoassay for theophylline. (A) Effect of theophylline rabbit antiserum (O) and normal rabbit serum (A) on the enzymatic hydrolysis of 8-[3-(7-/l-galactosylcoumarin-3-carboxyamido)propyl]theophylline. (B) Effect of various concentrations of theophylline on the fluorescence resulting from enzymatic hydrolysis of 8-[3-(7-/)-galactosyl-coumarin-3-carboxyamido)propyI]theophylline. (Reprinted from Ref. 3, with permission from the American Association for Clinical Chemistry.)...

Native fluorescence of a protein is due largely to the presence of the aromatic amino acids tryptophan and tyrosine. Tryptophan has an excitation maximum at 280 nm and emits at 340 to 350 nm. The amino acid composition of the target protein is one factor that determines if the direct measurement of a protein s native fluorescence is feasible. Another consideration is the protein s conformation, which directly affects its fluorescence spectrum. As the protein changes conformation, the emission maximum shifts to another wavelength. Thus, native fluorescence may be used to monitor protein unfolding or interactions. The conformation-dependent nature of native fluorescence results in measurements specific for the protein in a buffer system or pH. Consequently, protein denatur-ation may be used to generate more reproducible fluorescence measurements. 

In one of the first experimental studies where ion radical annihilation in solution was considered as an emissive possibility, Yamamato, Nakato, and Tsubomura61 found that Y,Y,Y, Y -tetramethyl-p-phenyl-enediamine (TMPD) and pyrene when irradiated in the ultraviolet in a glass at low temperature formed Wurster s blue cation radical, pyrene anion radical, and solvated electrons. When the glass was warmed, thermoluminescence was observed. A similar emission was observed when a previously irradiated mixture of TMPD and 2-methylnaph-thalene was warmed. The emission in both instances was ascribed to charge-transfer fluorescence resulting from combination of a cation radical with an anion radical. 

Kawakubo s fluorescence results 86> for methyl- and dimethylnaphthalene solids can be similarly related to the crystal structure. Both 2-and 2,6-substituted naphthalenes retain the same close-packed layer structure as seen in naphthalene. The only effect of the methyl substitution is to increase the crystal dimension along the naphthalene long axis87 . Less is known about the crystal structures of 1- and 1,6-substituted naphthalenes, except that the 1-substituent requires a different packing pattern than naphthalene and that 1- and 1,6-substituted naphthalenes have much lower melting points than the 2-substituted naphthalenes. The absence of sandwich pairs in 2- and 2,6-substituted naphthalene crystals certainly explains the lack of excimer fluorescence in the crystal spectra. Presumably, such pairs are also absent in crystalline 1-methylnaphthylene, but they seem to be present in glassy 1-methyl-naphthalene and in 1,6-dimethylnaphthalene solid. 

Figure 13.5—Several approaches that allow recording of spectra or X-ray fluorescence results. In the figure, references are indicated by their sections in the text.

For the gases which were not quenched by NO, the fluorescence resulting from irradiation at 3660, 4070, and 4358 A was in some cases... 

Chlorophyll itself shows a short-lived red fluorescence in solution. The green plants show a delayed fluorescence of approximately the same spectrum under specific conditions in which the electron transport chain is blocked. This delayed fluorescence results from the recombination of the charges (a process well known in electroluminescence), and its kinetics are complex and the decay quite long (several seconds). 

Hie distinction of chemiluminescence from fluorescence results from two lactois ... 

Cardamone and Puri (1992) stated that ANS binding and resultant Ka measured by a Scatchard plot or Kloz plot (and to a lesser extent quantum yield) may be used as a measure of the relative surface hydrophobicity of proteins. Titration of protein solutions with increasing concentrations of the fluorescent probe can provide information on both the number and the affinity of binding sites. This may be useful in determining whether the high value of fluorescence resulted from the presence of many binding sites of only moderate hydrophilic character, or from the existence of a high-affinity site with considerable hydrophilic character. 

Table III. Colorant Binder Combinations and Fluorescing Results...

An ongoing study of sulfur materials taken from an Icelandic Norse-trading site context (55) has used this combination of simultaneous co-incident X-ray micro-fluorescence and micro-diffraction analyses. The compositional data from the fluorescence results are used to constrain the multi-phase analysis of the diffraction data. This approach reduces the requirement for accuracy in the... 

It is possible to permeabilize the outer membrane of normal cells (with detergent or alcohol) in order to allow propidium iodide to enter the nuclei. If we then treat the normal cells with RNase in order to ensure that any fluorescence results from their DNA content (without a contribution from double-stranded RNA), we find that the nuclei fluoresce red with an intensity that is more or less proportional to their DNA content. By the use of a red filter and a linear amplifier on the photomultiplier tube, we can detect that red fluorescence. The channel number of the fluorescence intensity will be proportional to the DNA content of the cells. The method is simple and takes about 10 minutes. Flow cytometric analysis of the red fluorescence from the particles in this preparation of nuclei from normal, nondividing cells will result in a histogram with a single, narrow peak (see the first histogram in Fig. 8.1) all the particles emit very nearly the same amount of red fluorescence. This supports our knowledge that all... 

Figure 19.6 illustrates how we process the two-energy data and generate the ASAXS profiles. First, we average the set of all DNA and buffer intensity profiles at each of the two energies. The averaged data are displayed in Fig. 19.6A. Note that both DNA and buffer profiles, taken at Eon, have slighdy elevated background relative to Eofr data this is due to X-ray fluorescence. Most of this fluorescence background is removed by buffer subtraction (see Fig. 19.6B). The remaining on-edge fluorescence results...

One of the most interesting features of natural fluorescence results from the fact that the fluorescence response of a given molecule depends very much on their microenvironment. This feature can be used in order to gather information about the structure of complex molecules such as polypeptides and proteins, which may integrate several fluorescent amino acids residues such as tryptophan, tyrosine, and phenylalanine. Among these, tryptophan is the one that exhibits the highest quantum yield, which makes it a good candidate to be used as an intrinsic fluorescence reporter. 

Also, upon binding of a ligand to a protein, Trp observables (intensity, polarization, and lifetime) can be altered, and so one can follow this binding with Trp fluorescence. In proteins, tryptophan fluorescence dominates. Zero or weak tyrosine and phenylalanine fluorescence results from energy transfer to tryptophan and/or neighboring amino acids. 

Although fluorescent assays are very useful in HTS, the classic issue with these assays is that they are susceptible to interference from compounds that either absorb light in the excitation or emission range of the assay (known as inner filter effects) or that are themselves fluorescent, resulting in false negatives. At typical compound screening concentrations between 1 and 10 pM, these types of artifacts can become significant. 

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