Phenol fluorescence efficiency

If one of the substances has a known fluorescence efficiency, the value of the other is then simply obtained. Convenient standard solutions are rhodamine B in ethanol with fluorescence in the yellow and efficiency 0.69, quinine bisulfate in 0.1 N sulfuric acid with fluorescence in the blue and efficiency 0.55. anthracene in ethanol with fluorescence in the violet and efficiency 0.27 in the ultraviolet region, naphthalene ( = 0.19), phenol (0 = 0.19), or benzene (0 = 0.042) can be used. With the last four compounds the solution must be deaerated by passing a current of nitrogen before measurement. To minimize the effect of errors in the spectral sensitivity curve it is desirable to use as the standard a solution... 

Thus, knowledge of the transition moment direction of a phenol band could help in interpreting the fluorescence spectrum of a tyrosine chromophore in a protein in terms of orientation and dynamics. The absorption spectrnm of the first excited state of phenol was observed around 275 nm with a fluorescence peak aronnd 298 nm in water. The tyrosine absorption was reported at 277 nm and the finorescence near 303 nm. Fluorescent efficiency is about 0.21 for both molecules. The fluorescent shift of phenol between protic and aprotic solvents is small, compared to indole, a model for tryptophan-based protein, due to the larger gap between its first and second excited states, which resnlts in negligible coupling . ... 

Dns-amides are stable to acid hydrolysis, and fairly stable even to hydrolysis with bases. However, transfer of dansyl groups to other bases occurs in mild alkali this reaction must be borne in mind when hydrolysis is carried out in alkaline media. In Table 1 some data concerning the stability of Dns derivatives to hydrolysis are summarized. Phenolic esters are much less stable than the sulphonamides, therefore it is possible to split off the 0-E)ns from 0,N-bis-Dns-aminophenok with melhanolic KOH 128]. This is of analytical interest because the N-Dns-derivatives of tyramine, synephrine etc. have much higher fluorescence efficiencies than the corresponding 0,N-bis-Dns-deiivatives. The derivatives of imidazoles and esters are unstable, both in acid and alkaline media. Dns-deiivatives of imidazoles, can, however, be selectively cleaved with formic acid [29]. This feature of Dns-imidazole derivatives can be exploited in the deler-mination of histamine and methylhistamines [30]. 

The quantum yields of amine and amino acid Dns derivatives are of the same order of magnitude (Table 2). Phenol and imidazole derivatives, however, have much lower fluorescence efficiencies. In Table 3, the spectral characteristics of the Dns derivatives of an amine, an aminophenol and a phenol are compared. Interestingly, the aminophenol derivative (0,N-bis-Dns-p-tyramine) shows much lower fluorescence efficiency than the analogous amine derivative although it bears two apparently independent fluorophores, and possesses... 

We have also investigated other oxalate esters as a potential means to improve the efficiency. The most commonly used oxalates are the 2,4,6-trichlorophenyl (TCPO) and 2,4-dinitrophenyl (DNPO) oxalates. Both have severe drawbacks namely, their low solubility in aqueous and mixed aqueous solvents and quenching of the acceptor fluorescence. To achieve better solubility and avoid the quenching features of the esters and their phenolic products, we turned to difluorophenyl oxalate (DFPO) derivatives 5 and 6 (Figure 14). Both the 2,4- and the 2,6-difluoro esters were readily synthesized and were shown to be active precursors to DPA chemiluminescence. In fact, the overall efficiency of the 2,6-difluorophenyl oxalate 5 is higher than for TCPO in the chemical excitation of DPA under the conditions outlined earlier. Several other symmetrical and unsymmet-rical esters were also synthesized, but all were less efficient than either TCPO or 2,6-DFPO (Figure 14). 

The synthesized CPMV-alkyne 42 was subjected to the CuAAC reaction with 38. Due to the strong fluorescence of the cycloaddition product 43 as low as 0.5 nM, it could be detected without the interference of starting materials. TMV was initially subjected to an electrophilic substitution reaction at the ortho-position of the phenol ring of tyrosine-139 residues with diazonium salts to insert the alkyne functionality, giving derivative 44 [100]. The sequential CuAAC reaction was achieved with greatest efficiency yielding compound 45, and it was found that the TMV remained intact and stable throughout the reaction. 

INTRINSIC AND EXTRINSIC FLUORESCENCE. Intrinsic fluorescence refers to the fluorescence of the macromolecule itself, and in the case of proteins this typically involves emission from tyrosinyl and tryptopha-nyl residues, with the latter dominating if excitation is carried out at 280 nm. The distance for tyrosine-to-tryp-tophan resonance energy transfer is approximately 14 A, suggesting that this mode of tyrosine fluorescence quenching should occur efficiently in most proteins. Moreover, tyrosine fluorescence is quenched whenever nearby bases (such as carboxylate anions) accept the phenolic proton of tyrosine during the excited state lifetime. To examine tryptophan fluorescence only, one typically excites at 295 nm, where tyrosine weakly absorbs. [Note While the phenolate ion of tyrosine absorbs around 293 nm, its high pXa of 10-11 in proteins typically renders its concentration too low to be of practical concern.] The tryptophan emission is maximal at 340-350 nm, depending on the local environment around this intrinsic fluorophore. 

On the other hand, when CPO was modified with citraconic, maleic, and phthalic anhydrides, catalytic efficiencies for phenol oxidation were higher compared to native CPO. Those modifications also improved their thermostability by 1-2-fold and tolerance to dimethylformamide (DMF). Circular dichroism studies showed no changes in the secondary structure of CPO, but changes in the environment of the aromatic residues were demonstrated by fluorescence studies [67]. Those findings are in agreement with those obtained for HRP modification using the same compounds. Modification with citraconic, maleic, and phthalic anhydrides represents a simple and powerful method to enhance catalytic properties, thermostability, and organic solvents tolerance of hemeperoxidases. 

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