Chromophoric markers

Chromophoric markers were afforded by the synthesis, through Bt technology, of azo dye-labeled terpenes, sugars, and steroids (2009S1708), and fluorescent detection of biological thiols such as L-cysteine, glutathione, and L-penicillamine was achieved by reaction with N-coumarin-3-carbonyl benzotriazoles (2011S1494). 

Quinolone antibiotics are now used for various diseases other than bacterial infections, and tagging with a chromophoric marker may help to understand the mode of action. Thus coumarin-3-ylcarbonyl benzotriazoles... 

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

Figure 4 presents an example of rapid pKa measurement using a pressure-assisted system in combination with a photodiode array (PDA) detector. The migration time of DMSO (EOF marker) was measured at 220 nm, whereas the migration time of the analyte, naphazoline, was measured at 270 nm. The CE run time as well as data analysis time was drastically reduced. Consequently, this system allows the analysis of more than 96 compounds in one day. The limitation of this method is the application to drugs without UV chromophore at more than 250 nm. In some cases, it was effective to remove DMSO by evaporation under vacuum followed by the addition of methanol or acetonitrile as a neutral marker. 

By applying Fermi s golden rule, Forster derived a very important relation between the critical transfer distance R0 and experimentally accessible spectral quantities (Equation 2.35),° 67,68 namely the luminescence quantum yield of the donor in the absence of acceptor A, orientation factor, k, the average refractive index of the medium in the region of spectral overlap, n, and the spectral overlap integral, J. The quantities J and k will be defined below. Equation 2.35 yields remarkably consistent values for the distance between donor and acceptor chromophores D and A, when this distance is known. FRET is, therefore, widely applied to determine the distance between markers D and A that are attached to biopolymers, for example, whose tertiary structure is not known and thus... 

The GFP from the jellyfish Aequorea victoria, although not an enzyme, has become widely used as a marker for gene expression and localization. Although the fluorophore of GFP is not technically a protein-derived cofactor, it is a protein-derived fluorophore. This is another example of posttranslational modifications, which endow amino acid residues with a new function. In this case, the new function is not one which assists in catalysis. Instead, the results of these posttranslational modifications create new fluorescent properties, which serve a different biological function. As with most of the protein-derived cofactors discussed earlier, the presence and identity of the fluorophore is not evident from the amino acid sequence of the protein. The structure of the GFP fluorophore and mechanism of its biosynthesis were deduced from structural analyses. The X-ray crystal structure of GFP revealed that the covalently bound fluorescent chromophore is derived from three adjacent amino acids, serine-tyrosine-glycine on the polypeptide chain (Figure 13). ... 

FIGURE 3.16 Dependence of the absorbance of PMMA films containing (left) diarylethene and (right) spiropyran on the angle, 4, between UV irradiation and probe beam polarizations. The normalized absorbance is defined as (Abs - Abs n)/(Abs - Abs ,, where Abs j and Abs , are the maximum and minimum absorbances, respectively. The H and signs stand for the directions parallel and perpendicular to the UV polarization, respectively. The markers are experimental data points, and the full and dashed lines are cos 4 theoretical fits. The analysis wavelengths are indicated. Note the apparent n/2 angle-shift between the UV and visible orientational distributions of the diarylethene chromophore. After reference 29, redravm by permission. 

By identifying the functional groups present in a molecule, a molecular formula provides insight into numerous properties. These include the molecule s water and lipid solubility, the presence of fracture points for gas chromatography (GC) determinations, sources of potential markers such as chromophores, an indication as to the molecule s UV absorbance, whether derivatization is likely to be required when quantifying residues of the compound, and the form of ionization such as protonated ions or adduct ions when using electrospray ionization. The molecular formulas of the antimicrobial agents described in this chapter are shown in Tables 1.2-1.15. 

Fluorescent proteins are a class of proteins that have a distinguishing property of forming their chromophore without involvement of any additional cofactors and ferments (autocatalytic reaction), except for molecular oxygen. In recent years, FPs have gained enormous popularity as genetically encoded fluorescence markers that enable to visualize a broad range of biological processes in cells and tissues. The most popular for practical applications are FPs whose fluorescence is shifted to the red (red FPs) and whose molecules are monomers (Piatkevich et al, 2010). The mRFFl protein possesses these properties (Cambel et al., 2002), which made it an object of the research. 

Figure 2.8 Models of the QD-b-PE conjugate structure/conformation. (A) b-PE is parallel to the QD surface, and (B) b-PE is fully extended away from the QD. The central QD with a radius of 29 A shown in blue is surrounded by a crimson shell of 25 A thickness representing the DHLA-PEG-biotin. The intermediary streptavidin (SA) protein is shown in yellow with biotin binding sites highlighted in purple. The b-PE ring structure is shown in white, with the multiple chromophores highlighted in red. The inner concentric white circle corresponds to the predicted 53 A Fdrster distance (i ) for the 540 nm QD-b-PE assembly. The second outer white circle is a visual distance marker set at 95 A from the QD center and represents the closest approach of the b-PE to the QD. (Reproduced with permission from I. L. Medintz, T. Pons, K. Susumu, K. Boeneman, A. M. Dennis, D. Farrell, J. R. Deschamps, J. S. Melinger, G. Bao and H. Mattoussi,/. Phys. Chem. C, 2009,113,18552. Copyright (2009) American Chemical Society.) ...

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