Fluorescent substrates

One of the long-standing criticisms of Bis is that the incubation period required in order to confirm a satisfactory sterilization process imposes an undesirable delay on the release of the product. This problem has been overcome, with respect to steam sterilization at least, by the use of a detection system in which a spore enzyme, a-glucosidase (reflective of spore viability), converts a non-fluorescent substrate into a fluorescent product in as little as Ihour. 

The fl-galactosidase complementation assay has also been adapted for use in mammalian cells (Rossi et al., 1997). The availability of fluorescent substrates for (3-galactosidase allows for fluorescence microscopy and FACS analysis of mammalian cells expressing the fusion proteins of interest. Therefore, similar to the mDHFR system, fl-galactosidase complementation assays may prove useful for genome-scale studies of protein-protein interactions in mammalian cells. 

Instrumental measurement of whiteness has been the subject of much research. The parameters needed for unambiguous characterisation in the assessment of whiteness and tint of fluorescent substrates have been reviewed [21]. The importance of seeking good correlation between different instruments is stressed [20]. Various trials have demonstrated that it is possible to adjust modern instruments used to measure the optical characteristics of FBA-treated samples of paper so that the results agree with a standard deviation of the order of one CIE whiteness unit [22]. 

Trubetskoy, O.V., Gibson, J.R., and Marks, B.D. 2005. Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes. J. Biomol. Screen. 10 56. 

Huang H, Fleming CD, Nishi K, Redinbo MR, Hammock BD (2005) Stereoselective hydrolysis of pyrethroid-like fluorescent substrates by human and other mammalian liver carboxylesterases. ChemRes Toxicol 18 1371-1377... 

Gentest (now BD Biosciences) was the first to develop spectrophotometric assays to study CYP inhibition [98]. These assays are based on the turnover of mildly fluorescent substrate probes to moderately fluorescent metabolites, where metabolite formation is monitored by an increase in fluorescence using a plate reader [99,100]. Problems with these methods include background interference due to low signal-to-noise ratio, chemotype-specific interference and fluorescence quenching. Aurora Biosciences (now Vertex) has designed probes that exhibit larger fluorescence... 

The use of fluorescent substrates or products permits sensitive kinetic measurement of enzyme reactions to be undertaken and although there are relatively few natural fluorescent substrates, analogues can sometimes be used (Table 8.7). Some products can be converted to fluorescent compounds and can be used in fixed time assays. 

Chemifluorescence detection relies on the conversion of a fluorogenic compound to a fluorescent product by an enzymatic reaction. The conversion of the nonfluorescent or weakly fluorescent substrates to... 

Fig. 5.7 AChE-catalyzed hydrolysis of the fluorescent substrate AMQI in volatile buffer monitored by mass spectromet. Line 1 Start of the substrate pump delivering AMQI. Line 2 Start of the enzyme pump delivering AChE. Peak 3 Injection of 0.1 pM galanthamine. Peak 4 Injection of 1.0 pM galanthamine. MS instrument Q-ToF2 (Waters) equipped with a Waters Z-spray electrospray (ESI) source, (a) Mass chromatogram of m/z 288 (galanthamine) (b) mass chromatogram of m/z 104...

To make these substrates suitable for biological assays, the introduction of functional groups that can be traced with the proper analytical techniques is essential. The use of radio-, fluorescent-, and biotin-labeled lipidated peptides has been reported. The synthesis of fluorescent substrates is chemically straightforward and allows for production of larger quantities than the enzymatic synthesis used for radiolabeled peptides and is thus preferred over the use of radioactive compounds. [1 21] Common fluorescent probes can be introduced by conjugation to a free functional group present in the peptide. The fluorescent moiety is... 

This enzyme is deficient in Niemann-Pick disease type A/ (Table 4.4.1, Fig. 4.4.1). The assay with radioactive natural substrate is based on the method described by Wenger [59]. The assay with fluorescent substrate is based on the method described by van Diggelen et al. [54]. These assays have not yet been validated in the authors laboratory for use with dried blood spots. For this application the reader is referred to Chamoles et al. [9]. 

R. Griesser Assessment of Whiteness and Tint of Fluorescent Substrates with Good Inter-Instrument Correlation , ColorRes. Appl. 19 (1994) no. 6, 446-460. 

M Meldal, K Breddam. Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin. Anal Biochem 195 141-147, 1991. 

Chloramphenicol acetyl transferase (CAT). This bacterial enzyme was the first reporter protein used for studying the transcriptional activity of eukaryotic regulatory sequences (Gorman et al., 1982). CAT inactivates chloramphenicol, an inhibitor of prokaryotic protein synthesis, by converting it to the mono- or di-acetylated species. Measurement of CAT activity requires a 14C-radiolabeled chloramphenicol or acetyl-CoA and, therefore, an additional step is neccessary to separate the radio-labeled reactant from the product. Novel detection methods based on fluorescent substrates or ELISA assays, which do not use radiolabeled reagents, have been described more recently (Bullock and Gorman, 2000). 

The most critical and demanding requirement is that the fluorescent product generated by an enzyme is retained quantitatively within the cell. A number of ingenious strategies for product retention have been developed including intracellular precipitation (the Enzyme-Linked Fluorescence or ELF family of fluorescent substrates), attachment of lipophilic anchors, substrates that yield insoluble chromophores, and retention via formation of complexes with intracellular proteins (for examples, see footnote 3 and Zlokamik et al., 1998). 

Schalk, M., Batard, Y., Seyer, A., Nedelkina, S., Durst, F., and Werck-Reichhart, D., Design of fluorescent substrates and potent inhibitors of CYP73As, P450s that catalyze 4-hydroxylation of cinnamic acid in higher plants, Biochemistry, 36, 15253-15261, 1997. 

Watanabe, T. et al. 1998. Synthesis of fluorescent substrates for protein tyrosine phosphatase assays. Bioorg. Med. Chem. Lett. 8, 1301-1302. 

Of particular relevance to the current section, //-lactamase is a bacterial enzyme that normally cleaves //-lactam antibiotics such as the penicillins and cephalosporins. CCF4 is a FRET-based fluorescent substrate of //-lactamase that contains two fluorophores, coumarin and fluorescein, that are linked by a cephalosporin backbone. When CCF4 is intact, the coumarin and fluorescein are in close proximity and excitation of coumarin by light at wavelength 409 nm results in efficient transfer of energy to fluorescein, leading to emission of green fluorescence (peak at 520 nm). When the... 

Shaughnessy et al. (155) reported the rapid, sequential screening of three phosphine ligand libraries L22-L24 in a Heck coupling reaction involving a fluorescent substrate 9.92 and the supported aryl bromide 9.93, whose structure and synthesis are reported in Fig. 9.36. 

These results were slightly better than those obtained employing f-butyl acrylate to produce 9.97 (GC determination after cleavage). When 9.93 was treated with fluorescent substrate 9.92 without catalyst, the washed resin beads showed no fluorescence, proving the overall reliability of the fluorescent screening and the absence of interferences. 

---