Fluorogenic assays

Two major difficulties must be considered in any assay for acid phosphatase. The enzyme is subject to surface inactivation (23, 24). Accordingly, reproducible initial hydrolytic rates are not always obtained, and the kinetic behavior should be checked in any new assay developed. Discrepancies between the amount of inorganic phosphate produced and phenol liberated from phenolic phosphates may be substantial if extensive phosphotransferase activity occurs because of phosphoryl acceptor action on the part of hydroxylic buffers or other constituents of the incubation mixture (25, 26). Fluorogenic assays have been developed with very high sensitivity (27). Reference will be made to particular assays in the discussion of the specific enzymes. 

Kerby, M., Chieng, R.-L., A fluorogenic assay using pressure-driven flow on a microchip. Electrophoresis 2001, 22(18), 3916-3923. 

MIC. The resulting concentrations were adsorbed on Amberlite XAD-2 and analyzed by desorbing and reacting with fluorescamine and subsequent fluorogenic assay. An average recovery of 101.3 percent was obtained. The excellent recoveries and the small precision statistic indicate that high relative humidities do not significantly effect sample collection and retention of MIC on XAD-2. Table X displays the results. 

Scheme 1.15 Fluorogenic assay for BVMOs in whole cells.

Klein, G. and Reyniond, J.L (1999) Enantioselective fluorogenic assay of acetate hydrolysis for detecting lipase catalytic antibodies. Helvetica Chimica Acta, 82, 400-407. 

J.L. (1998) A stereoselective fluorogenic assay for aldolases detection of an anti-selective aldolase catalytic antibody. Tetrahedron Letters, 39, 9415-9418. 

Sevestre, A., Helaine, V., Guyot, G., Martin, C. and Hecquet, L. (2003) A fluorogenic assay for transketolase from... 

Badalassi, F., Wahler, D., Mein, G., Crotti, P. and Reymond, J.L (2000) A versatile periodatehydrolytic enzymes. Angewandte Chemie-International Edition in English, 39, 4067-4070. 

P. and Reymond, J.L. (2000) A versatile periodate-coupled fluorogenic assay for... 

T. and Fabrias, G. (2007) Synthesis of a novel ceramide analogue and its use in a high-throughput fluorogenic assay for ceramidases. Chemhiochem, 8, 642-648. 

Scheme 23. An enantioselective fluorogenic assay for alcohol dehydrogenases and esterases...

Fluorescence intensity assays detect an increase or a decrease in the strength of a fluorescence emission. Based on the origin of this change in fluorescence, the two main classes are fluorogenic assays and fluorescence quench assays. 

Embree, L. McErlane, K.M. Development of a high-performance liquid chromatographic-post-column fluorogenic assay for digoxin in serum. J.Chromatogr., 1989, 496, 321-334... 

Key Words 5 fluorogenic assay fluorogenic probes TaqMan allelic discrimination high-throughput genotyping insertion/deletion polymorphisms polymerase chain reaction. 

Scope and Limitations of the TK Fluorogenic Assay The designed fluorogenic probe 1 was used as a potential donor substrate of yeast TK. Indeed, TK was able to cleave the C2-C3 bond of 1 to generate the aldehyde 4. A fluorescence signal appeared because 4 proved rather unstable and, in the presence of BSA, underwent a p-elimination to release umbeUiferone, a highly fluorescent compound (Scheme 15.6). 

Caspase activity in the cell lysate was determined by a fluorogenic assay. 50 pg of total protein was pre-incubated in buffer containing 25 mM HEPES pH7.5, 5 mM MgClz, 5 mM EDTA, 0.1 M NaCl and 5 mM DTT at 30 C for 30 min. Then 25 pM of various caspase substrates, such as Ac-YVAD-AMC for caspase-... 

Ninkovic, M. Riester, D. Wirsching, E Dietrich, R. Schwienhorst, A. Fluorogenic assay for penicillin G acylase activity. Anal. Biochem. 2001, 292, 228-233. 

Ferguson, C. Prestwich, G. Madan, D. Fluorogenic assay for lysophospholipase D activity using fluorogenic lysophospholipid derivatives as substrates, and diagnostic and screening applications. U.S. Pat. Appl. Publ. US 20100260682, 2010. 

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