Protease fluorescence assays

Hennig A, Florea M, Roth D, Enderle T, Nau WM (2007) Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore. Anal Biochem 360 255-265... 

Recently, a SAMDI-MS assay was described by means of which endogenous caspase protease activities in cell lysates can be determined [26], Similar to the assay used to determine anthrax lethal factor inhibitors, peptide substrate SAMs for either caspase-3 or -8 were treated with cell lysates. In contrast to fluorescence assays, also longer peptide substrates could be used, thus enabling a better resolution of the two caspase activities. 

QM Wang, RB Johnson, JD Cohen, GT Voy, JM Richardson, LN Jungheim. Development of a continuous fluorescence assay for rhinovirus-14 3C protease using synthetic peptides. Antiviral Chem Chemother 8 303-310, 1997. 

Mao, S.S. et al. 2008. A time-resolved, internally quenched fluorescence assay to characterize inhibition of hepatitis C virus nonstructural protein 3-4A protease at low enzyme concentrations. Anal Biochem. 373, 1-8. 

Inhibitor kinetics The IC50 determinations described herein provide a simple method of comparing protease inhibitors. However, it may be desirable to characterize the interactions of inhibitors with the enzyme in more detail. Both the chromogenic and fluorescence assays can be read in kinetics mode on the machines described to give real-time rate measurements for use in determining kinetic parameters. 

Mao, S. S. Lewis, S. D. Time-resolved fluorescence assay for HCV NS3 protease using europium-labeled substrate containing quenching group, and drug screening use. PCT Int. Appl. WO 2006102087, 2006 Chem. Abstr. 2006, 145, 371745. 

Proteases are enzymes that break peptide bonds in proteins. As such they lend themselves to a variety of homogeneous assay techniques. Most employ labeling both ends of the substrate with a different tag, and looking for the appearance (disappearance) of the signal generated in the intact substrate (product). As an example, for a fluorescence quench assay, the N-terminal of a peptide is labeled with DNP and the C-terminal with MCA. As such, the peptide is fluorescently silent since the fluorescence from DNP is quenched by absorption by the MCA. Another very popular donor/acceptor pair is EDANS 5-[(2-aminoethyl)amino] naphthalene-1-sulfonic acid and DABCYL 4-(4-dimethylaminophenylazo)benzoic acid) (a sulfonyl derivative (DABSYL) [27], Upon peptide cleavage, the two products diffuse, and due to a lack of proximity, the fluorescence increases. 

Kohl, T., Heinze, K. G., Kuhlemann, R., Koltermann, A. and Schwille, P. (2002). A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins. Proc. Natl Acad. Sci. USA 99, 12161-6. 

Doering K, Meder G, Hinnenberger M, Woelcke J, Mayr LM, Hassiepen U (2009) A fluorescence lifetime-based assay for protease inhibitor profiling on human kallikrein 7. J Biomol Screen 14 1-9... 

Jones LJ, Upson RH, Haugland RP, Panchuk-Voloshina N, Zhou M, Haugland RP. Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement. Anal Biochem 1997 251(2) 144-152. 

Meldal et al. developed a novel protease assay based on the long-range resonance energy transfer (FRET) [25] fluorescence quenched (EQ) pair 3-nitrotyro-sine/2-aminobenzoic acid. This served to characterize enzyme specificity by direct visual inspection of the resin beads (33). 

In a different approach, fluorescence-based DNA microarrays are utilized (88). In a model study, chiral amino acids were used. Mixtures of a racemic amino acid are first subjected to acylation at the amino function with formation of A-Boc protected derivatives. The samples are then covalently attached to amine-functionalized glass slides in a spatially arrayed manner (Fig. 10). In a second step, the uncoupled surface amino functions are acylated exhaustively. The third step involves complete deprotection to afford the free amino function of the amino acid. Finally, in a fourth step, two pseudo-Qn nX. om.Qx c fluorescent probes are attached to the free amino groups on the surface of the array. An appreciable degree of kinetic resolution in the process of amide coupling is a requirement for the success of the ee assay (Horeau s principle). In the present case, the ee values are accessible by measuring the ratio of the relevant fluorescent intensities. About 8000 ee determinations are possible per day, precision amounting to +10% of the actual value ((S(S). Although it was not explicitly demonstrated that this ee assay can be used to evaluate enzymes (e.g., proteases), this should in fact be possible. So far this approach has not been extended to other types of substrates. 

---