Design and application of a fluorogenic assay for monitoring inflammatory caspase activity
| Autor: | Cathrine A. Southern, Raj Ranganathan, Caitlin E. Karver, Nicholas M. Tassone, Gena Lenti, Brian J. Scannell |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Biophysics Peptide Cleavage (embryo) Biochemistry Substrate Specificity 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Coumarins Drug Discovery Side chain Humans Molecular Biology Caspase Fluorescent Dyes Inflammation Caspase activity chemistry.chemical_classification Molecular Structure biology Chemistry Tryptophan Cell Biology Coumarin In vitro 030104 developmental biology Microscopy Fluorescence Caspases biology.protein 030217 neurology & neurosurgery |
| Zdroj: | Analytical Biochemistry. 543:1-7 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2017.11.023 |
| Popis: | Various fluorogenic assays exist for monitoring the activity of inflammatory caspases. However, there are no continuous assays that provide C-terminal substrate sequence specificity for inflammatory caspases. As a first step towards this, we have developed a continuous in vitro assay that relies on monitoring emission from tryptophan after cleavage of a quenching coumarin chromophore. The coumarin can be attached as an amino acid side chain or capping the C-terminus of the peptide. When the coumarin is a side chain, it allows for C-terminal and N-terminal sequence specificities to be explored. Using this assay, we obtained Michaelis-Menten kinetic data for four proof-of-principle peptides: WEHD-AMC (KM = 15 ± 2 μM), WEHD-MCA (KM = 93 ± 19 μM), WEHDG-MCA (KM = 21 ± 6 μM) and WEHDA-MCA (KM = 151 ± 37 μM), where AMC is 7-amino-4-methylcoumarin and MCA is β-(7-methoxy-coumarin-4-yl)-Ala. The results indicate the viability of this new assay approach in the design of effective fluorogenic substrates for inflammatory caspases. |
| Databáze: | OpenAIRE |
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