Design and application of a fluorogenic assay for monitoring inflammatory caspase activity.
Anal Biochem
; 543: 1-7, 2018 02 15.
Article
en En
| MEDLINE
| ID: mdl-29198574
ABSTRACT
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 ß-(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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Caspasas
/
Cumarinas
/
Descubrimiento de Drogas
/
Colorantes Fluorescentes
/
Inflamación
Límite:
Humans
Idioma:
En
Revista:
Anal Biochem
Año:
2018
Tipo del documento:
Article
País de afiliación:
Estados Unidos