Design and application of a fluorogenic assay for monitoring inflammatory caspase activity

Anal Biochem. 2018 Feb 15;543:1-7. doi: 10.1016/j.ab.2017.11.023. Epub 2017 Dec 1.

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 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.

Keywords: Caspase-1 substrate; Fluorogenic peptides; Tryptophan fluorescence.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Caspases / analysis
  • Caspases / metabolism*
  • Coumarins / chemical synthesis
  • Coumarins / chemistry*
  • Coumarins / metabolism
  • Drug Discovery*
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / metabolism
  • Humans
  • Inflammation / metabolism*
  • Microscopy, Fluorescence
  • Molecular Structure
  • Substrate Specificity

Substances

  • Coumarins
  • Fluorescent Dyes
  • Caspases