Assembly and selective "in synthesis" labeling of quenched fluorogenic protease substrates

Alberto Chersi, Silvia Ferracuti, Giuliana Falasca, Richard H. Butler, Doriana Fruci

Research output: Contribution to journalArticle

Abstract

Because impaired cellular protease activities are linked to many diseases, such as cancer, inflammation, neurodegeneration, and infection, internally quenched fluorescent peptides have recently been developed as tools for analyzing the specificities of these enzymes. Here we report convenient and cost-effective approaches for the selective "in synthesis" assembly of such substrate peptides for protease assays. Fluorescein and Dabcyl groups were covalently and selectively attached during synthesis to ε-amino groups of internal lysines. Functionality was then tested by digestion with leucine aminopeptidase, chymotrypsin, and microsomal vesicles. All peptides proved to be appropriate substrates of the enzymes tested and of the endogenous peptidases in the microsomal vesicles. In summary, we describe an innovative and cheap method to develop completely functional quenched fluorescent peptides that are usable in specific detection of individual proteases, in particular aminopeptidases, in both in vitro and in vivo systems.

Original languageEnglish
Pages (from-to)194-199
Number of pages6
JournalAnalytical Biochemistry
Volume357
Issue number2
DOIs
Publication statusPublished - Oct 15 2006

Fingerprint

Fluorescent Dyes
Labeling
Peptide Hydrolases
Peptides
Substrates
Leucyl Aminopeptidase
Aminopeptidases
Chymotrypsin
Enzymes
Fluorescein
Lysine
Digestion
Assays
Inflammation
Costs and Cost Analysis
Infection
Costs
Neoplasms

Keywords

  • Fluorescence
  • Peptide synthesis
  • Quenching
  • Selective labeling

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Assembly and selective "in synthesis" labeling of quenched fluorogenic protease substrates. / Chersi, Alberto; Ferracuti, Silvia; Falasca, Giuliana; Butler, Richard H.; Fruci, Doriana.

In: Analytical Biochemistry, Vol. 357, No. 2, 15.10.2006, p. 194-199.

Research output: Contribution to journalArticle

Chersi, Alberto ; Ferracuti, Silvia ; Falasca, Giuliana ; Butler, Richard H. ; Fruci, Doriana. / Assembly and selective "in synthesis" labeling of quenched fluorogenic protease substrates. In: Analytical Biochemistry. 2006 ; Vol. 357, No. 2. pp. 194-199.
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