A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates

Hidetaka Nakata, Takashi Ohtsuki, Masahiko Sisido

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

We developed novel substrates for protease activity evaluation by fluorescence correlation spectroscopy (FCS). Substrates were labeled in a position-specific manner with a fluorophore near the N terminus and included a C-terminal, 30 kDa, highly soluble protein (elongation factor Ts [EF-Ts]). The C-terminal protein enhanced the substrate peptide solubility and increased the molecular weight, enabling sensitive detection by FCS. Using the labeled substrates, caspase-3 and matrix metalloproteinase-9 (MMP-9) activities were confirmed by FCS. To demonstrate the suitability of this FCS-based assay for high-throughput screening, we screened various chemical compounds for MMP-9 inhibitors. The screening results confirmed the inhibitory activity of one compound and also revealed another potential MMP-9 inhibitor. Thus, this combination of position-specific labeled protein substrates and FCS may serve as a useful tool for evaluating activities of various proteases and for protease inhibitor screening.

Original languageEnglish
Pages (from-to)121-125
Number of pages5
JournalAnalytical Biochemistry
Volume390
Issue number2
DOIs
Publication statusPublished - Jul 15 2009

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Keywords

  • Fluorescence correlation spectroscopy
  • Four-base codon
  • Position-specific labeling
  • Protease assay
  • Protease substrate

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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