A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates
| Autor: | Masahiko Sisido, Hidetaka Nakata, Takashi Ohtsuki |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Proteases
Fluorophore Matrix metalloproteinase inhibitor medicine.medical_treatment Biophysics Fluorescence correlation spectroscopy Peptide Matrix Metalloproteinase Inhibitors Hydroxamic Acids Biochemistry Substrate Specificity chemistry.chemical_compound medicine Protease Inhibitors Molecular Biology Fluorescent Dyes chemistry.chemical_classification Protease Caspase 3 Substrate (chemistry) Cell Biology Dipeptides Peptide Elongation Factors Caspase Inhibitors Protease inhibitor (biology) Spectrometry Fluorescence chemistry Matrix Metalloproteinase 9 lipids (amino acids peptides and proteins) medicine.drug Peptide Hydrolases |
| Zdroj: | Analytical biochemistry. 390(2) |
| ISSN: | 1096-0309 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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