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A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
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A Facile Method to Determine Prolidase Activity Using a Peptide-Specific Fluorometric Reaction.

Tsutomu Kabashima1, Nana Hamasaki1, Keiko Tonooka2

  • 1Faculty of Pharmaceutical Sciences, Nagasaki International University.

Chemical & Pharmaceutical Bulletin
|January 3, 2023
PubMed
Summary

A new fluorescence method accurately measures prolidase activity, an enzyme vital for wound healing. This technique is simpler and safer than existing assays, offering a facile way to determine enzyme levels in cells.

Keywords:
fluorescence reactioniminodipeptideprolidaseproline

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Area of Science:

  • Biochemistry
  • Enzymology
  • Cell Biology

Background:

  • Prolidase is a key enzyme for cleaving specific peptide bonds, essential for physiological processes like wound healing and cell proliferation.
  • Existing methods for measuring prolidase activity often involve harsh chemicals, pre-incubation steps, or heating, limiting their ease of use and safety.

Purpose of the Study:

  • To develop a novel, facile, and specific fluorescence-based method for determining prolidase activity.
  • To establish a safer and more efficient alternative to traditional prolidase activity assays.

Main Methods:

  • A new fluorescence (FL) reaction utilizing 3,4-dihydroxyphenylacetic acid (3,4-DHPAA) was developed.
  • 3,4-DHPAA selectively reacts with N-terminal glycine (Gly)-containing peptides, specifically the prolidase substrate Gly-Pro.
  • Prolidase activity is quantified by monitoring the decrease in FL intensity resulting from the enzymatic cleavage.

Main Results:

  • The proposed method successfully measured prolidase activities in fibroblast and HeLa cell lines.
  • The new assay demonstrated specificity for the Gly-Pro substrate.
  • Compared to the Chinard method, the developed assay avoids caustic acids, enzyme pre-incubation, and heating for reagent reaction.

Conclusions:

  • A novel and facile fluorescence-based method for measuring biogenic prolidase activity has been successfully developed.
  • This method offers significant advantages over classical assays, including enhanced safety and simplicity.
  • The technique provides a specific and efficient tool for assessing prolidase activity in cellular contexts.