Enzyme Activity Assays using Fluorogenic Peptide Substrates

Introduction

The fluorogenic peptide substrate Mca-Y-V-A-D-A-P-K(Dnp)-OH (R&D Systems, Catalog # ES007) can be used to measure the activity of certain endopeptidases and carboxypeptidases including several R&D Systems enzymes (Table 1). When an active protease cleaves the substrate at a peptide bond between the Mca (fluorophore) and Dnp (quencher) the fluorescence will increase. The increase in fluorescence can be quantified on a fluorescence plate reader and correlates with the amount of protease activity.

Warnings and Precautions

• This substrate is light sensitive. Store aliquots at < -20° C in the dark.
• In general, handle enzymes on ice unless temperature is specified.
• This assay is designed for measuring the activity of a purified enzyme preparation. If used with cell culture media or cell lysates, contaminating proteolytic enzymes may be present that can cleave the substrate. Additional experiments using more specific reagents such as antibodies and inhibitors are required to show that the activity is due to the enzyme of interest.

Materials

Note: The materials vary depending on the enzyme being assayed.

• All Assays Fluorogenic Peptide Substrate VI (R&D Systems, Catalog # ES007)
  Uncoated black 96-well Microtiter Plate (F16 Black MaxiSorp™ Plate, Nunc, Catalog # 475515)
• ACE-2 Activity Assay:
  ACE-2 Assay Buffer: 75 mM Tris (Amresco, Catalog # 0826), 1 M NaCl (J.T. Baker, Catalog # 3624-07), pH 7.5
• BMP-1/PCP Activity Assay:
  BMP-1/PCP Activity Assay Buffer: 25 mM HEPES (Sigma, Catalog # H3375 or Amresco,
  Catalog # 0511), 0.1% Brij^® 35 (Sigma, Catalog # B4184), pH 7.5
• Meprin alpha Subunit/MEP1A Activity Assay:
  Meprin alpha Subunit/MEP1A Activity Assay Buffer: 50 mM Tris, pH 7.5
  TCNB Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂ (Sigma, Catalog # C-5080),
  0.05% Brij 35, pH 7.5
  Trypsin (Sigma, Catalog # T1426)
  AEBSF (R&D Systems, Catalog # EI001)

Assay Protocols

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