Enzyme Assays

Fluorogenic Peptide Substrates to Measure Enzyme Activity

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 Assay Buffer: 25 mM HEPES (Sigma, Catalog # H3375 or AMRESCO, Catalog # 0511) , 0.01% Brij ® 35 (Sigma, Catalog # B4184), pH 7.5
• Meprin alpha Subunit/MEP1A Activity Assay
  Meprin alpha Subunit/MEP1A Buffer: 50 mM Tris, pH 7.5
  TCNB Buffer:50 mM Tris, 150 mM NaCl, 10 mM CaCl 2 (Sigma, Catalog # C-5080), 0.05% Brij ® 35, pH 7.5
  Trypsin ( Sigma, Catalog # T1426)
  AEBSF (R&D Systems, Catalog # EI001)

Assay Protocol

1. Follow activation and assay conditions for the enzyme of interest (Table 2).

Table 2. Activation and assay conditions for specific enzymes.

Recombinant enzyme	Activation conditions	Assay conditions
Human ACE-2	No activation	Dilute hACE-2 to 0.2 µg/mL in ACE-2 assay buffer Dilute substrate to 20 µM with ACE-2 assay buffer.
Mouse ACE-2	No activation	Dilute mACE-2 to 0.5 µg/mL in ACE-2 assay buffer. Dilute substrate to 20 µM with ACE-2 assay buffer
Human BMP-1/PCP	No activation	Dilute hBMP-1 to 20.0 µg/mL in BMP-1 assay buffer. Dilute substrate to 20 µM with BMP-1 assay buffer.
Human Meprin alpha Subunit/MEP1A	Dilute Meprin MEP1A to 0.1mg/mL with TCNB buffer containing 0.1 µg/mL Trypsin. Mix well by gentle vortex. Incubate mixture at 37°C for 3 hours in a heating block covered with foil. Stop trypsin activity by adding 1 mM AEBSF (R&D Systems, Catalog # EI001)	Dilute activated mMEP1A to 0.4 µg/mL in 50 mM Tris, pH 7.5 assay buffer. Note: Include a control that has not been activated with trypsin if the activity of the non-activated enzyme needs to be determined. Dilute substrate to 20 µM with 50 mM Tris, pH 7.5 assay buffer.

2. Load the plate in the plate reader.
3. Add 50 µL of enzyme to each well.
4. Add 50 µL of substrate to each well.
5. Read in kinetic mode on a fluorescence plate reader (excitation 320 nm, emission 405 nm) for 5 minutes.
6. Calculate the specific activity (pmole/min/µg).
   Example:

   Rate =	4.2 Relative Fluorescence Units (RFU)/sec
   Conversion factor =	0.18 pmole/RFU
   Enzyme amount =	0.1 µg
   Specific Activity =	Rate x Conversion factor x 60 sec/min
   	Enzyme amount
   =	4.2 RFU/sec x 0.18 pmole/RFU x 60 sec/min
   	0.1 µg
   =	453.6 pmole/min/µg

Calculating the Conversion Factor

The conversion factor will depend on the instrument and settings. To determine the conversion factor, a standard curve should be constructed using a peptide fragment linked with only the fluorescent group. Add the peptide fragments in wells at different amounts (for example, for MCA peptide vary between 0 to 200 pmoles). Read RFUs at the correct excitation and emission wavelengths (320 nm/405 nm for MCA peptide). Plot RFUs (y-axis) versus the amount of peptide (x-axis) and fit by linear regression. In the resulting equation, y = mx + b, the slope of the line (m) corresponds to RFUs at 1 pmole. In the example above, 1 pmole = 5.56 RFU. Then 1 RFU = 0.18 pmole. Ideally, the conditions for constructing the standard curve should be as close to the assay conditions as possible, which include pH, salt concentration, buffer, etc. The peptide fragment should be similar to the cleavage product containing the fluorescent group. We use Mca-Pro-Leu (Bachem, Catalog # M1975) for all our Mca-containing protease substrates (ES001, ES002, ES003, ES004, ES005, ES007, and ES010).