Z-Gly-Pro-AMC

Protocol for assay conditions for the kinetic measurements^[1]:

1. The 4.6 mM stock solution of Z-Gly-Pro-AMC was made in 100% DMSO and the 6.9 mM stock solution of Z-Gly-Pro-AMC was made in 100% methanol.
2. Enzymatic activity was determined kinetically in a 96-well plate measuring the initial velocities of AMC release (λex = 380 nm, λem = 465 nm) from the substrate, using a Tecan microtiter plate reader.
3. First, 5 μL of plasma sample was pre-incubated with 10 μL of 250 nM FAP inhibitor, 10 μL of 250 nM PREP inhibitor or 10 μL of 0.0025% (v/v) DMSO for 15 min at 37℃.
4. Next, 35 μL pre-heated Z-Gly-Pro-AMC (380 μM diluted in buffer) was added to obtain a final concentration of 266 μM and fluorescence was measured kinetically for 30 min at 37℃.
5. Fluorescence intensity was related to an AMC standard curve in the same buffer. One unit of enzymatic activity is the amount of enzyme that catalyzed the release of 1 μmol AMC from the substrate per minute under assay conditions.
6. The following assay buffers were used: PREP assay buffer (100 mM potassium phosphate, pH 7.7, 1 mM EDTA), FAP assay buffer (100 mM Tris-HCl, pH 8, 300 mM NaF, 1 mM EDTA, 50 mM salicylic acid) and Tris buffer (100 mM Tris-HCl, pH 8, 1 mM EDTA). 5 mM DTT was added to the assay buffers.

Protocol for FAP activity measurements^[1]:

1. Standards and plasma samples were diluted 26-fold in FAP assay buffer.
2. 150 μL of diluted samples was mixed with 6 μL Z-Gly-Pro-AMC substrate (6.9 mM in 100% methanol) and incubated for 120 min at 37℃ in a warm water bath.
3. Reactions were stopped by adding 500 μL of 1.5 M acetic acid.
4. Next, 500 μL of this mixture was mixed with 2 mL of deionized water and the fluorescence was measured (λex = 380 nm, λem = 465 nm).
5. Fluorescence intensity was related to an AMC standard curve in the same assay conditions.

This protocol only provides a guideline, and should be modified according to your specific needs.

References:

[1].  Bracke A, Van Elzen R, et,al. The development and validation of a combined kinetic fluorometric activity assay for fibroblast activation protein alpha and prolyl oligopeptidase in plasma. Clin Chim Acta. 2019 Aug;495:154-160. doi: 10.1016/j.cca.2019.04.063. Epub 2019 Apr 11. PMID: 30981844.