ACE2 Activity Assay in Biological Fluids Using Mca-Ala-Pro-Lys(Dnp)-OH
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
1.Reagent Preparation
(1) ACE2 Substrate Solution: Dilute Mca-Ala-Pro-Lys(Dnp)-OH (15mM stock solution in 1% NH₄OH) with protease inhibitors NEM (100mM) and PMSF (100mM) in pre-chilled MES buffer (50mM MES, 300mM NaCl, 10μM ZnCl₂, pH=6.8) to final concentrations of 15μM, 1mM, and 1mM, respectively.
(2) ACE2 Standards: Reconstitute recombinant human or mouse ACE2 in PBS to 10μg/mL, followed by serial dilution to generate a standard curve ranging from 1.56 to 50ng/mL.
(3) ACE2 Inhibitors: Prepare MLN-4760 (10μM) or DX600 (10μM) for specificity validation.
2.Biological Sample Pretreatment
(1) Urine: Collect samples on ice, centrifuge (12,000×g, 5min, 4°C), aliquot supernatant, and store at -80°C. Dilute samples at least 1:2 with assay buffer before use.
(2) Plasma: Separate plasma from heparin-anticoagulated blood by centrifugation (3,000×g, 10min, 4°C), aliquot, and store at -80°C. Dilute 1:7.5 prior to assay.
(3) Cell Culture Supernatant: Centrifuge serum-free conditioned medium and use the supernatant directly without dilution.
3.ACE2 Activity Detection Procedure
(1) Add 10μL of inhibitor (MLN-4760 or DX600) or ddH₂O (control) to each well of a black 96-well plate.
(2) Add 75μL of ACE2 substrate/buffer mixture to all wells. Add 15μL of diluted biological sample or ACE2 standard to each well, achieving a total reaction volume of 100μL.
(3) Seal the plate with adhesive film and aluminum foil to protect from light. Incubate with shaking at room temperature for 16 hours.
(4) Measure fluorescence using a microplate reader (excitation: 320nm, emission: 405nm) and record relative fluorescence units (RFU).
Precautions:
(1) Samples with high background fluorescence (e.g., concentrated urine) require further dilution. Glucose, urea, creatinine, and albumin do not interfere with assay results.
(2) Avoid using EDTA-containing blood collection tubes or serum (which contains endogenous ACE2 activity). Maintain pH at 6.8±0.2 throughout the assay.
(3) For your safety and health, please wear a lab coat and disposable gloves during operation.
References:
[1] Xiao F, Burns KD. Measurement of Angiotensin Converting Enzyme 2 Activity in Biological Fluid (ACE2). Methods Mol Biol. 2017;1527:101-115. |
