I need the protocol for measuring of ACE inhibition using HHL as substrate. thanks
Dear Ahmed,
Herein the requested protocol:
ACE Activity Assay
Angiotensin-converting enzyme (ACE) activity can be measured using the
substrate hippuryl-L-histidyl-L-leucine (HHL). ACE cleaves the substrate to expose a
free N-terminus, which can be fluorogenically labeled with o-phthaldialdehyde (OPA).
HHL Sigma #H4884 FW=429.5
Prepare a 50mg/ml stock solution (116.4mM) in acetic acid. Store -20º for up to
1 year.
OPA Sigma #P1378 FW=134.1
Prepare a 20mg/ml stock solution in Ethanol. Store +4ºC.
Use a final amount of 1mg per 100μl reaction
NaB (Sodium Borate) Buffer -final concentration is 0.4M Boric Acid, 0.3M NaCl, pH
8.3. Prepare a 2x stock solution.
Protocol:
Use 2.5 μg lysate and 1mM HHL in a 35μl final volume of 1x NaB buffer.
1. Preincubate the enzyme with any inhibitors (the ACE inhibitor captopril, for example)
for 20 min in a 25μl volume in 1x buffer (no substrate).
2. Prepare 3.5mM HHL substrate in 1x NaB buffer.
Neutralize HHL by adding approximately 1.9 times the HHL volume of 10M NaOH.
Adjust pH to 8.3 (proper pH is critical for ACE activity).
3. Mix the 25μl of prepared enzyme from Step 1 with 10μl of 3.5mM HHL for a 35ul
total volume. Incubate 37ºC for desired timepoints (10-60 min, depending on activity of
sample).
4. Stop reaction by adding 150ul 0.34M NaOH, invert to mix.
5. Add 20μl 20mg/ml OPA. Invert to mix and incubate 10 min at RT.
6. Stop reaction by adding 50ul 3M HCl
8. Read on a fluorescent plate reader at 355nm excitation; 535nm emission
http://www.alzforum.org/sites/default/files/protocol_ACE_activity_assay_0.pdf
Hoping this will be helpful,
Rafik
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