ACE-inhibitory activity assay: IC50

This protocol describes the procedure for the determination of the IC₅₀ in inhibition on angiotensin-I converting enzyme (ACE) activity. ACE is also known as peptidyl dipeptidase A because it removes C-terminal dipeptides from a wide variety of peptide substrates. In the assay described here, the chosen substrate is the intramolecularly quenched fluorescent tripeptide o-aminobenzoylglycyl-p-nitrophenylalanylproline (Abz–Gly–Phe(NO2)–Pro). Hydrolysis of this substrate by the action of ACE generates the fluorescent product o-aminobenzoylglycine (Abz–Gly).

Prepare the necessary reagents carefully.

1U/mL

• Dissolve the (peptidyl-dipeptidase A, EC 3.4.15.1) in a solution of 50% glycerol in ultrapure water, to obtain a final concentration of 1 U/mL.
• Make aliquots of 200µL of the solution and store at -20°C .

0.150Molarity (M) 8.3

• Dissolve 1.817g or 2.364g in approx. 90mL.
• Titrate to 8.77 at the lab temperature of 20Room temperature with monovalent strong base or acid as needed.
• Make up volume to 100mL with ultrapure water. Buffer will be8.3at 37°C.

42mU/mL

• Prepare a solution of 1millimolar (mM) , dissolving 1.4mg in 10mL. Store at -20°C.
• Prepare 0.1millimolar (mM) 0.150Molarity (M) 8.3 (Enzyme buffer). Dilute 1/10 the previous solution (0.1millimolar (mM)) and add 25µL of this solution to 25mL of 0.150Molarity (M) 8.3. Store at 4°Cfor a maximum of one week, or at -20°C for a maximum of six months.
• Dilute 1/24 the 1U/mL with the Enzyme buffer. Prepare daily.

0.45millimolar (mM)

• Prepare 1.125Molarity (M) 0.150Molarity (M) 8.3 (Substrate buffer). Dissolve 3.2872g in 50mL of 0.150Molarity (M) 8.3. Store at 4°Cfor a maximum of one week, or at -20°C for a maximum of six months.
• Dissolve 3.6 mg of substrate in 16mL (for 96 wells). Prepare at the moment.