TITLE: ACE-inhibitory activity assay: IC50
AUTHORS: Ezequiel Coscueta, & Maria Manuela Pintado

[DESCRIPTION]
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).

[BEFORE_START]
Prepare the necessary reagents carefully.

  1 U/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 200 µL of the solution and store at -20 °C .

0.150 Molarity (M)  pH 8.3 
Dissolve 1.817 g  or 2.364 g  in approx. 90 mL. 
Titrate to pH 8.77 at the lab temperature of 20 Room temperature with monovalent strong base or acid as needed. 
Make up volume to 100 mL with ultrapure water. 
Buffer will bepH 8.3at 37 °C.

42 mU/mL 
Prepare a solution of 1 millimolar (mM) , dissolving 1.4 mg in 10 mL. Store at -20 °C.
Prepare 0.1 millimolar (mM)  0.150 Molarity (M)  pH 8.3 (Enzyme buffer). Dilute 1/10 the previous solution (0.1 millimolar (mM)) and add 25 µL of this solution to 25 mL of 0.150 Molarity (M)  pH 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 1 U/mL  with the Enzyme buffer. Prepare daily.

0.45 millimolar (mM) 
Prepare  1.125 Molarity (M) 0.150 Molarity (M) pH 8.3 (Substrate buffer). Dissolve 3.2872 g in 50 mL of 0.150 Molarity (M)  pH 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 16 mL  (for 96 wells). Prepare at the moment.


[GUIDELINES]
ACE-inhibitory activity is measured by fluorescence using the method of Sentandreu & Toldrá (2006) with some modifications (Coscueta, Campos, Osório, Nerli, & Pintado, 2019). A total of 40 µL of ultrapure water or ACE working solution are added to each microtiter-plate well, then adjusted to 80 µL by adding ultrapure water to blank, control or samples. For direct or 1/2 diluted samples a sample blank  is also made. The enzyme reaction is started by adding 160 µL of substrate solution and the mixture is incubated at 37 °C. Serial dilutions are made of each sample, usually 1/1 to 1/32. The fluorescence generated is measured at 30 min using a Multidetection plate reader (Synergy H1, Vermont, USA). The assay is performed in a black 96-well microplate (Nunc, Denmark). Excitation and emission wavelengths are 350 and 420 nm, respectively.

Note: In some cases it is necessary to extend the range to 1/5 or 1/10 serial dilutions.

Note: Generally the most concentrated dilutions are the ones that can fluoresce as the sample itself, so in the example sample blanks (SPLB) are used only for the two most concentrated dilutions. In the case of highly fluorescent samples, SPLB should be used for all dilutions.


 

 


[STEPS]
SECTION: Analysis
1.    


SECTION: Analysis
1.1. Add 80 µL to blank (BLK)

SECTION: Analysis
1.2. Add 40 µL to control (CTL) and sample blank (SPLB)
SECTION: Analysis
1.3. Add 40 µL to sample (SPL) and sample blank (SPLB)
SECTION: Analysis
1.4. Add 40 µL to control (CTL) and sample (SPL)
SECTION: Analysis
1.5. Add 160 µL to control all wells
SECTION: Analysis
1.6. Incubate at 37 °C 30 min and read fluorescence with 350 nm excitation wavelength and 420 nm emission wavelength

SECTION: Result treatment
2. Inhibitory activity is expressed as the peptide concentration required to inhibit the original ACE activity by 50% (IC50). The formula applied to calculate de percentage of ACE-inhibitory is:
  
Non-linear fitting to the data is performed to calculate the IC50 values, using the 5 Parameter curve fit method and then Interpolating to 50.