Assay for Angiotensin-Converting Enzyme Inhibition

We implemented the method used by Cushman & Cheung (1971) with some modifications [30 (link),31 (link)]. We added 40 mL of each sample to 100 mL of HHL substrate (hippuryl-L-histidyl-L-leucine) in a 0.1 M sodium borate buffer solution with 0.3 sodium chloride at a pH of 8.3. Subsequently, we added 2 mU of the ACE (ACE 3.4.15.1, 5.1 U/mg) (Sigma; Germany), which was dissolved in 50 % glycerol. The reaction was run at 37 °C for 30 min. We lowered the pH by adding 150 mL of 1 N HCl to inactivate the enzyme and the resulting hippuric acid was extracted with 1000 mL of ethyl acetate. 750 mL of the organic phase was collected after stirring and subsequent centrifugation at 4000×g for 10 min at room temperature. This volume was evaporated by heating it at 95 °C for 15 min. The hippuric acid residue was dissolved in 800 mL of distilled water. After stirring, absorbance was measured at 228 nm.
ACE inhibitory activity is calculated using Equation (4), while the EC₅₀ value is calculated as the amount of soluble protein required to inhibit 50 % of the enzyme. The activity of each sample was determined in triplicate. We used Equation (4) to calculate the inhibitory activity of each sample. $Inhibitoryactivity(\%)=\frac{{Abs}_{Control}-{Abs}_{sample}}{{Abs}_{Control}-{Abs}_{blank}}*100$ ${Abs}_{Control}$ represents the absorbance of hippuric acid after ACE action without inhibitors. ${Abs}_{blank}$ corresponds to the absorbance of unreacted hippuryl-L-histidyl-L-leucine (HHL) extracted with ethyl acetate. ${Abs}_{sample}$ is the absorbance of the hippuric acid formed after ACE action in the presence of inhibitory substances.