2.2. Assay for ACE inhibitory activity

ACE inhibitory activity was measured according to the method described by Shalaby et al. (2006) with minor modifications. Briefly, 10 μL of the ACE solution (final activity of 12.5 U/L) was mixed with 40 μL of the peptide solution (final concentrations were 100 to 700 μg/mL, dissolved in ultrapure water) in the wells of a 96-well microtiter plate at room temperature. Then, 150 μL of 0.88 mmol/L FAPGG (dissolved in 50 mmol/L Tris-HCl buffer containing 0.3 mol/L NaCl, pH 7.5, preheated at 37 ℃, for 15 min) was added to each well in less than 1 min. Next, the microtiter plate was immediately transferred to the microplate reader (Synergy H1, BioTek Instruments Inc., Vermont, USA). The rate of the decrease in absorbance at 345 nm was recorded for 30 min in 1-min intervals at 37 ℃. For control groups, 40 μL of buffer (50 mmol/L Tris-HCl buffer with 0.3 mol/L NaCl, pH 7.5) was used instead of the peptide solution. ACE activity was expressed as the slope of the decrease in absorbance (ρA), and the inhibitory activity was calculated according to the formula:

where ρA _inhibitor and ρA _control are the slopes obtained in the presence and absence of peptides, respectively. The IC₅₀ values of tested samples were determined by the plot of ACE inhibition (%) against lg(sample concentration).

To determine the modes of ACE inhibition, the experiment was carried out with a set of concentrations of FAPGG substrate (0.88, 0.44, 0.22, and 0.11 mmol/L). V ₀ is defined as the amount of FAPGG hydrolysed by ACE in 1 min at 37 ℃. The values of V ₀ were measured in the absence and presence of peptide I (0.255 and 0.340 μmol/L), peptide II (0.205 and 0.307 μmol/L), peptide III (0.273 and 0.410 μmol/L), and peptide IV (0.273 and 0.410 μmol/L). The inhibition patterns of peptides were determined by Lineweaver-Burk plots. Non-competitive inhibitor, which does not necessarily need the presence of substrate, will change both the slope and the y-intercept. Uncompetitive inhibitor, which requires the presence of substrate, changes the y-intercept but not the slope. Competitive inhibitor changes the slope but not the y-intercept.