MATERIALS AND METHODS
Salmon Protamine were treated with a single protease, with an enzyme to protein ratio of 1:50 (w/w) using
different temperatures which were based on the enzymes' activities: trypsin (37 °C), α-chymotrypsin (37 °C),
pepsin (37 °C), and thermolysin (60 °C). The enzymatic digestions of the salmon protamine were kept at pH 8,
except for pepsin, which was adjusted to pH 1.3. After incubation for 16 hours, the hydrolysis was stopped by
centrifugation at low temperature (14,000 rpm, 10 min, 4 °C) in ultrafiltration membrane (3 kDa MWCO). The
filtrate (<3 kDa) was lyophilized and kept at −20 °C for further assay or analysis.
The ACE inhibitory activity was determined according to the method reported by Cushman et al [9] with
partial modification. The sample solution containing 30 μl of 2.5 mM hippuryl-L-histidyl-L-leucine (HHL) as a
substrate and 10 μl of inhibitor (at an indicated concentration) in 200 mM borate buffer containing 300 mM NaCl
(adjusted to pH 8.3) was pre-incubated at 37 °C for 5 minutes. The control solution was prepared using the same
buffer but without inhibitor. Afterwards, 20 μl of 2 mU/ml ACE in 200 mM borate buffer was added to the sample
solution and control solution, individually. The reaction mixture was incubated statically at 37 °C for 30 minutes
and then shaken in a thermostatically controlled shaker incubator (200 rpm) at 37 °C for 30 minutes. The
reaction was quenched under acidic conditions by adding 1 M HCl (60 μl). Ferulic acid 0.2 mg/ml (10 μl) was used
as an internal standard for normalizing variation derived from different samples. HHL and its hydrolyzed product,
hippuric acid (HA) were analyzed using an HPLC equipped with a C18 column. The resulting HA was detected
using a UV detector fixed at 228 nm. The ACE inhibition (%) was determined based on the following equation:
[1–(ΔAinhibitor/ΔAcontrol) ] x 100
where ΔAinhibitor was the peak area of HA in the reaction mixture by the presence of peptide as ACE
inhibitor and ΔAcontrol was the peak area of HA in the reaction mixtures without peptide as ACE inhibitor.
Definition of ACE activity: One unit (U) of ACE activity was defined as the amount of enzyme required to catalyze
formation of 1 µmol of HA from HHL per minute at 37 ⁰C.
The peptide sequences in the lyophilized hydrolysate were further identified using LC-MS/MS analysis and
database matching. Freeze dried peptides were dissolved in 5% ACN (Acetonitrile) and 0.2% FA (Ferulic acid) in
deionized water for LC–MS/MS analysis. LC–MS/MS analysis was performed using a Thermo LCQ DECA XP MAX
system with an electrospray ionization (ESI) source (Thermo Scientific Inc., USA). Samples were loaded onto a
BioBasic C18 column with diameter 150 × 2.1 mm, particle size 5 μm. The mobile phase consisted of Solution A
(100% deionized water and 0.1% FA) and Solution B (100% ACN and 0.1% FA) and was kept at a flow rate of 200
μl/min. The MS/MS raw data were acquired using Thermo-XCalibur™ Thermo-Scientific) then processed into
MGF files using Mascot Distiller v2.3.2.0 (Matrix Science, London, UK). The resulting MGF files were searched
using the Mascot search engine v2.3 (Matrix Science, UK).
RESULTS AND DISCUSSIONS
ACE inhibitory activity of each hydrolysates is shown in Figure 1. Captopril is used as positive control. All
hydrolysates have potential to inhibit ACE, but compared to other hydrolysates, the highest inhibition was shown
in tryptic hydrolysate with 94.82% followed by chymotrypsin, thermolysin and pepsin with the inhibition of
79.11%, 70.20%, 48.66% respectively.
Tryptic hydrolysate, because it has the highest ACE inhibition, further was analyzed for IC50 of ACE
inhibition activity. The IC50 or the half maximal inhibitory concentration represents the concentration of a peptide
that is required for 50% inhibition of its target enzyme. To find out the IC50 value of crude hydrolysates, the
relative ACE inhibition was first determined for various concentrations of peptide; afterwards, the IC50 was
evaluated by plotting the curves of relative ACE inhibition against six different peptide concentrations (Figure 2).
The IC50 value of tryptic hydrolysate was considered as a low inhibition. The low IC50 value may be due to
cumulative and synergistic effects of various active peptides present in each hydrolysate [10].
To characterize the peptide identities, the lyophilized tryptic hydrolysate was subjected into LC-MS/MS for
analysis of ACE inhibitory peptides. Two major peaks were observed in the LC-MS chromatogram. Through LC–
MS/MS analysis and database-assisted identification, peptides derived from Salmon Protamine are compared to
the predicted peptides forecasted by peptide sequence application. All the sequences are summarized in Table 1.
LC-MS/MS analysis indicated two major peaks with three peptide sequences. A peptide was located at the
first peak with retention time at minute 1.74, whereas two other peptides were located at the second peak with
retention time at minute 10.70 and 10.95 respectively. Based on Mascot Distiller database search, for triply
To cite this paper: Rasyad F, Huang TC, Hsu JL, Fadjar M. 2016. Screening of Novel Angiotensin I Converting Enzyme Inhibitory Peptides Derived From Enzymatic
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