ACEIP ACE inhibitory peptide PSEP Proline specifc endopeptidase SHRs Spontaneously hypertensive rats SBP Systolic blood pressure HBP High blood pressure NPARCTR Crude peptide isolate ARTCR N-terminal cleavage pentapeptide
Hepatopancreas from fresh shrimp was used as a raw material for serine proteinase purification. The enzyme was purified to homogeneity by ammonium sulfate fractionation followed by a series of column chromatography assays. Active enzyme peak was observed at 0.4 mol/L NaCl during linear gradient elution on the DEAE-Sepharose column (Fig. 1a). The most positive peak was pooled, concentrated by membrane filtration, and further purified by passing through Sephacryl S-200 HR gel filtration (Fig. 1b) and HiTrap DEAE-Sepharose Fast Flow column (Fig. 1c). The whole purification process resulted in a purification fold of 23.5 and a yield of 6.4%, respectively (Table 1). The enzyme revealed a single band with a molecular weight of approximately 28 kDa on SDS-PAGE and a single active band on gelatin zymography (Fig. 1c), suggesting the target protein was highly purified.
Figure 1. Column chromatography purification of trypsin from Pacific white shrimp. a DEAE-Sepharose chromatography (4 ml per tube). b Sephacryl S-200 HR from DEAE-Sepharose chromatography (2 ml per tube). c DEAE-Sepharose Fast Flow column from Sephacryl S-200 HR (2 ml per tube). (dashed line) Spectrometry absorbance at 280 nm; (filled circle) Boc-Phe-Ser-Arg-MCA hydrolyzing activity. Fractions under the bars were pooled. The SDS-PAGE and Gelatin zymography of finally purified trypsin are shown in the inset of c. Lane M, Protein marker; Lane 1, SDS-PAGE of purified trypsin; Lane 2, Gelatin zymography of purified trypsin. The gels were stained with Coomassie Brilliant Blue
Purification steps Total protein (mg) Total activity (U) Specific activity (U/mg) Purification (fold) Yield (%) Crude enzyme 2823 188, 667 66.8 1.0 100 Ammonium sulfate precipitation 258 101, 172 392.8 5.9 53.6 DEAE sepharose fast flow 61.9 27, 011 436.4 6.5 14.3 Sephacryl S-200 HR 44.0 26, 141 594.1 8.9 13.8 HiTrap DEAE Sepharose fast flow 7.7 12, 103 1572 23.5 6.4
Table 1. Purification of serine proteinase from Pacific white shrimp
The N-terminal amino acid sequence of the purified serine proteinase was determined to be IVGGTDAKPGELPYQLSFQDI, sharing 95.2%, 76.2% and 95.2% identities to trypsinogen 1 (JQ277721.1), trypsinogen 2 (JQ304272.1), and trypsin (CAA60129) from Litopenaeus vannamei, respectively. High identity to trypsins from Panulirus argus (ADB667115) (95.2%) and Fenneropenaeus chinensis (ACQ45455) (90.4%) were also obtained (Fig. 2a). The purified protein was further analyzed by MALDI-TOF/TOF mass spectrometry. Several peptide fragments were observed in the m/z range of 800-4000 Da, which was compared to the NCBI non-redundant protein sequence database. Subsequently, peaks having signal-to-noise ratios (SNR) > 50 were analyzed by MS/MS. Four peptide fragments with 89 amino acid residues in total were obtained (Fig. 2b), which were 100% identical to trypsinogen (JQ277721.1) from Pacific white shrimp (Litopenaeus vannamei), strongly suggesting the purified enzyme is a trypsin (Fig. 2c). The enzyme was active at a broad alkaline pH range from 7.0 to 12.0 with an optimum temperature of 40 ℃ and pH of 9.0 (data not shown).
Figure 2. Sequence analysis of Pacific white shrimp trypsin. a Alignment of the N-terminal amino acid sequences of trypsin from different species. The trypsin sequence of Pacific white shrimp was compared with those of Litopenaeus vannamei (tryp 1 JQ277721.1; tryp 2 JQ304272.1; Try CAA60129), Panulirus argus (ADB66715), Fenneropenaeus chinensis (ACQ45455), Marsupenaeus japonicus (ACE80257), and Pontastacus leptodactylus (AAX98287). A chart of the amino acid sequences is shown in the box. b The mass spectrometry (MS/MS) map of the trypsin and active peptides from MS compared with standard data in NCBInr. c Protein sequence alignment of the target serine proteinase (trypsin) was carried out, followed by comparison with the complete sequence of trypsin from Pacific white shrimp (Litopenaeus vannamei). Identical amino acid residues are shown in black shadow
Recently, different methods were proposed to enable a better understanding of the utilization of aquatic processing by-products. The most recognized one is enzymatic hydrolysis, which is used to produce bioactive peptides. Acid-solubilized collagen (ASC) hydrolysates were obtained by treatment with trypsin, purified from the hepatopancreas of Pacific white shrimp. Gel filtration chromatography analysis showed that small peptides (< 3 kDa) occupied 99.7% in the trypsin hydrolysate (data not shown), suggesting enzymolysis by trypsin can be initially used to break down collagen into peptides. The prepared hydrolysate revealed its ACE inhibitory activity with an IC50 value of 2.34 mg/ml.
Ultrafiltration is a fast, simple, and reliable way for an enrichment of bioactive peptides and is suitable for industrial-scale applications (Yan et al. 2019). In the separation and purification steps, ASC hydrolysates were fractionated according to their molecular weights by ultrafiltration, obtaining fractions < 3 kDa, and > 3 kDa. The permeate fraction (< 3 kDa), which represented low molecular weight peptides, revealed 74.3% ACE inhibitory activity, which was higher than that (58.5%) of the retentive fraction (> 3 kDa). Thus, ultrafiltration could be used as a processing technique to generate a crude pool of bioactive peptides. The low molecular weight fraction (< 3 kDa) was collected for further assay.
The permeate fraction was lyophilized and loaded to SP-Sepharose (Fig. 3a). The major active peak was pooled and further purified by passing through Sephadex G-15 gel filtration (Fig. 3b). The fractions that revealed highest ACE inhibitory activity (84.7% inhibition) were lyophilized for further purification by reverse-phase high-performance liquid chromatography (RP-HPLC), using a Zorbax SB-C18 column. Figure 3c displays active fraction, separated into 14 peaks (designated as fractions a-n), with most fractions revealing ACE inhibitory activity. Among them, fraction g (Fig. 3d) was the most potent, with an IC50 value of 532.5 μg/ml, thus was further characterized.
Figure 3. Purification of ACE inhibitory peptides. a SP-Sepharose purification of ACE inhibitory fractions (4.0 ml per tube). b Sephadex G-15 gel filtration purification of active fractions (1.8 ml per tube). c Purification chromatogram of active fractions. d ACE inhibitory activity of each fraction from Reversed-phase HPLC
Peptide sequence using mass spectrometry showed fraction g was not composed of a single peptide, but three peptides, having amino acid sequences NPARTCR, GEAGTPGENGTPGAMGPR, and GEAGTPGENGTPGAMGPRGLPGER (Fig. 4). When searching in our protein database, we discovered all three peptides were identical to Nile tilapia (Oreochromis niloticus) collagen alpha-1 (I) chain precursor (NP_001266373.1), strongly suggesting these three peptides are derivatives of tilapia collagen.
Figure 4. Peptide profile of Fraction g performed by mass spectrometry analysis and the peptide sequences
Gastrointestinal hydrolysis is of particular importance in the bioavailability of ACE inhibitory peptides. After oral intake, peptides with high molecular weights may be easily digested by gastrointestinal enzymes. Studies have shown that ACE inhibitory peptides are mainly associated with low molecular weight peptides (Barbana and Boye 2011; He et al. 2013; Yu et al. 2006). Therefore, comparing with the other two peptides (GEAGTPGENGTPGAMGPR and GEAGTPGENGTPGAMGPRGLPGER), the heptapeptide NPARTCR would be more resistant against digestion in the gastrointestinal tract and is thus synthesized and used for following experiments. The ACE inhibitory activity of the synthesized NPARTCR was determined with IC50 value of 61.4 μmol/L.
On the other hand, in our previous study, a proline specific endopeptidase (PSEP) was purified to homogeneity from the skeletal muscle of common carp (Wang et al. 2012). We identified this endopeptidase as having a unique ability to hydrolyse peptide bonds on the carboxyl side of a proline residue, consistent with properties of such enzymes (Gass and Khosla. 2007). PSEP was characterized as a unique serine proteinase that contains a peptidase domain with an alpha/beta hydrolase fold. Its catalytic triad (Ser-554, His-680, Asp-641) is shielded by the central tunnel of an unusual beta propeller (Fulop et al. 1998). Further digestion of the heptapeptide NPARTCR by PSEP produced a pentapeptide ARTCR and a dipeptide NP. Both peptides, ARTCR and NP, were then synthesized for biological activity assay. The pentapeptide ARTCR showed ACE inhibitory activity with IC50 of 77.0 μmol/L, while the dipeptide NP did not reveal any inhibitory activity, indicating the two amino acid residues (Asn-Pro) in the N-terminal of NPARTCR did not contribute to ACE inhibition.
Biological functions of peptides are closely related to their sequences. Several reports suggest peptides with high ACE-inhibitory activity contain alphatic amino acid(s) at the N-terminal. Two examples of such peptides have been identified in cuttlefish muscle (Ala-Phe-Val-Gly-Tyr-Val-Leu-Pro, IC50 of 18.02 μmol/L) (Balti et al. 2015) and hen egg white lysozyme (Ala-Met-Lys, IC50 of 2.8 μmol/L) (Rao et al. 2012). In the present study, ARTCR exhibited higher ACE inhibitory activity with IC50 of 77.0 μmol/L. Though its inhibitory activity was lower than peptides derived from cuttlefish and egg white, its inhibitory activity was higher than a peptide (Ala-Met-Asn) from abalone gonads (IC50 of 378.7 μmol/ml) (Wu et al. 2015a, b). All these peptides share the same amino acid residue Ala at the N-terminal. In contrast, peptides MNPPK, VPAAPPK, and PPK, which share similar peptide sequence at their C-terminals, possess far different inhibition capacities with IC50 values of 945.5, 0.45 and > 1000 μmol/L, respectively (Forghani et al. 2016). In the present study, ARTCR, an N-terminal truncated form of NPARTCR, showed better ACE inhibitory activity. This inhibitory effect may be because ARTCR has alphatic amino acids at the N-terminal, which promotes its ACE inhibitory activity.
Figure 5 shows Lineweaver-Burk plots, obtained by examining the effect of NPARTCR and ARTCR on ACE inhibition as a function of peptide concentration. Lines with the coinciding intercept at the x axis were generated, indicating both NPARTCR and ARTCR are non-competitive inhibition types. The inhibition constant values are shown in the inner table of Fig. 5. The Km of the inhibitors did not change, but Vmax decreased. This suggests the peptides were unable to bind to the active site of ACE but may instead bind to other sites on the ACE molecule to produce an inactive complex, irrespective of substrate binding. It has been reported that peptide TPTQQS, which showed non-competitive inhibition pattern, could bind to the catalytic area of ACE, as well as, chelate zinc ion away from the ACE active site (Ni et al. 2012). In the present study, the ACE inhibitory peptides NPARTCR and ARTCR exhibited a non-competitive inhibition, which could be attributed to the same potent amino acid sequence (Ala-Arg-Thr-Cys-Arg) while the two amino acid residues (Asn-Pro) in the N-terminus of heptapeptide (Asn-Pro-Ala-Arg-Thr-Cys-Arg) are unnecessary.
Figure 5. Kinetics study of the inhibitory activity of peptides against ACE. Lineweaver-Burk plot analysis of the ACE inhibitory activity was determined in the absence or presence of different concentrations of the peptides. a NPARTCR: 120 μmol/L (purple colored open triangle), 90 μmol/L (green colored filled triangle), 60 μmol/L (red colored open circle), 0 μmol/L (blue colored filled circle), using value of 1/v against 1/[S]; b ARTCR: 150 μmol/L (purple colored open triangle), 100 μmol/L (green colored filled triangle), 60 μmol/L (red colored open circle), 0 μmol/L (blue colored filled circle), using value of 1/v against 1/[S]
It is of interest to notice that ACE inhibitory peptides (ACEIPs) isolated from porcine hemoglobin (Yu et al. 2006) and small red bean (Rui et al. 2013) fit the competitive inhibition pattern. In contrast, peptides obtained from lentil protein hydrolysates exhibited non-competitive inhibition (Barbana and Boye 2011). Furthermore, ACEIPs isolated from rapeseed hydrolysates exhibited uncompetitive inhibition (Mäkinen et al. 2012), suggesting that ACEIP isolated from different sources has the potential to display different substrate inhibition patterns, despite having inhibitory properties.
Antihypertensive activity of NPARTCR and ARTCR were evaluated by measuring the change in systolic blood pressure (SBP) at 0, 2, 4, 6, 12 and 24 h after oral administration of 10 mg/kg of body weight. In the negative control group, no significant changes were found in the SBP 24 h after administration (Fig. 6). Administration of NPARTCR, however, also did not cause a decrease in SBP during the investigation period. This suggests that gastrointestinal enzymes had further digested NPARTCR in the digestive tract producing smaller peptides or amino acids that had no antihypertensive effect. In vitro digestion tests were performed and the results revealed that NPARTCR could be further cleaved by trypsin to form two peptides, NPAR and TCR, and completely lose its ACE inhibitory activity. On the other hand, after oral administration of ARTCR, SBP clearly decreased and was maintained for about 12 h which is comparable to the positive control captopril (Fig. 6). The maximal decrements in SBP after oral administration of ARTCR and captopril were 25 and 31 mmHg at 4 h, respectively. Blood pressure gradually recovered in 24 h for all groups. Furthermore, no allergic reactions or coughing were observed on the day of the experiment or the following day. The results showed that pentapeptide ARTCR had a substantial effect on reduction of SBP in SHRs, suggesting it could be directly absorbed in the digestive tract.
Figure 6. Time course of oral NPARTCR and ARTCR administration in SHR. Captopril was used as a positive control. Single oral administration of the drug was performed with a dose of 10 mg/kg of body weight, and SBP was measured at 0, 2, 4, 6, 12 and 24 h after administration. Indicates significant difference between test and control, P < 0.05