Identification of New Angiotensin-Converting Enzyme Inhibitory Peptides Isolated from the Hydrolysate of the Venom of Nemopilema nomurai Jellyfish

Abstract

Recently, jellyfish venom has gained attention as a promising reservoir of pharmacologically active compounds, with potential applications in new drug development. In this investigation, novel peptides, isolated from the hydrolysates of Nemopilema nomurai jellyfish venom (NnV), demonstrate potent inhibitory activities against angiotensin-converting enzyme (ACE). Proteolytic enzymes-specifically, papain and protamex-were utilized for the hydrolysis under optimized enzymatic conditions, determined by assessing the degree of hydrolysis through the ninhydrin test. Comparative analyses revealed that papain treatment exhibited a notably higher degree of NnV hydrolysis compared to protamex treatment. ACE inhibitory activity was quantified using ACE kit-WST, indicating a substantial inhibitory effect of 76.31% for the papain-digested NnV crude hydrolysate, which was validated by captopril as a positive control. The separation of the NnV-hydrolysate using DEAE sepharose weak-anion-exchange chromatography revealed nine peaks under a 0-1 M NaCl stepwise gradient, with peak no. 3 displaying the highest ACE inhibition of 96%. The further purification of peak no. 3 through ODS-C18 column reverse-phase high-performance liquid chromatography resulted in five sub-peaks (3.1, 3.2, 3.3, 3.4, and 3.5), among which 3.2 exhibited the most significant inhibitory activity of 95.74%. The subsequent analysis of the active peak (3.2) using MALDI-TOF/MS identified two peptides with distinct molecular weights of 896.48 and 1227.651. The peptide sequence determined by MS/MS analysis revealed them as IVGRPLANG and IGDEPRHQYL. The docking studies of the two ACE-inhibitory peptides for ACE molecule demonstrated a binding affinity of -51.4 ± 2.5 and -62.3 ± 3.3 using the HADDOCK scoring function.

Keywords: Neophilia nomurai; angiotensin-converting enzyme (ACE) inhibitor; chromatography; papain enzyme hydrolysate; peptide identification.

Figure 1

Degree of enzyme hydrolysis upon NnV based on the Ninhydrin test and preliminary ACE inhibition. (A) Effect of different temperatures of enzyme incubation upon NnV; (B) Effect of enzyme concentrations on NnV; (C) Effect of reaction time of enzymes upon NnV; (D) Log-inhibitory curve of the percent ACE inhibition of papain-hydrolyzed NnV (positive control: captopril). Red arrow denotes highest hydrolysis condition. Results are expressed as mean ± standard deviation (SD), with significance denoted by * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 2

Purification of ACE-inhibitory fractions using DEAE sepharose anion-exchange chromatography. (A) Chromatogram of fractions that were isolated from hydrolyzed crude NnV. (B) ACE-inhibitory activity of separated fractions. The red arrows denote the active peak. Results are expressed as mean ± standard deviation (SD), with significance denoted by *** p < 0.001. The numbers 1–9 in (A) denote isolated peaks.

Figure 3

Further separation of ACE-inhibitory fractions using RP-HPLC. (A) Chromatogram of sub peaks from active peak no. 3 isolated (B) ACE-inhibitory activity. The red arrow marks denote the active peak. Results were expressed as mean ± standard deviation (SD), with significance denoted by *** p < 0.001. The numbers 3.1–3.5 in (A) denotes isolated peaks.

Figure 4

Mass spectrum of fraction 3.2, obtained using MALDI–TOF/MS.

Figure 5

Identification of ACE-inhibitory peptide sequence. (A,B) MS/MS peaks of identified peptides IVGRPLANG and IGDEPRHQYL from NnV-hydrolysate.

Figure 6

Molecular docking simulations of ACE (PDB: 1O8A) protein against the isolated peptides. (A,B) Depicting the optimal docking poses at the active site for IVGRPLANG (red) and IGDEPRHQYL (blue). (C,D) The peptides’ (IVGRPLANG and IGDEPRHQYL) interactions with ACE protein residues.