Isolation And Purification Of Proline Endopeptidase From Skeletal Muscle Of Blue Trevally And Its Effect On Collagen Peptides
Keywords
Round Scad Skeletal Muscle Prolyl Endopeptidase Purification Collagen Peptide Liquid Chromatography-Electrospray Ionization Mass Spectrometry (Lc-Esi-Ms)
Abstract
Taking blue trevally as the research object, the effect and mechanism of prolyl endopeptidase (PEP) on fish muscle collagen was explored. A proline endopeptidase was isolated and purified from the skeletal muscle of blue round trevally through ammonium sulfate fractionated salting out, DEAE-Sephacel anion exchange, Phenyl-Sepharose hydrophobic chromatography and Q-Sepharose anion exchange. The SDS-PAGE results showed that the molecular weight of PEP was 82 ku, and peptide mass fingerprint analysis yielded 16 peptide fragments with a total of 169 amino acid residues. The fragment sequence has 98.8% homology with Mexican snapper (Neolamprologus brichardi) PEP, proving that the purified enzyme is PEP. The optimal temperature of PEP is 35¡ãC, but its thermal stability is poor; its optimal pH is 6.0, and it has good stability between pH 5.0 and 7.5. PEP was reacted with synthetic small fish collagen peptides, and the product was separated using reversed-phase high-performance liquid chromatography. Electrospray mass spectrometry analysis showed that the hydrolysis site of PEP was at the carboxyl end of the proline residue. The above results show that PEP in fish muscles can cooperate with metalloproteinases to further degrade small collagen peptides by cleaving proline residues and thus participate in the metabolism of collagen in fish muscles. It is an important enzyme involved in collagen degradation after death of fish. . The action and mechanism of a prolyl endopeptidase (PEP) isolated from round scad was explored. The PEP was obtained from the skeletal muscle of blue scad via separation and purification by ammonium sulfate fractionation and a series of column chromatographies, including anion-exchange chromatography using a DEAE-Sephacel column, hydrophobic interaction chromatography using a Phenyl-Sepharose column, and anion-exchange chromatography using a Q-Sepharose column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the molecular weight of the PEP was 82 kDa. Peptide mass fingerprinting revealed that there were 16 peptide fragments, including a total of 169 amino acid residues. The sequence homology between the obtained peptide fragment and PEP from Neolamprologus brichardi was 98.8%, suggesting the purified enzyme was indeed a PEP. The PEP had an optimal temperature of 35¡ãC, but showed poor thermal stability. The optimal pH of the purified enzyme was 6.0, and good stability was observed in the pH range of 5.0 to 7.5. Purified PEP was allowed to react with three synthetic fish collagen peptides, then the degraded peptides were further separated by reverse phase-high performance liquid chromatography (RP-HPLC), and electrospray ionization mass spectrometry (ESI/MS) results showed that the PEP hydrolysis site was at the carboxyl terminus of prolyl residues. The results indicate that PEP in fish muscle can collaborate with matrix metalloproteinases to further degrade collagen peptides by cleaving peptide bonds at the carboxyl side of prolyl residues, thus participating in the metabolism of fish muscle collagen. PEP is an important enzyme that participates in post-mortem fish collagen degradation.
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Original research was done by Tang Jun
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