Purification, Characterization And CDNA Cloning Of Trypsin From The Pancreas Of Snakehead (Channa Argus)

Trypsin （EC3.4.21.4） is an important endopeptidase of serine proteinase, whichspecifically hydrolyzes peptides at the carboxyl side of arginine and lysine residues. It playssignificant roles of physiological and biochemical function in fish. At present, many trypsinsfrom different fishes have been reported at home and abroad. However, the reports of trypsinsfrom fresh water fishes are much less than those from marine fishes. Snakehead （Channaargus） is an important economic fish in China. However, the trypsin from snakehead has notbeen researched. In order to research the theories of trypsins physiological and biologicalfunctions, the purification, characterization and cDNA cloning of the trypsin （trypsinogen）from the pancreas of snakehead （Channa argus） have been studied in this paper.Two trypsins （trypsin A and trypsin B） from the pancreas of snakehead （Channa argus）were purified to homogeneity by ammonium sulfate precipitation and columnchromatographies, including DEAE-Sepharose anion-exchange chromatography, SephacrylS-200gel filtration chromatography and Hi-Trap Capto-Q anion-exchange chromatography.SDS-PAGE revealed trypsins were highly purified，and the molecular weights of trypsin Aand trypsin B were both22kDa. Peptide mass fingerprinting （PMF） of the purified proteinsshowed that trypsin A was obtained2peptide fragments with25amino acid residues andtrypsin B was obtained3peptide fragments with38amino acid residues, and they were bothidentical to the trypsinogen from puffer fish （Takifugu rubripes）, these results indicated thepurified proteins were both trypsins. Using Boc-Phe-Ser-Arg-MCA as substrat, the optimaltemperature of trypsin A and trypsin B were both40°C and optimal pH were both9.0. Theactivation energy （Ea） of purified trypsins were24.65and22.27kJ·M^-1, respectively. Theenzyme activity of two trypsins were stable below45°C, and decreased sharply above50°C.The half-life of the two trypsins were about60min at40°C, demonstrated trpsin could behydrolyzed by itself at optimal temperature resulting in inactivation of enzyme. Both thetrypsins were stable up in the pH range from7.0to10.0, and unstable in acid pH range. Theenzyme activity of two trypsins were strongly inhibited by serine proteinase inhibitors, such asMBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Metalloproteinase inhibitors and cysteineproteinase inhibitor partially inhibited the enzyme activity, which indicated there were metalions and Cys residuals near the enzyme active site. These results showed that the purified proteins were both trypsins. And low concentration of Ca²⁺could enhance the enzymeactivity，but other metal ions could restrain the enzyme activity. The substrate specificityexperiment showed the two trypsins could effectively hydrolyze trypsin-like substrates, andhardly hydrolyze other substrates, which demonstrated the purified enzymes were trypsins.Both of the trypsins hydrolyzed Boc-Phe-Ser-Arg-MCA most effectively, and trypsin A couldmore efficaciously hydrolyze substrates with Arg at P₁site, while trypsin B could hydrolyzesubstrates with both Arg and Lys at P₁site. Hydrolysis of different substrates were determinedin kinetic studies. The kinetic constants showed that trypsin B would be more efficient incatalytic efficiency to most substrates than trypsin A. For the substrates, which have the sameamino acid at P₁site and the different amono acids at P₂or P₃, trypsins showed differenthydrolysis, indicated that the amino acid at P₂or P₃site could affect the hydrolysis of trypsins.It could be infered that when the substrate was food proteins, the two trypsins would showdifferent hydrolysis. Kinetic constants of trypsins for different substates were detected.Apparent K_ms of trypsin A and trypsin B were2.020and0.663μM and Kcats of the twoenzymes were148and116S^-1for Boc-Phe-Ser-Arg-MCA, respectively.According to the result of PMF and conservative sequence of trypsins from differentfishes, the primers were designed for cloning of trypsins from the pancreas of snakehead byRT-PCR,3’-RACE and5’-RACE. One trypsinogen gene was cloned containing916bp, whichcould code247amino acids. The GenBank accession number was JN558641, and the proteinaccession number was AEM91638. The cDNA clone contained a9bp5’ non-coding region,744bp open reading frame and163bp3’ non-coding region. The open reading frame encodeda protein of247amino acid residues with a signal peptide of15amino acid residues, activepeptide of9amino acid residues and a mature trypsin of223amino acids. The deducedmolecular masses of the mature trypsin was24.45kDa. Three residues (His-64, Asp^-107andSer-201) formed the typical catalytic site of serine proteinases, and12Cys could form6disulfide bonds, which were conserved in vertebrate trypasins. After analysis the compositionof amino acid of the trypsinogen, the most was Ser （35） and next was Gly （23）, acounting for14.2%and9.3%of total amino acid, respectively. We also analyzed the enzyme cutting sitesof the trypsinogen for trypsin and chymotrypsin. The trypsinogen contained16trypsin cuttingsites and22chymotrypsin cutting sites, and had14and17cutting sites with more than80%cutting probability, respectively. The secondary structure of trypsin was consisted of α-helix,β-sheet and loop. The trypsin contained two domains which were similar. Both domainsshowed a β-sheet central core and a α-helice flanked by random coils.