Isolation, characterization, and some applications of trypsin from Greenland cod (Gadus ogac)

Simpson, Benjamin Kofi (1984) Isolation, characterization, and some applications of trypsin from Greenland cod (Gadus ogac). Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Trypsin E. C. 3. 4. 21: 4, was isolated from the pyloric ceca or the intestines of the Greenland cod (Gadus ogac) and purified by the successive steps of ammonium sulfate fractionation, acetone precipitation, and affinity chromatography using soybean trypsin inhibitor coupled to CNBr-activatod Sepharose 4B. Some of the physical and catalytic properties of the Greenland cod trypsin were compared with those of commercially available bovine pancreatic trypsin. The Greenland cod trypsin was shown to be homogeneous by analytical polyacrylamide gel electrophoresis and also by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Although certain properties of Greenland cod trypsin were similar to those of bovine trypsin there were also some significant differences between the two trypsins. -- Greenland cod trypsin and bovine trypsin were alike with respect to various criteria. The pH activity profile of Greenland cod trypsin was similar to that of bovine trypsin. Likewise the amino acid composition of Greenland, cod trypsin revealed that it was rich in potential acidic amino acid residues as has been reported for trypsin from bovine and other sources. The Greenland cod trypsin was similar to bovine trypsin in being able to hydrolyze ester and amide linkages involving the carboxyl group of arginine. The two trypsins were both inhibited by phenyl methyl sulfonyl fluoride, trasylol and soybean trypsin inhibitor and also by the thiol reagents. 2-mercaptoethanol and dithioerythritol, and were both effective in preventing milk oxidation induced by copper. The molecular weight of Greenland cod trypsin, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, was similar to values reported for tripsin from bovine and other sources. -- The Greenland cod trypsin differed from bovine trypsin in the following respects : the Greenland cod trypsin was most stable at alkaline pH, unlike bovine tripsin which was stable at acid pH; the Greenland cod trypsin was heat abile while bovine trypsin was heat stable. The temperature coefficients and activation energies for the hydrolysis of amide, ester and protein subtrates were considerably lower for Greenland cod trypsin than bovine trypsin. The apparent Michaelis-Menten constants (Km') and molecular activities for the hydrolysis of substrates were considerably higher for the cod enzyme than the bovine enzyme. Based on the amino acid compositions, the calculated average hydrophobicity of Greenland cod trypsin was considerably lower than that of bovine trypsin and the cod enzyme contained fewer cysteine residues than bovine trypsin. Greenland cod trypsin activity was depressed to a greaterextent by thiol reagents than that of bovine trypsin. Finally, the peptide maps of the two trypsins resulting from the cleavage by papain and cyanogen bromide were different.

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