Purification and characterization of trypsin from the Greenland cod ( Gadus ogac ). 1. Kinetic and thermodynamic characteristics
Trypsinogen was isolated from the pyloric ceca of Greenland cod by ammonium sulfate fractionation followed by acetone precipitation, and the trypsin(ogen) thus obtained was purified by affinity chromatography on soybean trypsin inhibitor – Sepharose 4B. The purified trypsin migrated as a single zone...
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crcansciencepubl:10.1139/o84-114 2024-04-28T08:19:36+00:00 Purification and characterization of trypsin from the Greenland cod ( Gadus ogac ). 1. Kinetic and thermodynamic characteristics Simpson, B. K. Haard, N. F. 1984 http://dx.doi.org/10.1139/o84-114 http://www.nrcresearchpress.com/doi/pdf/10.1139/o84-114 en eng Canadian Science Publishing http://www.nrcresearchpress.com/page/about/CorporateTextAndDataMining Canadian Journal of Biochemistry and Cell Biology volume 62, issue 9, page 894-900 ISSN 0714-7511 General Medicine journal-article 1984 crcansciencepubl https://doi.org/10.1139/o84-114 2024-04-09T06:56:24Z Trypsinogen was isolated from the pyloric ceca of Greenland cod by ammonium sulfate fractionation followed by acetone precipitation, and the trypsin(ogen) thus obtained was purified by affinity chromatography on soybean trypsin inhibitor – Sepharose 4B. The purified trypsin migrated as a single zone during polyacrylamide gel electrophoresis and its identity as trypsin (EC 3,4.21.4) was established by its catalytic specificity for amide or ester bonds involving the carboxyl group of arginine, its sensitivity to serine protease inhibitors and soybean trypsin inhibitor, and its molecular weight of 23 500. With tosylarginine methyl ester (TAME) as substrate, the turnover number of the hydrolytic reaction was about three times greater for the cod trypsin than for bovine trypsin at 5 °C. The Michaelis–Menten constant (K m,app ) for cod trypsin and TAME increased from 0.14 mM at 5 °C to 0.26 mM at 35 °C, while the K m,app for bovine trypsin – TAME was about 0.05 mM at all assay temperatures. The free energy of activation (ΔG*) for the hydrolysis of TAME was about 600 cal/mol (1 cal = 4.1868 J) lower for the cod trypsin than for bovine trypsin at 5 °C. The contribution of enthalpy of activation (ΔH*) and entropy of activation (ΔS*) to ΔG* differed considerably for the two enzymes. The "physiological efficiency" (V max /K m , app ) of the two enzymes with TAME was similar at 5 °C, but was much greater for bovine trypsin than cod trypsin at warmer temperatures. With N-α-benzoylarginine-p-nitroanilide (BAPA) as substrate, the turnover number was about eight times greater for the cod trypsin at 25 °C. The K m , app for cod trypsin – BAPA increased from 1.67 mM at 25 °C to 1.84 mM at 35 °C, whereas the K m , app for bovine trypsin – BAPA decreased from 0.97 mM at 25 °C to 0.90 mM at 35 °C. The ΔG* for hydrolysis of BAPA was about 1800 cal/mol lower for cod trypsin than it was for bovine trypsin at 25 °C. V max /K m,app was three to four times greater for cod trypsin than for bovine trypsin at 25 and 35 °C. These results show ... Article in Journal/Newspaper Gadus ogac Greenland Greenland cod Canadian Science Publishing Canadian Journal of Biochemistry and Cell Biology 62 9 894 900 |
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General Medicine Simpson, B. K. Haard, N. F. Purification and characterization of trypsin from the Greenland cod ( Gadus ogac ). 1. Kinetic and thermodynamic characteristics |
topic_facet |
General Medicine |
description |
Trypsinogen was isolated from the pyloric ceca of Greenland cod by ammonium sulfate fractionation followed by acetone precipitation, and the trypsin(ogen) thus obtained was purified by affinity chromatography on soybean trypsin inhibitor – Sepharose 4B. The purified trypsin migrated as a single zone during polyacrylamide gel electrophoresis and its identity as trypsin (EC 3,4.21.4) was established by its catalytic specificity for amide or ester bonds involving the carboxyl group of arginine, its sensitivity to serine protease inhibitors and soybean trypsin inhibitor, and its molecular weight of 23 500. With tosylarginine methyl ester (TAME) as substrate, the turnover number of the hydrolytic reaction was about three times greater for the cod trypsin than for bovine trypsin at 5 °C. The Michaelis–Menten constant (K m,app ) for cod trypsin and TAME increased from 0.14 mM at 5 °C to 0.26 mM at 35 °C, while the K m,app for bovine trypsin – TAME was about 0.05 mM at all assay temperatures. The free energy of activation (ΔG*) for the hydrolysis of TAME was about 600 cal/mol (1 cal = 4.1868 J) lower for the cod trypsin than for bovine trypsin at 5 °C. The contribution of enthalpy of activation (ΔH*) and entropy of activation (ΔS*) to ΔG* differed considerably for the two enzymes. The "physiological efficiency" (V max /K m , app ) of the two enzymes with TAME was similar at 5 °C, but was much greater for bovine trypsin than cod trypsin at warmer temperatures. With N-α-benzoylarginine-p-nitroanilide (BAPA) as substrate, the turnover number was about eight times greater for the cod trypsin at 25 °C. The K m , app for cod trypsin – BAPA increased from 1.67 mM at 25 °C to 1.84 mM at 35 °C, whereas the K m , app for bovine trypsin – BAPA decreased from 0.97 mM at 25 °C to 0.90 mM at 35 °C. The ΔG* for hydrolysis of BAPA was about 1800 cal/mol lower for cod trypsin than it was for bovine trypsin at 25 °C. V max /K m,app was three to four times greater for cod trypsin than for bovine trypsin at 25 and 35 °C. These results show ... |
format |
Article in Journal/Newspaper |
author |
Simpson, B. K. Haard, N. F. |
author_facet |
Simpson, B. K. Haard, N. F. |
author_sort |
Simpson, B. K. |
title |
Purification and characterization of trypsin from the Greenland cod ( Gadus ogac ). 1. Kinetic and thermodynamic characteristics |
title_short |
Purification and characterization of trypsin from the Greenland cod ( Gadus ogac ). 1. Kinetic and thermodynamic characteristics |
title_full |
Purification and characterization of trypsin from the Greenland cod ( Gadus ogac ). 1. Kinetic and thermodynamic characteristics |
title_fullStr |
Purification and characterization of trypsin from the Greenland cod ( Gadus ogac ). 1. Kinetic and thermodynamic characteristics |
title_full_unstemmed |
Purification and characterization of trypsin from the Greenland cod ( Gadus ogac ). 1. Kinetic and thermodynamic characteristics |
title_sort |
purification and characterization of trypsin from the greenland cod ( gadus ogac ). 1. kinetic and thermodynamic characteristics |
publisher |
Canadian Science Publishing |
publishDate |
1984 |
url |
http://dx.doi.org/10.1139/o84-114 http://www.nrcresearchpress.com/doi/pdf/10.1139/o84-114 |
genre |
Gadus ogac Greenland Greenland cod |
genre_facet |
Gadus ogac Greenland Greenland cod |
op_source |
Canadian Journal of Biochemistry and Cell Biology volume 62, issue 9, page 894-900 ISSN 0714-7511 |
op_rights |
http://www.nrcresearchpress.com/page/about/CorporateTextAndDataMining |
op_doi |
https://doi.org/10.1139/o84-114 |
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Canadian Journal of Biochemistry and Cell Biology |
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62 |
container_issue |
9 |
container_start_page |
894 |
op_container_end_page |
900 |
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1797583020640698368 |