KINETIC AND THERMODYNAMIC CHARACTERISTICS OF A DIGESTIVE PROTEASE FROM ATLANTIC COD, GADUS MORHUA

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Abstract
A 23,500 dalton protease was isolated from the pyloric ceca of Atlantic cod by the successive steps of ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The protein fraction recovered after affinity chromatography migrated as one band in both Davis and Laemmli gels. The protease was classified as trypsin (EC 3.4.21.4) on the basis of its substrate specificity, molecular weight and response to known trypsin inhibitors. For trypsin hydrolysis of benzoyl-DL-arginine p-nitroanilide, the substrate turnover number was 250 BAPA units per micromole trypsin (25.degree. C), Km'' was 1.48 mM, the Arrhenius energy of activation (Ea) was 8.9 kcal/mol, and the free energy of activation (.DELTA.G*) was 12.8 kcal/mol. The Vmax for the hydrolysis of tosyl arginine methyl ester was 18,210 TAME units per micromole trypsin and the Km'' for the same reaction was 0.22 mM at 25.degree. C.