On the activation of bovine chymotrypsinogen A. Preparation of alanine-neochymotrypsinogen and its activation to alpha-chymotrypsin.
Alanine-neochymotrypsinogen was prepared by incubating 20 parts bovine pancreas chymotrypsinogen A with one part alpha-chymotrypsin in a solution containing 1 M (NH4)2SO4, 0.1 M sodium acetate, 0.05 M Tris buffer (pH 8.0) and 0.5 mg/ml soybean trypsin inhibitor. Optimal yields of NH2-terminal alanine were obtained after 60 h incubation at 4 degrees C. Ala-neochymotrypsinogen was isolated from the reaction mixture by affinity chromatography and ion-exchange chromatography on carboxymethyl-cellulose. As expected, the purified preparation was enzymatically inactive and, compared to chymotrypsinogen, had one additional NH2-terminal group identified as alanine. Ala-neochymotrypsinogen was activated by incubating with trypsin at a zymogen : trypsin ratio of 30 : 1 in 0.1 M phosphate buffer, pH 7.6 at 4 degrees C for 1 h. The fully active, stable species was identified as alpha-chymotrypsin.
D000409:Alanine; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002918:Chymotrypsin / Q000378:metabolism*; D002919:Chymotrypsinogen / Q000378:metabolism*; D004355:Drug Stability; D004789:Enzyme Activation; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D014361:Trypsin Inhibitors
D014361:Trypsin Inhibitors; D002919:Chymotrypsinogen; C019633:neochymotrypsinogen; D002918:Chymotrypsin; D000409:Alanine