In human serum there are two protein fractions that are potent inhibitors to trypsin. These inhibitors travel with the a1-globulin and a2-globulin fractions when serum proteins are separated by electrophoresis (1-3). One ml of normal serum has sufficient amounts of trypsin inhibitor to neutralize the proteolytic effect of 1.15 (± 0.10) mg of crystalline trypsin. Approximately 90 per cent of the trypsin inhibitory proteins in serum migrate in the electrophoretic cell with the a1-globulin fraction, and the remainder with the a_2-globulin fraction (3).It is known that in many diseases of unrelated etiology the level of total serum trypsin inhibitor may be increased. This increase, therefore, is not specific for any disease, including acute pancreatitis. Also, it is likely that an increase in the a1-globulin trypsin inhibitor is no more specific in the various diseases than is a sedimentation rate. However, the results of a previous study indicate that in acute pancreatitis the a2-globulin inhibitor decreases, and in severe pancreatitis it frequently disappears. The divergent changes of the two serum globulin trypsin inhibitors in acute pancreatitis result in a marked increase in the ratio of the a,-to a2-globulin trypsin inhibitors. The decrease in the a2-globulin trypsin inhibitor in acute pancreatitis has been helpful in our laboratory in the diagnosis of acute pancreatitis (3). Continuing along this line of investigation, we determined that when trypsin was added to serum and the mixture subjected to electrophoresis, a substance with trypsin-like activity was detected, migrating with the a2-globulin fraction. This was an unexpected finding, since the serum to which trypsin was added contained sufficient trypsin inhibitor to completely neutralize many times the amount of trypsin added. Also, other known inhibitors of trypsin and plasmin failed to inhibit this activity.The aims of this study were: 1) to investigate the properties of the substance with the trypsinlike activity that appears in the a2-globulin fraction when trypsin is added to serum (this substance is designated trypsin-protein esterase) ; and 2) to determine whether the addition of chymotrypsin to serum will produce a substance with proteolytic activity which migrates with the a2-globulin fraction (chymotrypsin-protein esterase). METHODS AND MATERIALSThe following substances were used in this study: 1) Benzoyl-L-arginine-paranitroanilide (BAPNA), originally developed by Karmen, was synthesized as previously reported (4). 2) A supersaturated aqueous solution of BAPNA in a concentration of 1 mg per ml was prepared by heating to 85' C until dissolution was complete and then cooling in an ice bath. The solution may be stored at room temperature for at least 1 month without appreciable change. 3) 0.1 M Tris buffer, pH 7.67, 0.005 M in CaCl,; 0.005 M Tris buffer, 0.5 M in NaCl. 972

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