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
A B S T R A C T The alpha-2 macroglobulins from human serum and plasma were isolated by Bio-Gel P-300 and A5m gel filtration. The material showed a single peak on sedimentation velocity ultracentrifugation, a mol wt of 650,000 by sedimentation equilibrium ultracentrifugation, and a major precipitin arc in the alpha-2 macroglobulin region by immunoelectrophoresis against whole human serum. Two bands were observed in the alpha-2 macroglobulin region when acrylamide gel electrophoresis was performed with a pH 8.9 running gel. When a pH 7.8 gel was used, five electrophoretic species were observed. In both cases, the preaddition of stoichiometric amounts of trypsin or chymotrypsin added to alpha-2 macroglobulin resulted in disappearance of slower bands leaving only one band on acrylamide gel electrophoresis patterns.Preparative acrylamide gel electrophoresis separated alpha-2 macroglobulin obtained from Bio-Gel into five closely-spaced species. Separation was sufficiently adequate to show that those species of alpha-2 macroglobulin which bound trypsin and chymotrypsin were represented by slower moving species and that the fastest moving material had lost virtually all of the ability to bind these enzymes. Preparative acrylamide gel electrophoresis of a mixture of alpha-2 macroglobulin-trypsin complex and alpha-2 macroglobulin revealed that the fast moving component was alpha-2 macroglobulin-trypsin complex and that the slower moving material was unbound alpha-2 macroglobulin. The naturally occurring amidase activity of the alpha-2 macroglobulin using benzoylarginine-p-nitroanilide (BAPNA) as substrate was investigated and unlike its trypsin-binding activity, amidase activity was found to be of the same specific activity in all electrophoretic fractions.
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