250 IMMUNO-CATALYSIS 



munization of guinea pigs with purified trypsin preparations produced 

 sera which inhibited the tryptic activity of the preparations in vitro 

 and protected guinea pigs against their toxic effects. 



The purification of trypsin was carried out as follows: A five per cent 

 suspension of commercial powdered swine pancreatin was incubated 

 at 37°C. for 24 hours in the presence of chloroform to prevent contami- 

 nation and fermentation reactions. It was then filtered under sterile 



reported on the properties of the sera of rabbits which were daily treated with crude 

 trypsin via intramuscular, intravenous, subcutaneous and oral routes. He followed the 

 rise and fall of the antiproteolytic activity of these sera over a period of several weeks. 



Grob (Article I) believes that the possibility that antiproteolytic activities of the 

 above sera are due to antitrypsin antibody is unlikely. He stated that outside of a weak 

 precipitin reaction with serum of animals that had received trypsin intravenously, 

 they were all negative; and the positive precipitin reactions showed no parallelism to 

 antiprotease activity. According to him, the weak precipitin reactions may have been 

 due to impurities in crude trypsin used for injections. He refers to the observations 

 of Ten Broeck (1934, see also Part I of this treatise) who failed to obtain precipitin 

 reactions with sera prepared against crystalline trypsin. Ten Broeck reported, however, 

 having obtained positive Dale anaphylactic tests in guinea pigs sensitized with five 

 times crystallized trypsin, chymotrypsin and chymotrypsinogen. Ten Broeck stated 

 also having shown a differentiation in this manner between the enzymes as well as 

 between them and their respective precursors. These facts show that the actively 

 immunized guinea pigs contained specific antibodies against these crystalline enzymes. 



The failure to obtain a strong precipitin reaction may possibly be explained in the 

 following manner. It is a well-known fact that a small molecular weight hapten 

 inhibits the precipitin reaction between an antibody and the conjugated antigen of 

 which the hapten is a component. (For non-specific inhibitors of precipitin reactions 

 see also Goebel and Hotchkiss, 1937.) It would therefore appear possible that the 

 normal serum inhibitor (polypeptide) combines with trypsin, forming a compound 

 (see Kunitz and Northrop, 1936; Schmitz, 1938) which prevents the union between 

 trypsin and its homologous antibody. In such studies it would be necessary to separate 

 the inhibitor from the globulin fraction before performing the precipitin reaction. Such 

 a critical test is absent in the study by Grob. 



This study lacks also the following critical tests. An electrophoretic separation of 

 sera into the albumin fraction (containing inhibitor polypeptide) and the globulin 

 fraction (containing antitrypsin antibody), as performed by Smith and Lindsley 

 (1939), would have enabled Grob to determine the relative quantities of these two 

 substances. Or at least an attempt could have been made to separate them by some 

 means of fractionation. Another factor to be considered is the species specific relation- 

 ship of the trypsin used as antigen and the animals used for immunization (see Part 

 V). A study dealing with controversial questions necessitates likewise the use of 

 highly purified antigens. 



To evaluate the results and interpretation of Grob, it might be of interest to refer 

 the reader also to the observations of Maschmann (p. 256), Pozerski and Guelin, and 

 Smith and Lindsley (p. 261) who differentiated the trypsin inhibitor from the specific 

 antiproteolytic antibodies. Grob does not make reference to these workers. 



Further to interpret Grob's results (Article III) in an accurate manner, the reader 

 is referred to that section of this treatise which deals with the subject of 'The 

 Resistance of the 'Living' Protein Molecule to Proteolytic Enzymes," and the ready 

 hydrolysis of denatured proteins and the proteins of mechanically injured cells by 

 proteolytic enzymes. 



