4 o8 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



The Products of the Action of Trypsin. 



Proteins are hydrolysed by trypsin and converted into amino acids 

 (and a polypeptide). 



The demonstration of the amino acids can be shown in a digest of 

 protein, prepared by treating about 100 gm. of caseinogen, or other 

 protein, dissolved in 2000 c.c. of -iN ammonia or sodium carbonate at 

 37 for several days with about I gm. of dried pancreas preparation in 

 the presence of toluene or chloroform. 



(1) The solution will most probably contain a white precipitate, 

 which consists mainly of tyrosine. This is proved by filtering it off, 

 washing and dissolving it in dilute acetic acid and testing with Millon's 

 reagent (p. 268). 



(2) The filtrate from the tyrosine can be shown to contain trypto- 

 phan by acidifying a portion of about 5 c.c. with acetic acid and add- 

 ing bromine water, drop by drop, as described on p. 349. 



(3) On evaporating the filtered solution on a water- bath to a small volume 

 and allowing to stand for about 24 hours, a crystalline crust forms. This con- 

 sists mainly of tyrosine as can be shown by microscopic examination, especially 

 after solution in a drop of ammonia. The crystals will also give Millon's 

 reaction. 



(4) On further evaporation of the filtrate leucine and glutamic acid sep- 

 arate out on standing. Microscopic examination will show that the crystals 

 consist mainly of rounded cones with a radiating striation (leucine). If free 

 from tyrosine they will not give Millon's reaction. They dissolve in hot water 

 and form copper salts, as can be shown by adding a very little caustic soda and 

 a few drops of copper sulphate. The precipitate of cupric hydroxide dissolves 

 on warming, giving a blue solution. 



IX. Trypsinogen. 



Trypsinogen is present in the pancreas and is contained in pancreatic 

 juice; it may be prepared from the pancreas by the method de- 

 scribed by Mellanby and Wool ley : 



The pancreas is removed without contact with the intestines. It 

 is finely minced and treated with twice its weight of -5 per cent, 

 hydrochloric acid at room temperature for 12 hours. The solution is 

 strained through muslin and neutralised with sodium carbonate. The 

 precipitate is filtered off and the solution is kept under toluene. 



Preparation of Pancreatic Juice. 



The mucous membrane of the small intestine is ground up with sand and 

 boiled with dilute hydrochloric acid. The boiling solution is neutralised with 

 dilute alkali. Coagulable proteins are thus precipitated and filtered off. The 

 solution contains secretin. Secretin is not an enzyme as it can be boiled, but 

 belongs to the class of substances termed hormones by Professor Starling. 



A cannula is placed in the pancreatic duct and the solution of secretin is 

 slowly injected into the jugular vein. Pancreatic juice flows from the cannula 

 after each injection and is collected in a clean vessel. It is mixed with an 

 equal volume of 2 per cent, sodium fluoride to preserve it. 



