212 COMPOSITION OF THE DIGESTIVE JUICES. [CH. VIII. 



of the protein is not complete even with a very prolonged 

 period of digestion. It is much more complete if the pro- 

 tein has been previously submitted to peptic digestion, 

 indicating the presence of certain groupings in the protein 

 molecule that are attacked by pepsin, and not by trypsin. 

 It must be remembered that in the course of natural diges- 

 tion in the body the proteins are liable to the attack of 

 three proteolytic enzymes, pepsin, trypsin and erepsin, and 

 that it is possible that complete hydrolysis is most effec- 

 tively and rapidly attained if the three enzymes are pre- 

 sented in due order. 



The course of hydrolysis can be followed by Sorensen's 

 method of formal titration, or by the direct estimation of 

 the amino-acid nitrogen by the gasometric method of D. 

 Van Slyke. As stated above, it is most rapid at the com- 

 mencement of the reaction, falling off as the hydrolysis 

 reduces the concentration of the substrate. The process 

 is a very complicated one to follow mathematically, owing 

 to the variety of substances simultaneously present which 

 are liable to be attacked by the enzyme, and to the fact 

 that the amount of enzyme is continuously decreasing 

 owing to its instability. For the estimation of trypsin 

 it is preferable to rely on the method given in Ex. 259. 



Preparation of Trypsin from Pig's Pancreas. 



The following method is a modification of that of Mellanby and Wooley. 

 It must be noted that the extract obtained does not contain amylopsin or 

 lipase, both of which are rapidly destroyed by acids. 



Obtain the fresh pancreas of the pig (usually sold as the " internal sweet- 

 bread "). Free the glandular tissue from fat as completely as possible, mince 

 finely in a machine and weigh. For every gram, of the mince add 3 cc. of 

 0-5 per cent, hydrochloric acid (by weight). This can be prepared with 

 sufficient accuracy by diluting 13-7 cc. of pure concentrated hydrochloric 

 acid (Sp. Gr. 1-16) to make 1000 cc. with distilled water. Stir the mixture 

 well at intervals for 30 minutes. Then add 6-4 cc. of 5 per cent, soda (or 8 cc. 

 of N. soda) for every 100 cc. of the dilute hydrochloric acid originally used. 

 This gives a reaction of about P H = 4-7, which results in a readily filterable 

 mass. Stir thoroughly and filter on a large folded paper. To the filtrate, 

 which is usually quite clear, add 10 per cent, soda to reduce the acidity to 

 about P H = 5-5, the optimum reaction for the preservation of trypsin. This 

 can be obtained approximately by cautiously adding the soda until a 2 cc. 

 portion of the mixture gives only a faint reddish tinge with a few drops of 

 methyl red. Then add toluol (10 cc. per litre) as a preservative, shake and 

 store in a stoppered bottle in a cool, dark, cupboard. Should the bottle be 



