IQ4 PHYSIOLOGICAL CHEMISTRY 



it at 40C. Into each tube introduce a small piece of fibrin and note the progress 

 of digestion. In which tube does the most rapid digestion occur? What is the 

 reason? 



3. Influence of Bile. Prepare three tubes as follows : 



(a) 5 c.c. of pancreatic extract -f- 0.5-1 c.c. of bile. 



(b) 5 c.c. of pancreatic extract + 5 c.c. of bile. 



(c) 5 c.c. of pancreatic extract. 



Introduce into each tube a small piece of fibrin and^keep them at 4OC. 

 Shake the tubes frequently and note the progress of digestion. Does the pres- 

 ence of bile retard tryptic digestion? How do these results agree with those 

 obtained under gastric digestion? 



4. Quantitative Determination of Tryptic Activity. 1 Gross* Method. This 

 method is based upon the principle that faintly alkaline solutions of casein are 

 precipitated upon the addition of dilute (i per cent) acetic acid whereas its digestion 

 products are not so precipitated. The method follows : Prepare a series of tubes 

 each containing 10 c.c. of a o.i per cent solution of pure, fat-free casein, 2 which 

 has been heated to a temperature of 4OC. Add to the contents of the series of 

 tubes increasing amounts of the trypsin solution under examination, 3 and place 

 them at 4OC. for fifteen minutes. At the end of this time remove the tubes and 

 acidify the contents of each with a few drops of dilute (i per cent) acetic acid. 

 The tubes in which the casein is completely digested will remain clear when 

 acidified, while those tubes which contain undigested casein will become more 

 or less turbid under these conditions. Select the first tube in the series which 

 exhibits no turbidity upon acidification, thus indicating complete digestion of 

 the casein, and calculate the tryptic activity of the enzyme solution under 

 examination. 



Calculation. The unit of tryptic activity is an expression of the power of i 

 c.c. of the fluid under examination exerted for a period of fifteen minutes on 10 

 c.c. of a o.i per cent casein solution. For example, if 0.5 c.c. of a trypsin solu- 

 tion completely digests 10 c.c. of a o.i per cent solution of casein in fifteen 

 minutes, the activity of that solution would be expressed as follows : 



Tryptic activity = 1-1-0.5=2. 



Such a trypsin solution would be said to possess an activity of 2. If 0.3 c.c. 

 of the trypsin solution had been required the solution would be said to possess an 

 activity of 3.3 ; i.e., i -1-0.3 =3-3' 



EXPERIMENTS ON PANCREATIC AMYLASE 



i. The Most Favorable Reaction. Prepare four tubes as follows' 



(a) i c.c. of neutral pancreatic extract+i c.c. of starch paste +2 c.c. of 

 water. 



(b) i c.c. of neutral pancreatic extract+i c.c. of starch paste+2 c.c. of 

 i per cent sodium carbonate. 



1 For a discussion of Spencer's method for the quantitative determination of trypsin 

 in stomach contents see chapter on Gastric Analysis. 



2 Made by dissolving i gram of Gru bier's casein in a liter of o.i per cent sodium car- 

 bonate. A little chloroform may be added'to prevent bacterial action. 



3 The amount of solution used may vary from o.i-i c.c. The measurements may 

 conveniently be made by means of a i c.c. graduated pipette. 



