1 6 PHYSIOLOGICAL CHEMISTRY. 



may be prepared .from the intestinal worm, ascaris, according to the 

 directions given on page 15. 



4. Demonstration of Anti-Trypsin. Introduce into a test-tube 

 a few shreds of fibrin and equal volumes of an artificial tryptic 

 solution 1 and the ascaris extract made as described on page 15. 

 Prepare a control tube in which the ascaris extract is replaced by 

 water. Place the two tubes at 38 C. Ordinarily the fibrin in the 

 control tube will be completely digested in two hours. The fibrin 

 in the tube containing the ascaris extract may, however, remain 

 unchanged for days, thus indicating the inhibitory influence of the 

 anti-enzyme. 



Blood serum also contains anti-trypsin. This may be demon- 

 strated as follows : Introduce equal volumes of serum and artificial 

 tryptic solution (prepared as described above) into a test-tube and 

 add a few shreds of fibrin. Prepare a control tube containing boiled 

 serum. Place the two tubes at 38 C. It will be observed that the 

 fibrin in the tube containing the boiled serum digests, whereas that 

 in the other tube does not digest. The anti-trypsin present in the 

 unboiled serum has exerted an inhibitory influence upon the action 

 of the trypsin. 



C. Quantitative Applications. 



i. Quantitative Determination of Amy loly tic Activity. Wohl- 

 gemuth's Method. Arrange a series of test-tubes with diminishing 

 quantities of the enzyme solution under examination, introduce into 

 each tube 5 c.c. of a i per cent solution of soluble starch 2 and place 

 each tube at once in a bath of ice water. 3 When all the tubes 

 have been prepared in this way and placed in the ice water-bath they 

 are transferred to a water-bath or incubator and kept at 38 C. 

 for from thirty minutes to an hour. 4 At the end of this digestion 



1 Made by dissolving 0.04 gram of sodium carbonate and 0.015 gram of trypsin 

 in 8 c.c. of water. 



2 Kahlbaum's soluble starch is satisfactory. In preparing the i per cent solu- 

 tion, the weighed starch powder should be dissolved in the proper volume of 

 cold, distilled water and stirred until a homogeneous suspension is obtained. 

 The mixture should then be heated, with constant stirring, in a porcelain dish, 

 until it is clear. This ordinarily takes about 8-10 minutes. A slightly opaque 

 solution is thus obtained which should be cooled before using. 



3 Ordinarily a series of six tubes is satisfactory, the volumes of the enzyme 

 solution used ranging from i c.c. to o.i c.c. and the measurements being made 

 by means of a i c.c. graduated pipette. Each tube should be placed in the ice 

 water bath as soon as the starch solution is introduced. It will be found 

 convenient to use a small wire basket to hold the tubes.' 



4 Longer digestion periods may be used where it is deemed advisable. If ex- 

 ceedingly weak solutions are being investigated, it may be most satisfactory to 

 permit the digestion to extend over a period of 24 hours. 



