198 PHYSIOLOGICAL CHEMISTRY 



stand for 6-24 hours at room temperature, shaking occasionally, toluol being 

 added as a preservative. Strain and filter. 



2. Demonstration of Intestinal Nucleases. Prepare a 2 per cent solution 

 of yeast nucleic acid put in solution with the aid of just sufficient NaOH solution 

 to make the resulting mixture neutral to litmus. To each of two large test-tubes 

 add 20 c.c. of the intestinal extract prepared as above. Boil one for one to two 

 minutes. To each tube then add 10 c.c. of the 2 per cent nucleic acid solution. 

 Add 2-3 c.c. each of toluol and chloroform to each mixture. Keep at 38C. for 

 24 hours. 



Heat the tubes to boiling in a water-bath to coagulate protein. Add 5 c.c. of 

 5 per cent HC1 and allow to stand for one hour. This precipitates any unchanged 

 nucleic acid. Filter and take aliquots of the filtrate (about 20 c.c.). Precipitate 

 the phosphate from each mixture by adding 5 c.c. of magnesia mixture and 5 c.c. 

 of ammonia. Allow to stand over night. A heavy precipitate of magnesium 

 ammonium phosphate should be found in the test experiment indicating that the 

 phosphoric acid of the nucleic acid had been liberated by the nucleotidase of the 

 intestinal extract. The control should show only a slight precipitate. 



If desired the phosphorus of the precipitates may be determined quantitatively 

 by dissolving in 2 per cent HNOs, precipitating as the phosphomolybdate and 

 determining volumetrically according to the Neumann procedure (see 554). 



EXPERIMENTS ON EREPSIN 



1. Preparation of Erepsin. Grind the mucous membrane of the small intes- 

 tine of a cat, dog, or pig with sand in a mortar. Treat the finely divided membrane 

 with toluol- or chloroform-water and permit the mixture to stand, with occasional 

 shaking, for 24-72 hours. 1 Filter the extract thus prepared through cotton and 

 use the filtrate in the following experiment. 



2. Demonstration of Erepsin. To about 5 c.c. of a i per cent solution of 

 Witte's peptone in a test-tube add about i c.c. of the erepsin extract prepared as 

 described above and make the mixture slightly alkaline (o.i per cent) with sodium 

 carbonate. Prepare a second tube containing a like amount of peptone solution 

 but boil the erepsin extract before introducing it. Place the two tubes at 38C. 

 for two to three days. At the end of that period heat the contents of each tube 

 to boiling, filter and try the biuret test on each filtrate. In making these tests 

 care should be taken to use like amounts of filtrate, potassium hydroxide and 

 copper sulphate in each test in order that the drawing of correct conclusions may 

 be facilitated. The contents of the tube which contained the boiled extract 

 should show a deep pink color with the biuret test, due to the peptone still present. 

 On the other hand, the biuret test upon the contents of the tube containing the 

 unboiled extract should be negative or exhibit, at the most, a faint pink or blue 

 color, signifying that the peptone, through the influence of the erepsin, has been 

 transformed, in great part at least, into amino-acids which do not respond to the 

 biuret test. 2 



1 The enzyme may also be extracted by means of glycerol or alkaline "physiological" salt 

 solution if desired. 



2 Strictly speaking, this erepsin demonstration is not adequate unless a control test 

 is made with native protein (except casein, histones and protamines) to show that the 

 extract is trypsin-free and digests peptone but not native protein. 



