482 Journal of Agricultural Research voi. xiii, no. 10 



in Table III, i c. c. of serum 268 in mixture A contained 97.5 mgm. of 

 coagulable protein four days after the mixture had been prepared. In 

 mixture D digestion was going on, and i c. c. of serum in this mixture 

 contained but 4.7 mgm. of coagulable protein. The other 92.8 mgm., 

 95 per cent of the total, had been transformed into proteoses and pep- 

 tones which do not coagulate on heating. This figure, 95 per cent, 

 together with others similarly calculated, is to be foimd in Table VII. 



Amino nitrogen. — Although the determination of coagulable pro- 

 tein digested past the coagulable stage, as described, gives valuable infor- 

 mation regarding the extent of digestion, further information was ob- 

 tained by determining the amino nitrogen liberated at the same time. 

 Van Slyke's method and apparatus were used as described in a pre- 

 vious publication by the senior writer (j, p. 6g6). For analytic pur- 

 poses ammonia was removed in the following manner : At the close 

 of the digestion period lo-c. c. portions of the various mixtures were 

 transferred to porcelain crucibles, to each of which 0.5 c. c. of con- 

 centrated hydrochloric acid was added to destroy the trypsin at the 

 desired time. These were allowed to stand for one hour and then made 

 faintly but distinctly alkaline to litmus strips by the addition of about 

 0.3 gm. of sodium carbonate. The crucibles were then placed in a 

 vacuum desiccator provided with a shallow dish containing some NI5 sul- 

 phuric acid. No attempt was made to estimate the ammonia. That 

 the removal was complete was shown by numerous blanks with pure 

 ammonium sulphate in this and in the following procedure. The next 

 day the contents of the crucibles were used for the amino-nitrogen 

 determinations; 5 or 10 c. c. of digestion mixture were used for one 

 determination, according to the amount of amino nitrogen present. The 

 results are given in Table III, last column, and have been corrected for 

 amino nitrogen in the trypsin and pepsin. The amounts found in 

 mixture A represent the preformed amino nitrogen present in the serum 

 or antitoxin, and were subtracted from the amounts found in the other 

 mixtures, when calculating the amount liberated by the digestion. 



For total amino-nitrogen determination, 10 c. c. of serum or antitoxin 

 were boiled with 10 c. c. of concentrated hydrochloric acid for 24 hours 

 under a reflux condenser. The mixture was evaporated to dryness to 

 expel hydrochloric acid, made alkaline with sodium-carbonate solution, 

 keeping the total volume small, and set aside in an incubator room for 

 24 to 48 hours for the ammonia to escape. The mixtures usually evapor- 

 ated almost to dr>Tiess. They were taken up with water and 0.5 c. c. 

 concentrated hydrochloric acid, transferred to a 50-c. c. volumetric 

 flask and diluted to the mark. For a single determination in the amino- 

 nitrogen apparatus, 5 or 10 c. c. of the unfiltered solution were used, 

 containing i or 2 c. c, respectively, of hydrolyzed serum or antitoxin. 



Pepsin-acid mixtures were introduced directly into the Van Slyke 

 apparatus without removal of ammonia, as the amounts were too small 



