35 



The washed precipitate of barium phosphotungstate and filter 

 were subjected to Kjeldahl determination for any nitrogen that 

 might be held by absorption, adsorption, or occlusion, as was also 

 the filter and contents remaining after the final filtration of the 

 solution containing the basic nitrogen. In all cases some nitrogen 

 was found. This nitrogen is probably derived from the "unad- 

 sorbed humin carried down with the !^asic phosphotungstates" 

 mentioned by Van Slyke (1915, p. 284). Inasmuch as my work was 

 done prior to this publication, I added this nitrogen to the total 

 nitrogen content of the bases instead of to the humin. 



During the distillation after kjeldahling this precipitate the 

 cochineal indicator took on a color which made the acid solution 

 appear that complete neutralization might have occurred when in 

 fact it had not. This color change was noticed in every case with 

 the barium phosphotungstate distillate. This made titration diffi- 

 cult, since a new end point had to be established. Gortner and 

 Holm (private communication) have observed a similar color 

 change in the case of fibrin hydrolyzed in the presence of a large 

 excess of formaldehyde. They explain this finding on the assump- 

 tion that pyridine (or some similar base) is formed which is not 

 easily broken down in the Kjedahl process (cf. Dakin and 

 Dudley, 1914), and which greatly influences the color changes of 

 the indicator when the base is volatalized during the subsequent 

 distillation with alkali. Whether or not this is the cause of the 

 phenomenon observed in my materials cannot be ascertained, with- 

 out further investigation. 



In no case did I attempt to separate the basic nitrogen into 

 the usual fractions of "arginine," "cystine," "histidine," and "lysine" 

 nitrogen, because I am not dealing with pure protein. Instead in 

 each case the total nitrogen liberated as ammonia was determined 

 on 25 cc. of the solution containing the bases. This was determined 

 in exactly the same manner that Van Slyke used for the determina- 

 tion of arginine nitrogen. The volume of standard acid neutralized 

 by the ammonia indicated the amount contained in the 25 cc. of 

 solution used. The nitrogen found is listed as "basic nitrogen set 

 free as ammonia by 50 per cent potassium hydroxide." The solution 

 remaining" from this determination was used in the estimation of 

 the total nitrogen of the bases. This was performed according to 

 Van Slyke's directions. The quantity of acid neutralized in this 

 determination was added to that neutralized in the basic-nitrogen- 

 set-free-as-ammonia-by-50-per-cent-potassium-hydroxide, thus se- 

 curing- the "total basic nitrogen." 



The "amino nitrogen of the bases"" was determined in Van 

 Slyke's (1912) apparatus, using 10 cc. portions of the solution. 



The filtrate from the bases was treated with sodium hydroxide 

 solution until a slight turbid precipitate -of lime was formed, and 

 then cleared immediately by the addition of acetic acid. This was 

 concentrated under diminished pressure and on cooling was made 

 to 200 cc. volume. The solutions were more or less violet in color. 

 "Total nitrogen in the filtrate from the bases" was determined on 

 duplicate portions of 25 cc. each by the method of Kjeldahl. The 



