Nov. i8. 1918 Physiological Studies of Normal and Blighted Spinach 395 



Examination of Table V, first section, shows that the calculated 

 percentage of nonprotein nitrogen is usually higher than that found 

 directly by analysis. This is especially marked in the case of No. i to 10, 

 in which the filtrates from protein copper precipitate were concentrated 

 on the water bath without having previously been acidulated. Hov/- 

 ever, where acidulation of the filtrates did take place, as in No. 11 to 17, 

 the difference in the results is still not inconsiderable. It seems reasonable 

 to ascribe the loss of nonprotein nitrogen, at least in part, to the heating 

 of the spinach with copper hydroxid (incidental to the Stutzer method) 

 whereby the amids, which, as will be shown later, are contained in the 

 spinach, undoubtedly lose a part of their nitrogen as ammonia in addi- 

 tion to the loss of ammonia present as such in the plant. From the data, 

 which will be given later in this paper, it will be noticed that the sum of 

 ammoniacal and acid amide nitrogen in the healthy spinach tissues is, 

 as a rule, higher than the percentage present in the diseased tissues. 

 For this reason it could be expected that the difference between the 

 nonprotein nitrogen found and the nonprotein nitrogen calculated would 

 be greater in the case of the healthy plant tissues. Just why in the case 

 of the diseased spinach the nonprotein nitrogen calculated and found is 

 practically the same needs still further investigation. 



EXTRACTION OF THE NONPROTEIN NITROGEN OF THE SPINACH 



A preliminary experiment was made to ascertain how the nitrogen 

 can best be extracted from the spinach. Fifteen gm. of air-dry healthy 

 spinach were treated in a round-bottom flask with 100 cc. of boiling 

 hot ammonia-free water and digested on the steam bath with frequent 

 shaking for 15 minutes, when the substance was filtered through a Buch- 

 ner funnel provided with a filter, the extraction being repeated three 

 more times. The final cake remaining on the Buchner fu^nel was thor- 

 oughly washed with boiling hot water. The combined filtrates and wash- 

 ings were acidified with acetic acid, boiled for a few minutes, filtered, 

 and washed as usual on a filter. The filtrate and washings were cooled 

 and made up to i liter. Two more portions of 15 gm. each were treated 

 as outlined, with the difference that these two portions were extracted 

 six and eight times, respectively. Nitrogen estimations in aliquots of the 

 three extracts, showed that they contained, respectively, 55.86, 58.28, 

 and 59.31 per cent of the total nitrogen. Inasmuch as the healthy 

 spinach contains about 50 per cent of protein nitrogen (see Table IV), 

 the fact that the three extracts contained nitrogen in excess of what 

 could be expected, and more of it the more frequently the substance was 

 extracted, seemed to indicate that a part of the protein nitrogen went into 

 solution, probably through peptonization. The extraction was then 

 modified so as to use a smaller amount of water and to effect the extrac- 

 tion more rapidly. The procedure was as follows : 



