Nov. i8, 1918 Physiological Studies of Normal and Blighted Spinach 397 



spinach, where the amounts of soluble nitrogen plus protein nitrogen 

 range from 106 to no per cent. Giving due allowance for the inaccura- 

 cies of the operation involved, it is reasonable to ascribe the discrepancies 

 noticed, not merely to the high results of Stutzer's protein method, but 

 also to the possibility that the protein of the diseased spinach examined 

 may perhaps differ from that of the healthy spinach in undergoing changes 

 more readily. 



DISTRIBUTION OF THE WATER-SOLUBLE NITROGEN IN SPINACH 



The determination of the nitrogen of acid amids, diamino acids, and 

 monoamino acids was made essentially according to Hausmann's method 

 (jp, 20), as modified by Osborne and Harris {31), and as applied to 

 soils by one of the writers {Jodidi 21, 22, 23, 24). The estimation of 

 the nitrogen of compounds other than those mentioned was made 

 according to methods which will briefly be described subsequently in this 

 paper. 



Ordinarily to 250 cc. of spinach solution, prepared as outlined above and 

 corresponding to 10 gm. of air-dry spinach, concentrated hydrochloric 

 acid was added to a concentration of 20 per cent, and boiled under a 

 reflux condenser 30 minutes. The hydrolyzed substance was now quan- 

 titatively transferred to a porcelain dish and evaporated on the steam 

 bath practically to dryness. 



In order to ascertain whether or not all of the acid amid nitrogen was 

 split off as ammonia under the conditions outlined, and at the same time 

 to completely hydrolyze any polypeptids present, another portion of 

 250 cc. of the same spinach extract was treated with enough concentrated 

 hydrochloric acid to give a 20 per cent concentration and boiled under 

 a reflux condenser for 8 hours. The fact that not all of the nitrogenous 

 constituents of plants are known, and the consideration that some of 

 them might be decomposed by boiling with 20 per cent hydrochloric 

 acid, made it desirable to hydrolize the spinach extract with as dilute 

 an acid as possible, but strong enough to split off quantitatively in the 

 form of ammonia the nitrogen of acid amids present. Inasmuch as 

 asparagin and glutamin are probably the principal acid amids contained 

 in plants, their behavior toward hydrochloric acid of different strength 

 was here of special interest. Unfortunately, the writers had to confine 

 the experiment to asparagin only, not having any glutamin. Its chem- 

 ical behavior, however, is known to be very similar to that of asparagin, 

 its lower homolog. 



Two and five-tenths gm. of crystallized asparagin, with a nitrogen 

 percentage of 18.38 (the formula C^HgNjOg -f- HjO requires 18.66 per cent 

 N), were dissolved in water and made up to 350 cc. Of this solution por- 

 tions of 20 cc, each, were transferred to small round-bottom flasks to 

 which concentrated hydrochloric acid was added until the desired per- 



