1162 
Journal of Agricultural Research 
Vol. XXXI, No. 12 
30 c. c. of a solution containing 20 gms. of phosphotungstic acid and 
5 gm. of sulphuric acid per 100 c. c. After 24 hours, the precipitate 
was filtered off and washed with 200 c. c. of a solution containing 
5 gms. of sulphuric acid and 2.5 gms. of phosphotungstic acid per 
100 c. c., the washing being accomplished by rinsing the precipitate 
from filter into a beaker and returning to the filter three times. 
The washed precipitate was then oxidized according to the Kjeldahl 
method, yielding the nitrogen of any proteoses and peptones present, 
as well as of diamino acids. 
The filtrate from the phosphotungstic acid precipitate was treated 
with calcium hydroxide to slight acidity, then with barium hydrox¬ 
ide to distinct alkalinity, saturated with carbon dioxide, heated, 
filtered on Buchner, and thoroughly washed with boiling hot am¬ 
monia-free water. The residual cake was extracted at least once 
more with hot water. The filtrates and washings from sulphate, phos- 
photungstate, and carbonate of calcium and barium were ordi¬ 
narily saturated once more with carbon dioxide, and when clear con¬ 
centrated in a vacuum, filtered, and made up to 100 c. c., of which 
50 c. c. were used directly for the estimation of the amino nitrogen 
according to Van Slyke’s method, and jn the other 50 c. c. the amino 
nitrogen was estimated, on hydrolysis, as outlined above. By this 
method in which any proteoses and peptones present are removed, 
quantitatively, it was found that, on the average, after two days 
the seedling extract contained 22.08 per cent of amino nitrogen and 
33.83 per cent of peptide nitrogen; after four days it contained 
23.08 per cent of ammo nitrogen and 30.60 per cent of peptide 
nitrogen, calculated on the basis of the water-soluble nitrogen. 
These figures reasonably approximate those obtained by the tannic- 
acid method alone, as a glance at columns 8 and 9 of Table V shows. 
PROTEOLYTIC ENZYMES 
The appearance of polypeptides, amino acids, and acid amides— 
well known disintegration products of proteins—in the aqueous 
extracts of the corn seedlings is to be attributed to the action of 
proteolytic enzymes. In view of the doubt expressed by some 
writers (2) as to whether these enzymes appear for the first time 
during the process of germination or are preexistent in the resting 
seed, it should be emphasized that the proteases are unquestionably 
preexistent in the ungerminated corn seed, since the latter was 
shown by the writer (11) to contain polypeptides and amino acids. 
This is further confirmed by the observation that the resting seed 
respires like the growing plant, though in a very much lesser degree 
(4), and it is for this reason that the decrease in weight shown by 
cereal grains during their storage is to be ascribed not only to loss of 
water but also to loss of carbon dioxide. The acid reaction which „ 
has been shown in this paper gradually to increase as germination of 
the corn seed progresses is a powerful aid to the proteases in their 
degradation of the proteins, since the action of proteolytic enzymes 
is known to be very much more intensive in an acid medium than 
in one that is neutral or alkaline (26, p. 228). 
