May 15, 
Amino Acids and Polypeptides in Rye Kernels 
991 
dues were treated once more in the 
same manner. The filtrates were then 
made up to 1 liter, of which 400 c. c. 
portions were oxidized according to 
Kjeldahl’s method. In the case of 
alcohol, 100-gram portions of flour were 
extracted with boiling 92 per cent 
alcohol, the extracts being separated by 
suotion on Buchner funnels from the 
solid residues, which were extracted 
once more in like manner. Filtrates 
and washings were now made up to 
1,000 c. e., in 400 c. c. portions, from 
which the alcohol was first removed by 
distillation and the nitrogen then esti¬ 
mated in the residue according to the 
Kjeldahl method. The results are re¬ 
corded in Table II. 
Reference to Table II shows that 
both the aqueous and alcoholic extrac¬ 
tions were quite uniform, but that water 
extracted about three times as much 
nitrogen as did the alcohol. This is 
perhaps due to a greater proportion of 
protein matter taken up by the water. 
In order to demonstrate the presence 
of amino acids and polypeptides in the 
rye kernel a series of flasks containing 
definite quantities of flour were treated 
with boiling-hot ammonia-free water, 
which was followed by digestion on the 
steam bath for 15 to *30 minutes. The 
extract was now separated by filtration 
or centrifugalization from the solid 
residue, which was treated once more 
in like manner. The extracts were then 
concentrated under reduced-. pressure, 
the precipitates formed (proteins, bran, 
etc.) removed by centrifugalization, 
the supernatant liquid evaporated to 
dryness, extracted with 85 per cent 
alcohol, filtered, the alcohol distilled off, 
and finally evaporated to dryness. 
The dry residue, a dry yellow sirup, 
was taken up with hot water, filtered, 
cooled, and made up to 100 c. c. (or its 
multiple), of which two portions of 10 
c. c. each were oxidized according to 
Kjeldahl’s method. To the remaining 
solution sulphuric acid was added to a 
concentration of 5 per cent, which was 
followed by treatment with a phos- 
photungstic acid solution containing 5 
grams of sulphuric acid and 20 grams of 
phosphotungstic acid per 100 c. c., 
using but a slight excess of the pre¬ 
cipitant. After 24 hours the heavy 
precipitate was filtered out and thor¬ 
oughly washed with a solution contain¬ 
ing 5 gm. of sulphuric acid and 2.5 gm. 
of phosphotungstic acid per 100 c. c. 
The precipitate formed by phospho¬ 
tungstic acid ordinarily removes any 
proteins, proteoses, and peptones pres¬ 
ent. That it also contained diamino 
acids was shown in the following man¬ 
ner: The precipitate was treated with 
barium hydroxide, the excess of which 
was removed with carbon dioxide, the 
whole was filtered off with suction, 
and the remaining cake thoroughly 
washed with hot water. Filtrate and 
washings were evaporated in vacuo 
practically to dryness, taken up with 
a few cubic centimeters of water, and 
filtered. The filtrate, which had a 
light-yellow color, gave the following 
reactions: 
(1) Phosphotungstic acid gave at once a heavy 
White precipitate. 
(2) , Phosphomolybdic acid gave immediately 
a yellow precipitate. 
(3) Silver nitrate gave a grayish precipitate 
soluble in excess of ammonia. 
(4) Addition of neutralized formaldehyde caused 
the solution to become acid. 
(5) Mercuric chloride gave a grayish floeculent 
precipitate. 
The filtrate from the phosphotung¬ 
stic precipitate was freed from sulphuric 
and phosphotungstic acids by treat¬ 
ment with calcium hydroxide to slight 
acidity, then with barium hydroxide 
to alkalinity, the excess of barium 
hydroxide being removed with carbon 
dioxide. The Whole was now brought 
to a boil and filtered, the solid residue 
being extracted once more with hot 
ammonia-free water. The filtrate and 
washings were then concentrated under 
reduced pressure and made up to 100 
o. c., of which two portions of 20 c. c. 
each were oxidized by the Kjeldahl 
method, while 50 c. c. were (on being 
freed from carbon dioxide, phosphoric 
acid, and coloring matter) formol- 
titrated, which gave the nitrogen of 
monoamino acids. 
The presence of polypeptides was 
demonstrated as follows: The. flour 
extract and the phosphotungstic acid 
precipitate were obtained exactly in 
the manner just described. Equally, 
the filtrate from the phosphotungstic 
precipitate was freed from sulfuric 
and phosphotungstic acids and finally 
concentrated in vacuo to 100 c. c., 
as outlined above. In two 10 c. c. por¬ 
tions the nitrogen was estimated ac¬ 
cording to the Kjedahl method. To 
the remaining 80 c. c. hydrochloric 
acid was added to a concentration of 
20 per cent and hydrolyzed under a 
reflux condenser for 12 hours, in ac¬ 
cordance with the observations of 
Fischer (4). The hydrolysate was 
now evaporated on the water bath to 
dryness, taken up with hot water, 
and distilled with magnesium oxide 
in order to remove the ammonia. The 
residue was then thoroughly extracted 
with hot water (which was previously 
freed from ammonia by long boiling), 
evaporated, and made up to 100 c. c., 
of which two portions of 20 c. c. each 
