A STUDY 
OF THE 
Proteids of the Corn or Maize Kernel 
BY 
R. H. CHITTENDEN, Ph. D., 
Professor of Physiological Chemistry in Yale University, 
AND 
THOMAS B. OSBORNE, Ph. D., 
Chemist at the Connecticut Agricultural Experiment Station. 
Reprinted from the American Chemical Journal, Vol. 13, Nos. 7 and 8, 
and Vol. 14, No. x. 
THE FRIEDENWALD COMPANY, 
BALTIMORE, 
1892. Contributions from the Sheffield Biological Laboratory of Yale University. 
A STUDY OF THE PROTEIDS OF THE CORN OR 
MAIZE KERNEL. 
By R. H. Chittenden, Ph. D., Professor of Physiological Chemistry in Yale 
University, and Thomas B. Osborne, Ph. D., Chemist at the 
Connectictit Agricultural Experiment Station. 
The character of the proteids present in the kernel or seed of 
corn or maize, the most important of American cereals, has up to 
the present time never been investigated to any great extent. In 
fact, our knowledge of this subject has been practically limited to 
the observations of Ritthausen1 concerning “maize fibrin,” a body 
soluble in alcohol, and to the bare statement made by Th. Weyl3 
that the powdered seeds of maize yield to a 10-per cent, solution 
of sodium chloride a globulin-like substance which, after purifica- 
tion by repeated precipitation with water and re-solution in salt 
water (10 per cent.), coagulates at 750 C. It is a fair presumption, 
however, judging from the interesting results obtained by Sidney 
Martin, Vines and other workers among the vegetable proteids, 
that the proteids of the .corn kernel must doubtless be as numerous 
and complex as those of other seeds already investigated, and 
this we have indeed found to be the case. In fact, the subject, 
w'hile more or less fruitful in results, has proved an exceedingly 
complex one, on account of the large number of different proteids 
present in the seed, the small quantities present, and the ease with 
which several of the bodies, especially the globulins, are appa- 
rently converted into other forms. Again, in view of the probable 
widespread distribution of the vegetable proteolytic ferments, 
any study of the vegetable proteids normally present in seed or 
1 Die der Getreidearten, Hiilsenfriichte und Olsamen, Bonn, 1872, p. 113. 
2 Zlschr. fiir physiolog. Chem. 1, 84. 2 
fruit must necessarily be complicated by possible ferment changes, 
through which new bodies may be formed. 
Our study of the subject has naturally divided itself into three 
main parts: ist, a study of the proteids soluble in salt solutions, 
but insoluble in water ; 2d, a study of the proteids soluble both in 
water and in dilute salt solutions ; 3d, a study of the proteid matter 
soluble in alcohol, but insoluble in water and salt solutions. 
I. Proteids Soluble in Salt Solutions, but Insoluble in 
Water. 
Finely ground corn meal1 extracted with a 10-per cent, solu- 
tion of sodium chloride yields a slightly opalescent fluid, moder- 
ately rich in globulin-like bodies. A simple aqueous extract of 
ground corn likewise contains more or less globulin held in solu- 
tion by the salts dissolved from the corn. From both solutions 
the globulins are slowly precipitated by dialysis of the salts, and 
partially so by addition of water. They are, moreover, com- 
pletely precipitated, mixed with other proteids, on saturation of 
their solutions with ammonium sulphate. 
a. Direct extraction of corn meal with 10-per cent, solution of 
sodium chloride, and separation of the globulin by dialysis. 
In view of the well known action of water, in the presence of 
possible ferments, on the globulins of some seeds, the first extrac- 
tions of the corn kernel were made with salt solutions, with a view 
to diminishing the possibility of cleavage or dissociation of the 
normal proteids of the kernel, 5 kilos, of finely ground corn 
meal were therefore soaked in 8 litres of 10-per cent, solution of 
sodium chloride, with frequent stirring, for 24 hours, after which 
the fluid was strained off and the residue placed in a screw-press 
and squeezed dry, A second extraction of the meal was made 
with 4 litres of fresh salt solution, and the two extracts united. 
The solution was then filtered through thick filter-paper, yielding 
an almost perfectly clear yellowish filtrate. 
Tested by fractional heat-coagulation a series of coagulums 
were obtained commencing at about 35°-40°, a final one making 
its appearance as the solution was boiled. Further, the clear 
filtrate from this precipitate gave on addition of acetic acid a 
1A1! the corn used in these experiments was of one variety, known as White Dent, and was 
freshly ground to a fine powder prior to each experiment. 3 
flocculent precipitate of proteid matter, all of which may be taken 
as evidence of the presence of a variety of proteid bodies, coagu- 
lable and non-coagulable by heat. 
The entire salt solution, with its content of proteids and other 
substances, was then dialysed1 in a stream of running water for a 
period of six days, at the end of which time chlorides were entirely 
removed. As the percentage of salt in the solution began to 
diminish a heavy white precipitate made its appearance, which 
steadily increased in amount as the removal of the salt was con- 
tinued, leaving a perfectly clear fluid containing some coagulable 
proteids. The globulin precipitated in this manner was separated 
from the fluid by decantation, washed somewhat with distilled 
water, and then redissolved in a 10-per cent, solution of sodium 
chloride. This latter solution was somewhat turbid even after 
repeated filtration, A portion tested by heat-coagulation gave the 
following results : At 6o° the fluid became decidedly turbid, with 
formation of flocks at 64°. The temperature was then raised to 68° 
and the mixture filtered. The clear filtrate became turbid at 740, 
with formation of a flocculent precipitate at 790, which was filtered 
off when the temperature reached 82°. The filtrate from this 
precipitate became turbid at 83°, with separation of flocks at 
87°. The temperature was then raised to go° and the precipi- 
tate filtered off. This filtrate on being heated to boiling gave no 
precipitate at first, but as the boiling was continued considerable 
flocculent matter gradually made its appearance. The largest 
coagulum was obtained at 790. If we are to trust the results of 
this fractional heat-coagulation, the natural inference would be that 
the globulin-like substance separated by dialysis from the sodium- 
chloride extract of the corn meal is made up of several distinct 
globulins, or else that the one globulin possibly present is broken 
apart into several fragments by the action of heat. 
The main portion of this sodium-chloride solution of the globulin 
was dialysed until the salt was entirely removed, a process which 
took five days and which left the globulin completely precipitated 
on the sides of the parchment. The peculiar, somewhat granular, 
appearance of the deposit led us to examine it under the micro- 
scope, when it was seen to be composed entirely of spheroids, thus 
showing a close approach to crystallisation. The precipitate was 
1 During these long periods of dialysis, putrefactive changes were prevented by the occa- 
sional addition of a few drops ofa 20-per cent, alcoholic solution of thymol. 4 
collected on a filter, washed with water, alcohol and ether, and 
when air-dried was found to weigh 10. i grams, thus indicating 
about 0.2 per cent, of globulin in the air-dry kernel, on the 
assumption that the extraction and separation were complete. 
The filtrate from this last separation of the globulin was almost 
entirely free from proteid matter, giving only a very slight reaction 
with Millon’s reagent, no further separation of any matter by 
continued dialysis, neither any precipitate on addition of acetic 
acid or acetic acid and solution of salt, and only the faintest 
turbidity on boiling. 
A portion of the globulin dissolved in 10-per cent, solution of 
salt yielded by heat-coagulation much the same results as the 
previous salt solution of the proteid, viz. turbidity at 58°, forma- 
tion of flocks at 67°. In the filtrate from this coagulum a second 
turbidity appeared at 770, followed by flocks at 87°. Further, in 
this filtrate another coagulum appeared on boiling the solution, so 
that while the individual temperatures of coagulation were some- 
what different from the preceding, they point to essentially the 
same general conclusion. 
A portion of the globulin was dried at 1 io° C. until of constant 
weight, and then analysed with the following results : 
Analysis' of Corn Globulin, Preparation A. 
I. 0.3933 gram substance gave 0.2325 gram HsO —6.57 per cent. H, and 
0.7250 gram C02 ~ 50.42 per cent. C. 
11. 0.3693 gram substance gave 0.2182 gram II20 6.55 per cent. H, and 
0.6845 gram C02~ 50.55 per cent. C. 
111. 0.3038 gram substance gave 44.1 cc. N at io° C. and 761.4 mm. pres- 
sure ~ 17.62 per cent. N. 
IV. 0.3067 gram substance gave 44.1 cc. N at 8.5° C. and 757.8 mm. pres- 
sure zz 17.47 Per cent. N. 
V. 0.5 m gram substance fused with KOH + KNO3 gave 0.0406 gram 
BaS04 ~ 1.08 per cent. S. 
VI. 0.6773 gram substance fused with KOH-j-KN03 gave 0.0440 gram 
BaS04 = 0.89 per cent. S. 
VII. 0.5265 gram substance gave 0.0102 gram ash ~ 1.93 per cent. 
1 Carbon and hydrogen were determined by combustion in a current of oxygen, in an open 
tube, the vapors passing over a fairly long layer of coarsely granulated oxide of copper, a 
shorter layer of chromate of lead, and an anterior roll of freshly reduced metallic copper. 
Nitrogen was determined by combustion with oxide of copper after the Dumas method, while 
sulphur was estimated by Hammarsten’s modification of Liebig’s method. 5 
Percentage Composition of the Ash-free Substance. 
c 
5Mi 
5r-54 
Average. 
51.48 
H 
6.69 
6.67 
.. .. 6.68 
N 
17.97 
17.82 
17.90 
S 
1.to 0.91 1.01 
o 
22.93 
100.00 
b. Direct extraction of corn meal with 10-per cent, solution of 
sodium chloride, and separation of the globulin by ammonium 
sulphate and dialysis. 
25 kilos, of corn meal were extracted with 50 litres of 10-per 
cent, salt solution, and the residue of meal extracted a second 
time with 16 litres of salt solution. The two extracts were united, 
filtered through paper, and the clear fluid saturated with pure 
ammonium sulphate. This gave rise to a more or less sticky pre- 
cipitate composed of all the proteids present in the extract. The 
precipitate so obtained was filtered off, dissolved in water as far as 
possible, and then treated with 10-per cent, salt solution. The 
portion remaining insoluble was washed with salt solution as 
long as anything was removed, and then reserved for further 
treatment. 
The solutions of the ammonium-sulphate precipitate in water 
(or rather in dilute ammonium sulphate) and in 10-per cent, sodium 
chloride were united and dialysed for two weeks, at the end of 
which time a large amount of globulin had separated from the 
solution. The clear fluid, however, still contained some globulin, 
and was therefore reserved for further examination. The sepa- 
rated globulin was filtered off and redissolved in about 2 litres of 
ro per cent, solution of sodium chloride, leaving almost no in- 
soluble residue, and the solution again dialysed until the globulin 
had separated. This required seven days. The product, after 
being washed and air-dried, weighed 42 grams, and was composed 
wholly of spheroids. The filtrate from this latter deposit of 
globulin was again returned to the dialyser, but failed to give 
any further separation of globulin even after ten days’ continued 
dialysis. 
It is to be observed here that the separation of this globulin 
from a pure sodium-chloride solution is fairly rapid and quite 6 
complete, whereas the presence of ammonium sulphate interferes 
decidedly with its precipitation by dialysis, doubtless on account 
of the slower rate of diffusion characteristic of this salt. 
The globulin prepared in this manner was readily soluble in salt 
solution, leaving only a slight insoluble residue. Subjected to heat- 
coagulation, the following results were obtained with a moderately 
large amount of globulin in a 10-per cent, solution of sodium 
chloride: The solution became turbid at 62°, with separation of 
flocks at 76.5°. The mixture was then kept at 78° for half an 
hour, after which it was filtered, the filtrate yielding a further 
turbidity at 83°, with separation of flocks at 930. This latter 
coagulation was considerably heavier than the one at 76°. On 
boiling the solution, there was a slight increase in the amount 
of coagulum, which was still further increased by the addition of a 
drop or two of very dilute acid. Hence, this product shows 
approximately the same general range of heat-coagulation points 
as the preceding preparation, separated by simple dialysis from 
the original sodium-chloride extract. As the salt solution of the 
globulin showed a faint alkaline reaction, another test was made 
with a solution of approximately the same strength as the preced- 
ing, but carefully neutralised. This solution grew turbid at 64°, 
with formation of flocks at 76.5°. The mixture was filtered at 770, 
and on being heated further became turbid at 82.5°, with separa- 
tion of flocks at 91 °. The filtrate from this latter precipitate 
became turbid on boiling, and yielded a large precipitate on addi- 
tion of acetic acid. It is thus evident that the proteid dissolved 
in a neutral salt solution cannot be wholly coagulated by heat, 
even though the heating be continued for some time. In fact, the 
solution may be evaporated to dryness on a water-bath, the residue 
taken up again in water, and in the salt solution which results 
considerable proteid matter will be found dissolved and non- 
coagulable on further application of heat. In fact, the great 
bulk of this product appears to be non-coagulable by heat alone. 
Variations in the proportion of globulin dissolved in the 10-per 
cent, salt solution modify the temperatures of coagulation only 
slightly. 
A portion of the substance dried at no° C. until of constant 
weight was analysed with the following results, which, aside from 
a slightly higher percentage of carbon, show a close agreement in 
composition with the corresponding globulin A. 7 
Analysis of Corn Globulin, Preparation B. 
I. 0.3500 gram substance gave 0.2180 gram H20zz6.82 per cent. H. 
11. 0.3251 gram substance gave 0.1992 gram H2O zz 6.81 per cent. H, and 
0.6145 gram C02 zz 51.55 per cent. C. 
111. 0.3717 gram substance gave 0.7021 gram C02 51.52 per cent. C. 
IV. 0.3592 gram substance gave 50.5 cc. N at 2.50 C. and 769.1 mm. 
pressure zz 17.73 Per cent. N. 
V. 0.7523 gram substance fused with KOH + KNO3 gave 0.0472 gram 
BaS04 0.86 per cent. S. 
VI. 0.7871 gram substance fused with KOH + KNOs gave 0.0491 gram 
BaS04 0.85 per cent, S. 
VII. 0.4622 gram substance gave 0.0025 gram ash zz 0.54 per cent. 
Percentage Composition of Ashfree Substance. 
c 
51-83 
Average. 
51.80 . . . . . . 5I-82 
H 
6.85 
6.84 
6.85 
N 
17.82 .. .. 17.82 
S 
.. .. 0.86 0.85 0.86 
o 
22.65 
100.00 
Another sample of globulin was prepared as follows: 2.5 kilos, 
of ground corn were extracted with about 10 litres of 10 per cent, 
solution of sodium chloride, and the extract, together with the 
washings, after filtration, saturated with ammonium sulphate. 
The precipitate was dissolved as far as possible in water and in 10- 
per cent, salt solution, and the united filtrates dialysed until free 
from chlorides. The deposit of globulin, which gradually formed, 
was filtered off, washed with alcohol and ether, and dried at 1 io° 
C. until of constant weight. 
On analysis it gave the following results : 
Analysis of Corn Globulin, Preparation C. 
I. 0.2307 gram substance gave 0.1442 gram H2O 6.95 per cent. H, and 
0.4330 gram C02 zz 51.17 per cent, C. 
11. 0.3528 gram substance gave 51.5 cc. N at n.50 C. and 745.6 mm. pres- 
sure z= 17,26 per cent. N. 
111. 0.3000 gram substance gave 0.0028 gram ash —0.93 per cent. 8 
Percentage Composition of the Ash free Substance. 
Average. 
c 
51.64 
51.64 
H 
7.01 
7.01 
N 
17.42 
17.42 
0} 
23-93 
100.00 
Still another preparation of globulin was separated in the follow- 
ing manner: 2.5 kilos, of corn meal were extracted with 12 litres 
of 5-per cent, solution of ammonium chloride, the residue of 
meal re-extracted with more ammonium-chloride solution, and the 
united filtrates precipitated with ammonium sulphate. This pre- 
cipitate was dissolved as far as possible in water and 5-per cent, 
ammonium-chloride solution, leaving only a small insoluble residue, 
and the united fluids dialysed in running water for seven days. 
The globulin which separated was then dissolved in 10-per cent, 
solution of sodium chloride and redialysed until the salt was 
entirely removed. The precipitated globulin was then washed 
with water, alcohol and ether, and after being dried at no° C. 
was analysed with the following results : 
Analysis of Corn Globulin, Preparation D. 
I. 0.1289 gram substance gave 0.0781 gram H2Q = 6.74 per cent. H, and 
0.2432 gram C02 “51.43 per cent. C. 
11. 0.2639 gram substance gave 39.x cc. N at ii° C. and 746.5 mm. pres- 
sure 17.57 per cent. N. 
111. 0.2561 gram substance gave 0.0012 gram ash —0.47 percent. 
Percentage Composition of the Ash-free Substance. 
c 
51-67 
Average. 
51.67 
H 
6.77 
6.77 
N 
17-65 
I7-65 
S 
o 
}23-9! 
100.00 
In these four preparations we have a good illustration of the 
general character of the globulin obtainable from the corn or 
maize kernel by direct extraction with salt solution. The product 9 
appears to be the same whether directly separated from its saline 
solution by dialysis, or first precipitated by ammonium sulphate 
and then separated by dialysis. The chemical composition of the 
four preparations is essentially identical, as seen from the accom- 
panying table; 
Globulin A. 
Globulin B. 
Globulin C. 
Globulin D. 
Average. 
c 
SI.48 
51.82 
$1.64 
51.67 
SI-65 
H 
6.68 
6.85 
7.01 
6.77 
6.82 
N 
17.90 
17.82 
17.42 
17.65 
17.69 
S 
r.oi 
0.86 ) 
°-93 
O 
22.93 
22.65 j 
23-93 
23.91 
22.91 
100.00 
100.00 
100.00 
100.00 
100.00 
Many things, however, point to the view that the corn globulin 
separated from the kernel by the above methods is not a single 
substance, although we must continually keep in mind the possi- 
bility of unknown methods of cleavage which may perchance 
be facilitated by the very processes made use of in testing the 
globulin. Foremost among these is the peculiar and more or less 
variable range of coagulation-points obtained whenever a sodium- 
chloride solution of the globulin is subjected to fractional heat- 
coagulation, From the data thus obtained we might argue a 
variable mixture of phyto-myosin, vitellin, and some non-coagu- 
lable globulin. As to myosin, however, saturation of a salt solu- 
tion of the globulin with salt crystals gives only a small precipitate, 
indicating but little of this substance. The next point suggestive 
of mixture is the fact that a portion of the globulin readily under- 
goes change into an insoluble modification by contact with water 
or strong salt solutions, while another portion is apparently very 
resistant to such action. Thus, in the precipitation of the globulin 
by saturation with ammonium sulphate or by dialysis, more or 
less insoluble matter is formed (insoluble in 10-per cent, salt 
solution), but this amount grows less and less as the process is 
repeated, being apparently co-extensive with the amount of this 
changeable globulin present. The bulk of the globulin, however, 
retains its solubility in salt solutions by this treatment, under 
ordinary circumstances. 
c. Fractional separation of the above corn globulin by various 
methods. 
Treatment of the purified globulin with 10-per cent, salt solution, 
as stated above, almost invariably shows the presence of at least a small amount of substance insoluble in salt solution, possibly 
due to a partial conversion of the globulin into an albuminate by 
the action of water. It was further found that addition of water 
to a 10-per cent, salt solution of the globulin was followed by only 
a partial precipitation of the proteid. Hence an attempt was 
made to separate the globulin into three fractions for analysis. 
Accordingly, 5-6 grams of the air-dry globulin, Preparation A, 
were treated with about 300 cc. of a 10-per cent, solution of sodium 
chloride, and the insoluble matter collected on a filter and washed 
thoroughly with 10-per cent, salt solution. This residue was finally 
washed with water until the salt was entirely removed, then with 
alcohol and ether. It weighed 0.57 gram (Preparation A 1). 
The salt solution and washings containing the bulk of the 
globulin, amounting to 430 cc., were diluted with distilled water 
to 4300 cc., by which an abundant precipitate was obtained. This 
was allowed to settle, then collected on a filter, washed free from 
chlorides, and lastly with alcohol and ether. It weighed 1.87 
grams (Preparation A 2). 
The dilute salt solution containing the remainder of the globulin, 
representing the more soluble portion, was dialysed in running 
water until the salt was entirely removed, at the end of which time 
the globulin had separated wholly in the form of spheroids. It 
was collected on a filter, washed with water, alcohol and ether, 
and weighed 1.6 grams (Preparation A3). 
These three fractions were dried at no° C. until of constant 
weight, and analysed with the following results: 
Analysis of Preparation A'2. 
I. 0.5068 gram substance gave 0.3082 gram H2O~ 6.75 Per cent. H, and 
0.9540 gram C02 51.34 per cent. C. 
11. 0.3622 gram substance gave 53.5 cc. N at 8° C. and 759.6 mm. pres- 
sure 18.01 per cent. N. 
111. 0.2994 gram substance gave 0.0025 gram ash —0.83 per cent. 
Percentage Compositio7i of the Ashfree Substance. 
Average. 
c 
51.76 
S1'?6 
H 
6.S0 
6.80 
N 
18.16 
18.16 
S 
o 
} 23.28 
100.00 11 
Analysis of Preparation A3. 
I, 0.2220 gram substance gave 0.1382 gram HaO 22:6.91 per cent. H, and 
0.4132 gram C02 zzz 50.76 per cent. C. 
11, 0,3221 gram substance gave 47.5 cc. N at 6° C. and 762.6 mm. pres- 
sure 18.19 Per cent. N. 
111. 0.3318 gram substance gave 49.3 cc. N at 5.8° C. and 754.2 mm. 
pressure rz 18.14 Per cent. N. 
IV. 0.2977 gram substance gave 0.0022 gram ash 0.74 per cent. 
Percentage Composition of the Ashfree Substance. 
Average. 
c 
5J-!3 
S'-l3 
H 
6.96 
6.96 
N 
18.32 
18.27 
18.30 
S 
o 
} 23.61 
100.00 
Analysis of Preparation Al. 
I. 0.3030 gram substance gave 36.5 cc. N at 5.8° C. and 758.8 mm. pres- 
sure— 14-79 Per cent. N. 
11. 0.1914 gram substance gave 0.0x12 gram ash 5.85 per cent. 
Percentage of nitrogen in the ash-free substance rz: 15.59. 
The following table shows the relationship of the several pro- 
ducts : 
The original Portion soluble in Portion insoluble in Portion insol. in 
globulin. the dilute NaCl. the dilute NaCl. 10-per ct. NaCl. 
A. 
A3. 
A2. 
A1. 
c 
51.48 
51-13 
51-76 
H 
6.68 
6.96 
6.80 
N 
17.90 
18.30 
18.16 
55-59 
S} 
1.01 
22.93 
23.61 
23.28 
Ash 
i-93 
0.74 
0.83 
5.85 
From this it is to be seen that while there is no very radical 
point of difference in composition between the first three products, 
there are changes in the relative percentages which are strongly 
suggestive of admixture. Certainly that portion of the original 
globulin insoluble in 10-per cent, salt solution has a noticeably low 
percentage of nitrogen, surely low enough to account for the 12 
slightly higher percentages of nitrogen in the products A3 and A 2. 
From the data here offered there is no positive evidence as to 
whether A 1 is an alteration-product of the original globulin, or 
whether it simply represents a small impurity. The former view 
is certainly the more plausible one, however, especially when it is 
remembered that this globulin had been dissolved in a salt solu- 
tion and filtered two or three times previously. Again, it is to be 
noticed in comparing the two soluble products A 2 and A3, that 
the body containing the lower percentage of nitrogen contains the 
higher percentage of carbon ; and further, that in both products 
the percentage of nitrogen is by this treatment raised so as to 
correspond closely with the nitrogen content of pure phyto- 
vitellin. 
In view of these results a portion of corn globulin was next 
separated into a number of fractions by the solvent action of 
various strengths of salt solution, and the several fractions studied, 
especially with reference to their composition and heat-coagula- 
tion points. 
The globulin (Preparation E) was made from 5 kilos, of freshly 
ground corn by direct extraction with an abundance of 10-per 
cent, solution of sodium chloride, the proteids precipitated by 
saturation of the filtered solution with ammonium sulphate, this 
precipitate dissolved in water and salt solution, and the filtered 
fluid dialysed in running water for one week until the globulin 
had separated. 
The entire quantity of globulin, about 10 grams, was then 
treated directly with one litre of 0.25-per cent, solution of sodium 
chloride at the temperature of the room and kept in agitation for 
about 3 hours. The residue was then filtered off and extracted 
again with one litre of salt solution of the same strength, the 
solution being kept in contact with the globulin for about 18 hours. 
This was likewise filtered off and the residue again extracted with 
two litres of 0.25-per cent, salt solution. A portion of the com- 
bined extract obtained in this manner, on being heated, grew 
slightly turbid at 540 ; this turbidity was still slight at 63°, but at 
710 a flocculent coagulum appeared. No further coagulum 
appeared until the solution was boiled, and then only a slight one. 
The filtrate from this precipitate gave a faint reaction with acetic 
acid and potassium ferrocyanide. These united extracts were 
then placed in dialysers and dialysed in running water until the 13 
salt was entirely removed, when the deposited globulin was filtered 
off, and washed with alcohol and ether. It weighed air-dry 0.18 
gram (Preparation E1). 
That portion of the globulin insoluble in 0.25-per cent, salt 
solution was placed in two litres of 0.5-per cent, solution of sodium 
chloride, and kept in contact with it for 18 hours or longer at 
200 C., after which it was filtered off and treated with a fresh 
portion of salt solution of the same strength. The extract so 
obtained became turbid at 56°, and gave a flocculent coagulum at 
750. The filtrate from this coagulum became slightly turbid on 
boiling and gave a slight precipitate with acetic acid and 
potassium ferrocyanide. On dialysis of the 0.5-per cent, salt 
solution 0.527 gram of air-dry globulin was obtained (Prepara- 
tion E2). 
The remainder of the globulin was next treated with 0.75-per 
cent, salt solution at 20° C. until all soluble matter was removed. 
A portion of the solution so obtained, on being heated, became 
turbid at 590, with separation of flocks at 72.50. The filtrate from 
this coagulum became turbid again at 790 and flocked at 85°. 
On boiling this filtrate a little more precipitate appeared. By 
dialysis of the united filtrates 2.32 grams of globulin were obtained 
(Preparation E3). 
The globulin still undissolved was then treated, after the manner 
described, with 1.0-per cent, salt solution as long as anything dis- 
solved. This extract coagulated as follows; it became faintly 
turbid at 63°, with separation of flocks at 79.50. The filtrate 
gave a second turbidity at 85°-87°, which increased slightly 
on boiling. By dialysis, 2.9 grams of globulin were obtained 
(Preparation E4), 
The residue of the original globulin was next treated with two 
litres of 2.0-per cent, salt solution, in which it nearly all dissolved. 
The slight residue was rejected. The extract coagulated as fol- 
lows : turbid at 79°, with formation of flocks at 90°. The filtrate 
grew turbid again at 940, with separation of flocks at 990. On 
dialysis of the solution 2.2 grams of globulin were deposited 
(Preparation E5). This substance, on being tested anew with 1.0- 
per cent, salt solution, was found practically insoluble. 
The several products enumerated were dried at no° C. until of 
constant weight, and then analysed with the following results : 14 
Analysis of that Portion of the Globulin Soluble in 0.25-per cent 
Solution of Sodium Chloride (El). 
I. 0.1594 gram substance gave 21.6 cc. N at 4.00 C. and 772.3 mm. pres- 
sure zz 17.04 per cent. N, 
Not enough substance for an ash determination. 
Analysis of that Portion of the Globulin Soluble in oy-per cent. 
Solution of Sodium Chloride (Ea). 
I. 0.2470 gram substance gave 34.9 cc. N at 3,0° C. and 763.3 mm. pres- 
sure zz 17.63 per cent. N. 
11. 0.2170 gram substance gave 0.0075 gram ash zz 0.69 percent, ash. 
Percentage of nitrogen in the ash-free substance zz 17.74. 
Analysis of that Portion of the Globulin Soluble in 0.75-per cent. 
Solution of Sodium Chloride (E3'). 
I. 0.3303 gram substance gave 0.2018 gram H2t> —6.79 per cent. H, and 
0.6300 gram C02 zz 52.02 per cent. C. 
11. 0.3222 gram substance gave 0.1987 gram H2O 6.86 per cent. H, and 
0.6125 gram C02 zz 51.84 per cent. C. 
111. 0.4261 gram substance gave 61.5 cc. N at 4.2° C. and 750.2 mm. pres- 
sure = 17.62 percent. N. 
IV. 0.3042 gram substance gave 42.9 cc. N at 3.00 C. and 764.1 mm. pres- 
sure zz: 17.61 per cent. N. 
V. 0.4353 gram substance gave 0.0023 gram a®h 0.53 Per cent. 
Percentage Composition of the Ash-free Substance. 
c 
52-2S 
52.07 
Average. 
52,16 
H 
6.82 
6.89 
6.86 
N 
17.69 
17.68 
17.69 
S 
o 
:: f 
23.29 
100.00 
Analysis of that Portion of the Globulin Soluble in 1.0-per cent. 
Solution of Sodium Chloride (E4). 
I. 0.3057 gram substance gave 0.1900 gram H20zz6.90 per cent. H, and 
0.5814 gram C02 51.87 per cent C. 
11. 0.5543 gram substance gave 76.0 cc. N at 3.30 C. and 774.2 ram, 
pressure zz 17.33 per cent. N. 15 
111. 0.3593 gram substance gave 50.0 cc. N at 3.00 C. and 754.3 ram. 
pressure 17.16 per cent. N. 
IV. 0.4353 gram substance gave 0.0023 gram ash —0.53 per cent. 
Percentage Composition of the Ash-free Substance. 
c 
52.14 
Average. 
52.14 
H 
6.93 
6 93 
N 
.. 
17-43 
17.26 
T7-35 
S 
o 
} 23-58 
100.00 
Analysis of that Portion of the Globulin Soluble in 2.0-per cent. 
Solution of Sodium Chloride (A5). 
I. 0.3705 gram substance gave 0.2263 gram H2O zz 6.79 per cent. H, and 
0.6995 gram C02 2=51.50 per cent. C. 
11. 0.3632 gram substance gave 0.2209 gram H20i=6.76 per cent. H, and 
0.6850 gram C02 51.46 per cent. C. 
111. 0.4940 gram substance gave 70.3 cc. N at 3.00 C. and 755.4 ram. 
pressure 17.57 per cent. N. 
IV. 0.3434 gram substance gave 0.0028 gram ash = 0.81 per cent. 
Percentage Composition of the Ash-free Substance. 
c 
ST-92 
51.88 
Average. 
51.9° 
H 
6.84 
6.8x 
• . 
6.83 
N 
17.71 
17.71 
S 
o 
} 23-56 
100.00 
The first thing to be noticed from these results is that only a 
comparatively small amount of the original globulin is soluble in 
very weak salt solutions. It is not until the solution contains 0.75 
per cent, of salt that much globulin is dissolved. It is further 
noticeable that the weaker salt solutions have throughout a lower 
coagulation-point than the stronger salt solutions, and, moreover, 
that no one of the extracts coagulates completely at a given tem- 
perature, but shows some evidence, by a turbidity at least, of 
several different heat-coagulation points. As regards composi- 
tion, the nitrogen of the several fractions is practically the same, 
and as this is apt to be the most variable element in proteid bodies, 
it is probable that the several fractions have essentially the same composition. It is further noticeable that the composition of the 
fractions E3, E4 and E5 is practically identical with the composition 
of the globulins A and B, although the average content of carbon 
in the former products is a trifle higher. Of the 10 grams of air- 
dry globulin started with, in this experiment, 8.12 grams were 
recovered in the several fractions. There was obviously some 
loss, especially in the many filtrations, so that this deficiency is 
not to be considered as representing the amount of globulin or 
alteration product insoluble in the 2.0-per cent, salt solution. 
The actual amount of insoluble matter was certainly far smaller 
than this. 
It would have been interesting to have determined the percent- 
age of nitrogen in the insoluble portion, but the quantity was so 
small and it was so mixed with shreds of filter-paper that the 
result in this case could have had little value. 
The relationship of these several fractions or portions of the 
original globulin is clearly shown in the following table: 
Portion soluble in NaCl 
o. 
25 per ct. 
0.50 per ct. 
0.75 per ct. 
1.0 per ct. 
2.0 per ct. 
c 
52.16 
52.14 
51.90 
H 
6.86 
6-93 
6.83 
N 
17.04 
17-74 
17.69 
17-35 
17.71 
o} 
23.29 
23.58 
23-56 
Coagulation-points,1 
ISt. 
54°, 7i° 
56°, 75° 
59°. 72-5° 
63°. 79-5° 
790, 90° 
<< « 
2d. 
79°, 83° 
85°, 87° 
94°, 99° 
This experiment, then, so far as it goes, corroborates the idea 
that the original globulin is a mixture of closely allied bodies pos- 
sessed of different heat-coagulation points and of different degrees 
of solubility in dilute salt solutions. This method of treatment, 
however, is insufficient to bring about a complete separation of 
the individual bodies, the separate fractions obtained above being 
evidently themselves more or less mixtures, as indicated by their 
heat-coagulation points and their resemblance in chemical com- 
position. This might indeed be possible, even if there were only 
two globulins present, for, as is well known, it is often as diffi- 
cult to bring about a sharp separation of certain proteid bodies, 
having many points in common, as it is to separate a mixture of 
several fats. 
1 The first figure of each pair represents the appearance of a turbidity, the second figure the 
formation of flocks. Another and more successful separation of the globulin into 
its possible constituents was made by means of fractional heat- 
coagulation. Obviously, the products so obtained, or at least a 
portion of them, are coagulated by the process ; but if the sub- 
stance is composed simply of a single body, the several products 
or coagulums might naturally be expected to have the same 
composition. 
In this experiment, 10 grams of globulin B were dissolved in 
10-per cent, salt solution, filtered, the clear fluid heated to 50° in 
a roomy water-bath and a slight excess of very dilute hydro- 
chloric acid added, by which a copious precipitate was obtained, 
doubtless an acid compound, leaving the fluid perfectly neutral. 
This precipitate was washed with salt solution, water, alcohol and 
ether, and weighed, air-dry, 1 gram (Preparation B1). 
The perfectly neutral filtrate, on being heated further, became 
turbid at 570, with formation of flocks at 66°. The temperature, 
however, was raised to 70°, where it was held some time, and the 
precipitate finally filtered off and washed with salt solution, 
water, alcohol and ether. It weighed, air-dry, about 1 gram 
(Preparation B2). The filtrate became turbid again at 720, and at 
750 a flocculent precipitate made its appearance, which was finally 
filtered off when the temperature reached Bi°. This was treated 
like the preceding products, and when air-dry weighed 1 gram 
(Preparation B3). 
The clear filtrate from this latter coagulum was dialysed free 
from chlorides, when there resulted a separation of a globulin-like 
substance, which, after washing with water, alcohol and ether, 
weighed, air-dry, 1.43 grams (Preparation B4). 
These several products, on being dried at no° C. until of con- 
stant weight, gave on analysis the following results ; 
Analysis of the HCI Precipitate at 50° (A1). 
I. 0.3539 gram substance gave 48.7 cc. N at 2.5° C. and 767.8 mm. pres- 
sure 17.33 Per cent. N. 
11. 0.4731 gram substance gave 0.0016 gram ash m 0.34 per cent. 
Percentage of nitrogen in ash-free substance 17.39. 
Analysis of the Coagulum at 66°-jo° (Z?2). 
I. 0.3687 gram substance gave 47.3 cc. N at 2.50 C. and 767.8 mm. pres 
sure~ 16.15 Per cent. N. 11. 0.3855 gram substance gave 0.0017 gram ash =0.44 per cent. 
Percentage of nitrogen in the ash-free substance 16.21. 
Analysis of the Coagulum at 75°-Bi° (B3). 
I. 0.3361 gram substance gave 44.9 cc. N at 2.50 C. and 766.1 mm. pres- 
sure ~ 16.78 per cent. N. 
11. 0.2015 gram substance gave 0.0004 gram ash ~ 0.19 per cent. 
Percentage o£ nitrogen in the ash-free substance s 16.81. 
Analysis of the Globulin separated by Dialysis (A4). 
I. 0.2993 gram substance gave 0.1810 gram H2O —6.72 per cent. H, and 
0.5674 gram C02 51.72 per cent. C. 
11. 0.2595 gram substance gave 0.1596 gram II20 ~ 6.83 per cent. H, and 
0.4910 gram C02~ 51.60 per cent. C. 
111. 0.3300 gram substance gave 47.9 cc. N at 2.50 C. and 762.2 mm. pres- 
sures 18.14 Per cent, N. 
IV. 0.3265 gram substance gave 0.0016 gram ash s 0.49 per cent. 
Percentage Composition of the Ashfree Substance. 
c 
Si-97 
Si-85 
Average, 
5I-9I 
H 
6-75 
6.86 
. • 
6.8 x 
N 
, . 
. . 
18.22 
18.22 
S 
o 
• • 
23.06 
100.00 
A comparison of these results shows plainly that the globulin 
separable from corn meal by the process previously described, as 
in Preparation B, is composed of at least two dissimilar proteids, 
or, less probably, is broken into such bodies by the process of 
heat-coagulation. The products coagulable below Bo° are 
characterised by a comparatively low percentage of nitrogen, while 
the product (B4) separated by dialysis of the filtrate from the 
coagulum at Bo° has a higher content of nitrogen than the original 
globulin, resembling in this respect Preparation A 2, the more 
insoluble portion of globulin Al. This relationship in chemical 
composition is clearly seen from the following table : 19 
Globulin B. 
Prep. B1.* 
Prep. B2. 
Prep. B3, 
Prep. B4. 
Prep. A2. 
c 
51.82 
SI-94 
5I-76 
H 
6.85 
• . 
6.8l 
6.80 
N 
17.82 
17-39 
l6.2I 
16.81 
18.22 
l8.l6 
23-51 
•• 
23.06 
23.28 
*ln considering these results it must be remembered that Preparation B1 is probably nothing 
more than an acid compound of the mixed globulins. 
This close relationship in composition between the products 
A 2 and B4 is strongly suggestive of their being the same body, 
viz., a form of phyto-vitellin especially characterised by non-coagu- 
lation below Bo° and by very incomplete coagulation above that 
temperature, even when its solution in sodium chloride is heated 
to ioo°. 
Another point to be noted in the above experiment is, that of 
the 10 grams of globulin (B) started with, only 4.43 grams were 
recovered in the form of the products BlB4, just described. 
Naturally some loss must have occurred, especially through 
possible incomplete separation during dialysis of the final solu- 
tion, but evaporation of the salt-free fluid from which globulin B4 
had separated revealed the presence of about 1.5 grams of non- 
coagulable matter, either derived from, or present in, the original 
globulin. The substance so obtained gave a red color with cupric 
sulphate and potassium hydroxide, the usual proteid reactions 
with Millon’s reagent, no reaction with acetic acid, but a heavy 
precipitate with acetic acid and potassium ferrocyanide. Addition 
of acetic acid to a 10-per cent, salt solution of the substance gave 
a heavy precipitate, possibly in part of globulin, which was still 
further increased by saturation of the acid fluid with salt. Satu- 
ration of a neutral solution of the substance with salt gave no 
precipitate whatever. Nitric acid produced a distinct turbidity, 
and addition of an equal volume of alcohol yielded a heavy pre- 
cipitate. 
From this it is plainly evident that in the separation or breaking 
apart of the original globulin by heat-coagulation there is formed 
in small quantity a body, or bodies, non-coagulable by heat and 
readily soluble in water ; in other words, bodies resembling pro- 
teoses. That such soluble substances were not present in the 
original globulin was proved by boiling a small portion of globu- 
lin Bin water and then filtering the hot solution. The filtrate 20 
gave no reaction whatever for proteids with the biuret test. In 
other words, the original globulin contained no proteose-like sub- 
stances, but when dissolved in salt solution and subjected to heat- 
coagulation such bodies were apparently formed. This was 
demonstrated in several ways, among others as follows ; A portion 
of globulin B was suspended in water, and the emulsion quickly 
poured into a 10-per cent, salt solution heated to 750 C. A nearly 
complete solution resulted, the temperature not falling below 65°. 
The temperature was immediately raised to ioo° and the boiling 
continued for some minutes, thus killing any possible ferment 
present, after which the coagulum was filtered off. The some- 
what turbid filtrate was evaporated to dryness on a water-bath, a 
little coagulum forming during the process, and the residue taken 
up in water. Addition of acetic acid to the strong salt solution 
produced a heavy precipitate of the vitellin-like proteid, but there 
still remained in solution some non-precipitable proteid, giving a 
distinct pink color with the biuret test. While, then, this so-called 
corn globulin can be approximately separated by heat-coagulation 
into two dissimilar globulins, there is apparently formed at the 
same time, presumably by hydrolysis of the less resistant globu- 
lin, a small amount of proteose-like bodies, the quantity of which 
appears to be dependent in part upon the duration of the heating. 
The vitellin with 18 or more per cent, of nitrogen is especially 
characterised, as already indicated, by its pronounced insolubility 
in weak salt solutions, especially when cold. It is likewise more 
prone to separate in the spheroidal state. By making use of these 
facts we were able to separate it directly from the mixed globulin 
without recourse to coagulation. The method was as follows : 
8 grams of globulin B were dissolved in 300 cc. of 5-per cent, salt 
solution, and the fluid filtered. The clear solution was then diluted 
with distilled water to about 2 litres, whereby a heavy precipitate 
resulted. On warming to about 450 C., however, a nearly clear 
solution was obtained, which on gradual cooling to 8° C. deposited 
at the end of 48 hours a precipitate of spheroids. This precipitate 
was then filtered off, dissolved in as little salt solution as possible 
(warmed to 50° C), and diluted with distilled water at the same 
temperature, until a permanent turbidity began to make its appear- 
ance. The globulin which separated from this solution on cool- 
ing, was wholly soluble in the salt fluid when warmed to 50° C. 
As the amount of globulin which separated by this treatment was 21 
small, the entire mixture was further diluted with an equal volume 
of water and the resulting precipitate collected. It was then 
partly dissolved in about 2 litres of quite dilute salt solution, the 
mixture heated to 50°, and 20-per cent, salt solution added until 
the globulin had entirely dissolved. On cooling this solution very 
slowly, ultimately near to o°, a considerable deposit of very large 
spheroids resulted, which, after being washed with water, alcohol 
and ether, weighed, air-dry, 1.5 grams. This product (BB),onbeing 
dried at 1 io° C. until of constant weight, gave by analysis the 
following results: 
Analysis of the Globulin Bh. 
I. 0.4311 gram substance gave 0.2658 gram H2O 22:6.85 per cent, H, and 
0,8227 gram C02 22: 52.05 per cent. C. 
11. 0.3338 gram substance gave 49 cc. N at 30 C. and 754.2 mm. pressure 
18.10 per cent N. 
111. 0.4458 gram substance gave 0.0019 gram ash 22. 0,42 per cent. 
Percentage Composition of the Ashfree Substance. 
B5. 
BA 
AA 
Globulin B. 
c 
52.26 
52.26 
51.94 
5i-76 
51.82 
H 
6.88 
. . 
6.88 
6.8l 
6.80 
6.85 
N 
. , 
18.17 
18.17 
18.22 
18.16 
17.82 
S 
o 
:: } 
22.69 
23.06 
23.28 
0.86 
22.65 
100.00 
100.00 
100.00 
100.00 
Comparison of the analysis of this product with that of the 
original mixed globulin B shows that the nitrogen has been 
raised to correspond with the percentage in the preceding prep- 
arations of vitellin, notably B4 and A 2, while the carbon is a trifle 
higher than in the latter products. Evidently, then, this simple 
process is sufficient to separate the vitellin from the mixed globulin; 
the more soluble globulin, with its lower content of nitrogen, 
remaining in the several solutions. 
It is thus evident that the corn or maize globulin originally 
referred to by Weyl is a mixed substance, composed of at least 
two dissimilar globulins, one of which approximates in composi- 
tion to phyto-myosin, the other to phyto-vitellin, although both 
bodies possess peculiarities of reaction not exactly in accord with 
the reactions of these two substances as usually described. 22 
d. Extractio7i of corn meal with water, and separation of the 
globulin by ammonium sulphate and dialysis. 
When corn meal is extracted with water there results a solution 
rich in salts, especially phosphates of the alkalies, in which more 
or less globulin is dissolved. Direct dialysis of such a solution is 
followed by separation of the dissolved globulin. It can likewise 
be precipitated, together with the other proteids present, by satu- 
ration of the solution with ammonium sulphate. A concentrated 
solution, obtained by extracting corn meal with water, when heated 
grows turbid at about 50° C. (the exact point depending on the con- 
centration of the fluid), with formation of a flocculent coagulum at 
about 6o°. At 64° the solution again becomes turbid, with forma- 
tion of a second flocculent coagulum at 750. 
The globulin was separated as follows: 6.5 kilos, of corn meal 
were treated with 10 litres of water, with more or less stirring, for 
some hours, and residue of meal re-extracted in the same 
manner. The united solutions, filtered clear, were saturated with 
ammonium sulphate. The precipitate so obtained was dissolved 
as far as possible in water and in 10-per cent, salt solution, leaving 
quite an insoluble residue. The united aqueous and salt solu- 
tions, after filtration, were dialysed until the greater portion of the 
salt was removed, when there resulted a decided separation of 
globulin. This globulin was far more insoluble in 10-per cent, 
solution of sodium chloride than any previous product. In 
other words, a much larger proportion of this originally soluble 
globulin passed into an insoluble modification during the process 
of dialysis, etc., than any product previously separated. That 
portion still soluble in 10-per cent, salt solution coagulated as 
follows; turbid at 54°-6o°, with formation of flocks at 67°-7i°. 
A second turbidity appeared in the filtered fluid at but this 
was very slight, and was not much increased even when the solution 
was boiled. Addition of acetic acid, however, gave a distinct pre- 
cipitate. On dialysis of the sodium-chloride solution, the dissolved 
globulin was deposited. It amounted to only 0.3 gram when air- 
dry (Preparation F). Dried at no° C., it yielded by analysis 
16.80 per cent, of nitrogen, thus agreeing approximately both in 
content of nitrogen and in coagulation-point with that portion of 
the mixed globulin B coagulable below Bo°, viz., B2 and 83.B3. 
0.2593 §Fani substance gave 35.2 cc. N. at 2,5° C. and 754.9 
mm. pressures 16.80 per cent. N. 23 
A larger quantity of this globulin, sufficient for a more complete 
analysis, was obtained from another portion of the corn meal as 
follows: 5 kilos, of freshly ground corn were thoroughly extracted 
with water, and the filtered solution precipitated by the addition 
of ammonium sulphate to saturation. This precipitate was treated 
with water, and the globulin soluble in the dilute ammonium 
sulphate solution formed was, after filtration, separated from the 
fluid by dialysis. After washing the separated globulin with 
water, alcohol and ether, it weighed, air-dry, 2 grams (Prepara- 
tion G1)- 
There was not enough substance for an ash determination. 
Dried at no° C. it gave on analysis the following results : 
Analysis of Corn Globulin, Preparation Gl. 
I. 0.1973 gram substance gave 0.1243 gram II20 2=6.99 Per cent* H> and 
0.3784 gram C02 52.30 per cent. C. 
11. 0.3343 gram substance gave 0.2071 gram H2O = 6.88 per cent. H, and 
0.6392 gram C02 52.14 per cent. C. 
111. 0.3594 gram substance gave 50.5 cc. Nat 17.x° C. and 758.5 mm, 
pressure =: 16.57 per cent. N. 
IV. 0.459° Sram substance fused with KOH + KNOs gave 0.0433 gram 
BaSo4=: 1.28 per cent, S. 
V. 0.3159 gram substance gave 0.0025 gram ash =2 0.79 per cent. 
Percentage Composition of the Ashfree Substance. 
c 
52* 71 
52.56 
Average. 
52.64 
H 
7.04 
6.94 
6.99 
N 
16.70 
.. 16.70 
S 
I.28 I.28 
o 
22.39 
100.00 
A duplicate of this last product was prepared from another 
portion of corn meal (5 kilos.) in essentially the same manner. 
The meal was thoroughly extracted with water, the filtered extract 
precipitated by saturation with ammonium sulphate, the precipi- 
tated proteids filtered off and soaked in water, by which the 
globulin and some other substances were dissolved. From this 
dilute ammonium sulphate solution the globulin was separated by 
dialysis of the salt, and after being washed with water, alcohol and 
ether, weighed, when air-dry, 2 grams (Preparation H1). 
Dried at 1 io° C. and analysed, it yielded the following results: 24 
Analysis of Corn Globulin, Preparatioyi Hl. 
I. 0.2825 gram substance gave 0.1769 gram H20rr6.96 per cent. H, and 
0.5425 gram CC2 22 52.37 per cent. C. 
11. 0.1553 gram substance gave 0.0989 gram H2O 22:7.07 per cent. H,and 
0.2987 gram C02~ 52.45 per cent. C. 
111. 0.4200 gram substance gave 59.0 cc. Nat 17.z° C. and 765.1 mm. 
pressure 22: 16.71 per cent. N. 
IV. 0.3118 gram substance gave 0.0021 gram ash 220.67 percent, (ash 
mainly oxide of iron). 
V. 0.5500 gram substance fused with KOH + KNOs gave 0.0530 gram 
BaS04=: 1.32 per cent. S. 
Percentage Composiiioyi of the Ash-free Substance. 
c 
52.72 
52.8° 
Average. 
52.76 
H 
7.00 
7.IX 
7.05 
N 
16.82 
16.82 
S 
I.32 I.32 
o 
22.05 
100.00 
From this description it is obvious that only a portion of the 
globulins contained in the corn-kernel can be withdrawn by water; 
in other words, the dilute salt solution which results when corn 
meal is extracted with water is capable of dissolving only a portion 
of the globulins present. More important, however, is the fact that 
the globulin which is so dissolved represents simply that portion 
of the mixed globulins, previously described, coagulating under 
Bo°, or, to be more exact, it is apparently a body coagulating 
completely at 750 C. and with a content of nitrogen approximating 
16.8 per cent. Furthermore, it is a globulin excessively prone 
to become insoluble in salt solution by long contact with water or 
with strong solutions of salt. In other words, it agrees closely 
with the general descriptions of vegetable myosin except in its 
peculiar coagulation-points. This view is still further substantiated 
by the almost complete agreement in composition with animal 
myosin. This relationship in chemical composition can be clearly 
seen from the following table: 
Corn 
Corn 
Corn 
Coagulum 
at 750 from 
Animal 
myosin. 
myosin. 
myosin. 
mixed globulin. 
myosin.* 
c 
Fi. 
G'. 
52.64 
Hi. 
52.72 
B3. 
52.82 
H 
6.99 
7.05 
l6.8l 
7.11 
N 
16.80 
16 70 
16.82 
16.77 
S 
1.28 
I.32 
I.27 
o 
22.39 
22.05 
21.9O 
* Average of the analyses of 13 different preparations of animal myosin from various sources. 
See Studies from Laboratory of Physiological Chemistry, Yale University, 3, 133. 25 
Hence, by this method of extracting corn meal directly with 
water, and by the use of the method of separation already de- 
scribed, only one globulin is obtained, with a coagulation-point of 
about 70° and agreeing substantially in other respects with the 
general properties of vegetable myosin. Doubtless the trace of a 
coagulum obtained at higher temperatures and on the addition of 
acid is due to a slight admixture of the vitellin-like body. The 
above statement being correct, it is obvious that the residue of 
corn meal remaining after complete extraction with water should 
yield to 10-per cent, salt solution a globulin with 18 per cent, or 
more of nitrogen unmixed with myosin, providing the latter body 
has been completely withdrawn from the tissue of the seed. 
e. Extraction of corn meal with 10-per cent, solution of sodium 
chloride, after previous extraction with water, and separation of 
the globulin by direct dialysis, and by precipitation with ammonium 
sulphate and dialysis. 
Extraction of corn meal with 10-per cent, salt solution, after 
previous extraction with water, yields a solution containing con- 
siderable globulin, which can be separated in part by addition of 
water and completely, or nearly so, on dialysis. 
6.5 kilos, of corn meal, after thorough extraction with water 
(Extract F), were treated several times with an abundance of to- 
per cent, salt solution, and the dissolved proteids precipitated 
from the filtered extract by saturation with pure ammonium sul- 
phate. The precipitate was dissolved, so far as possible, in water 
and 10-per cent, salt solution, and the united fluids dialysed in 
running water until the bulk of the salt was removed. The 
globulin separated in this manner dissolved only partially in to- 
per cent, salt solution. The soluble matter coagulated as follows: 
slight turbidity at 6o°-63°, with separation of a few flocks at 
The filtrate from this coagulum grew turbid again at 
Bi°-82°, flocking at 9i°-95°. Addition of acetic acid to this 
filtrate gave quite an abundant precipitate. This globulin dis- 
solved by the 10-per cent, salt solution was again separated from 
the fluid by dialysis. The precipitate so obtained, when washed 
with water, alcohol and ether, weighed, air-dry, 4.12 grams (Prepa- 
ration F2). It was almost entirely soluble in 10-per cent, salt 
solution, and when subjected to heat-coagulation yielded only a 
slight turbidity at 63°, with separation of a few flocks at 730. On 
heating this filtrate nothing further appeared other than a trifling 26 
turbidity, even when the solution was boiled. Addition of acetic 
acid, however, gave a heavy precipitate. Evidently, therefore, 
this body is characterised especially by non-coagulation by heat, 
in a neutral solution, the trifling coagulum observed being unques- 
tionably due to a trace of myosin. 
Dried at no0 C. until of constant weight, and analysed, the 
following results were obtained : 
I. 0.3781 gram substance gave 0.2295 gram HaO = 6.74 per cent. H, axul 
o.72°5 gram €O2 = 51.96 per cent. C. 
11. 0.3293 gram substance gave 0.2031 gram H2O = 6.85 Per cent. H, and 
0.6253 giani C02= 51.79 per cent. C. 
111. 0.3401 gram substance gave 48.8 cc. N at 30 C. and 766.9 mm. pres- 
sure = 17.99 Per cent. N. 
IV. 0.3972 gram substance gave 57 cc. N at 30 C. and 756.6 mm. pres- 
sure = 17.96 per cent, N. 
V. 0.7561 gram substance fused with KOH + KN03gave 0.0337 gram 
BaS04 = 0.6 i per cent. S. 
VI. 0.8611 gram substance fused with KOH + KNO3 gave 0.0446 gram 
BaSO4 = 0.7 i per cent. S. 
VII. 0.3951 gram substance gave 0.0009 gram ash = 0.22 per cent. 
Analysis of Corn Globulin, Preparation F2. 
Percentage Composition of the Ashfree Substance. 
c 
52-°7 
51.90 
Average. 
5T.99 
H 
6-75 
6.86 
.. 6.81 
N 
18.03 
18.00 
18.02 
S 
. . 
. . 
0.61 
0.71 0.66 
o 
•• 
22.52 
100.00 
The character of this product was verified by another prepara- 
tion as follows: 
5 kilos, of finely ground corn, after being thoroughly extracted 
with water (Preparation G), were treated with an abundance of 
10-per cent, salt solution, and the filtered extract directly dialysed 
until the salt was entirely removed. The precipitate which sepa- 
rated was washed with water, then redissolved in 10-per cent, salt 
solution, filtered from the insoluble residue, and again dialysed 
until the globulin had completely separated once more. This 
final product was very soluble in salt solutions, and weighed, after 
being washed with alcohol and ether, 5 grams (Preparation G2). A portion dried at uo° C. gave on analysis the following 
results; 
Analysis of Corn Globulin, Preparation G\ 
I. 0.3063 gram substance gave 0.1857 gram II20 2=6.73 per cent. H, and 
0.5607 gram C02 = 49-9I Per cent. C. 
11. 0.3205 gram substance gave 0,1925 gram H20rc6.76 percent, H, and 
0.5882 gram Co22= 50.04 per cent. C. 
111. 0.4245 gram substance gave 63.3 cc. Nat 17.0° C. and 764.5 mm. 
pressure 17-73 Per cent. N. 
IV. 0.5045 gram substance fused with KOH + KNO3 gave 0.0355 gram 
BaSO* 2=0.96 per cent. S. 
V. 0.4490 gram substance fused with KOH + KNO3 gave 0.0315 gram 
BaS04 2=0.96 per cent. S. 
VI. 0.5435 gram substance gave 0,0125 gram ash 2= 2.30 per cent. 
VII. 0.2865 gram substance gave 0.0062 gram ash 2= 2.16 per cent. 
Percentage Composition of the Ash-free Substance. 
Average. 
c 
ST-°4 
Si-17 
.. 
• . 
51.10 
H 
6.88 
6.91 
, . 
6.90 
N 
.. 
18.13 
# , 
. . 
18.13 
S 
.. 
• • 
.. 
0.98 
0.98 
O.98 
o 
* * 
* * 
* * 
* ' 
22.89 
100.00 
A third preparation of the same order was made as follows: 5 
kilos, of corn meal, after extraction with water (Preparation H), 
were treated with io-per cent, salt solution, and the filtered extract 
directly dialysed until the salt was removed. The separated pro- 
teid was then filtered off, dissolved again in 10-per cent, salt solu- 
tion, the solution filtered, and once more dialysed until the proteid 
had completely separated. The globulin so obtained (Preparation 
H2), after being washed with alcohol and ether, weighed 5 grams, 
and on analysis (dried at 1 io° C.) gave the following results: 
Analysis of Corn Globulin, Preparation H*. 
I. 0.2844 gram substance gave 0.1728 gram H2O 2=6.75 Per cent. and 
0.5196 gram C02=2 49.82 per cent. C. 
11. 0.4564 gram substance gave 0,2699 gram H20=z6.57 per cent. H, and 
0.8283 gram C02zr 49.49 per cent. C. 
111. 0.3892 gram substance gave 57.4 cc. N at 16.3° C. and 759.8 mm. 
pressure 17.47 per cent. N. 
IV. 0.3767 gram substance gave 56.1 cc. Nat 17.40 C. and 755.0 mm. 
pressure = 17.47 per cent. N. 28 
V. 0.5075 gram substance gave 0.0156 gram ash = 3,07 per cent. 
VI. 0.5323 gram substance gave 0.0162 gram ash 3.04 per cent. 
VII. 0.5050 gram substance fused with KOH-j- KN03 gave 0.0338 gram 
BaS04 ~ 0.92 per cent. S. 
VIII. 0.5315 gram substance fused with KOH -f- KNO3 gave 0.0345 gram 
BaS04 0.89 per cent. S. 
Percentage Composition of the Ash-free Substance. 
Average. 
c 
S*-38 
5I>04 
.. 
51.21 
H 
6.96 
6.78 
.. 
. . 6.87 
N 
18.02 18.02 
.. 18.02 
S 
. • 
0.94 
O.9I O.93 
o 
* * 
- * 
• • • • 
* * 
. . 22.97 
100.00 
It is evident from these results that by this process of extraction, 
first with water and lastly with salt solution, a good separation of 
the two globulins present in the corn-kernel may be obtained ; the 
myosin-like body passing mainly into the first or aqueous extract, 
the vitellin dissolving in the 10-per cent, salt solution. When we 
recall how difficult it many times is to separate closely related 
proteid bodies, the above method seems fairly successful. In the 
body last described we have the same globulin (vitellin) which 
was previously separated from the mixed globulin B, both by heat- 
coagulation and by what we may term recrystallisation from dilute 
salt solutions. In their content of nitrogen all of these prepara- 
tions of vitellin show a close agreement, and, furthermore, a fairly 
close agreement in carbon. Moreover, the average composition 
of these products agrees very closely with the generally accepted 
composition of phyto-vitellin. 
The following table shows this relationship very clearly: 
Phyto- 
Prep. A 2. Prep. 84. Prep. 88.BB. Prep. F2. Prep. G2. Prep. H2. vitellin,* 
C 51.76 51.94 52.26 51.99 51.10 51.21 51.88 
H 6.80 6.81 6.88 6.81 6.90 6.87 7.51 
N 18.16 18.22 18.17 18.02 18.13 18.02 18.08 
S \ 2, 2o 2, o6 22 6q / 0.66 0.98 0.93 0.60 
0/3' 3- -9 22.89 22.97 21.93 
* Prepared from pumpkin seeds and analysed by Barbieri by Weyl’s method (J. prakt. 
Chem. 18, ioz-116). 
As is seen from the table of results, the vitellin is noticeably 
different from the corn myosin in having a smaller percentage of 
sulphur, the latter containing on an average 1.27 per cent, of 29 
sulphur, while the vitellin contains only about 0.85 per cent. It 
is also noticeable that preparations G2 and H2 contain a lower per- 
centage of carbon than the other products. What the explanation 
of this is we cannot say, although it is to be remembered that 
these two preparations of vitellin (unlike F2) were separated from 
the salt solution by direct dialysis, without previous precipitation 
with ammonium sulphate, and possibly are not quite so pure. 
As to the respective quantities of these two globulins present in 
the kernel, it would appear that the vitellin is greatly in excess, 
although it is to be remembered that the myosin is more readily 
changed into an insoluble modification by the process of separa- 
tion, and hence considerable loss occurs. 
From the foregoing it is plain that the mixed globulin first 
described, obtained by direct extraction of corn meal with 10-per 
cent, salt solution, as preparations A-D, is mainly a mixture of 
two globulins resembling in composition myosin and vitellin, the 
composition of the mixed product being readily explainable, in 
the main, on that hypothesis. The coagulation-points of these 
two globulins, however, are far from according with the coagulation- 
points of phyto-myosin and vitellin as usually given. What we 
have chosen to call corn myosin shows a tendency to coagulate at 
several points, as already described, but is mainly coagulable at 
about 70°, while the vitellin-like body in a neutral salt solution 
appears almost non-coagulable by heat. In fact, the latter body 
partakes in many respects of the character of hetero-albumose, 
and is perhaps as closely related to this body as to the true 
globulins. The myosin-like body is evidently much like the 
peculiar globulin (not analysed) described by Martin1 as present 
in papaw juice, the coagulation-temperatures and general reactions 
of the two bodies being apparently much the same. 
There is every indication of gradual changes being produced 
by the application of heat to the solutions of these globulins, and, 
in our opinion, the variable coagulation-points observed are an 
index of this fact. Sidney Martin2 has shown that the myosin 
present in the kernels of wheat and rye is transformed into an 
insoluble body by simple warming of the 10-per cent, sodium- 
chloride solution of the globulin at 35°-40° C. for some hours, 
and Vines3 claims that phyto-vitellin may undergo transformation 
into myosin. The peculiar heat-coagulation points of the two 
1 Journal of Physiology 6, 353. 2 Ibid. 8, “On two classes of vegetable globulins. ' 
3 Ibid. 3, 93-114. 30 
corn globulins may possibly be due in part to rapid conversion into 
the insoluble or albuminate stage and its separation as a coagulum. 
There is, however, evidence of still another globulin in the corn 
kernel, differing from all of the preceding preparations, but, 
doubtless, sometimes present in small quantity in the products 
already described. 
f, A more soluble globulin which separates from its solution 
only after long-contimied dialysis. 
The two most important characteristics of a globulin are its 
solubility in various strengths of salt solution and its insolubility 
in water; hence the removal of the salt from a globulin containing 
solution, by dialysis, constitutes one of the most effectual methods 
of separating such proteids from their solutions, provided the 
removal of the salts is complete. Under ordinary circumstances 
a globulin dissolved in a salt solution separates quite rapidly when 
the solution is subjected to dialysis. Obviously, the rate of 
separation will depend greatly upon the endosmotic equivalent of 
the salts present and the solubility of the globulin, but under most 
circumstances the separation is practically complete some time 
before every trace of the salt is removed. Sodium chloride dif- 
fuses much more rapidly than ammonium sulphate; indeed, the 
latter salt requires a very long time for its complete removal, even 
when dialysed in running water of a fair degree of purity. The 
same is true of the phosphates of the alkalies present in the corn 
kernel, so that an extract of corn meal, for example, made with 
10-per cent, salt solution, may be dialysed until every trace of 
chloride is removed and still give a reaction for phosphates. 
In the separation of the mixed globulins from the corn kernel 
as already described, it will be remembered that the usual pro- 
cedure was to precipitate the filtered salt extract of the meal with 
ammonium sulphate added to saturation. The precipitated pro- 
teids were collected on a filter, allowed to drain, and then dissolved 
so far as possible in water and 10-per cent, solution of sodium 
chloride. Obviously, such a solution after filtration when ready 
for dialysis, would contain, in addition to the proteids, anywhere 
from 5 to 8 per cent, of sodium chloride, and possibly i per cent, 
of ammonium sulphate. In separating the dissolved globulin by 
dialysis it was our custom to continue the dialysis until the solu- 
tion failed to give any reaction for chlorides with silver nitrate and 
nitric acid, considering that when that point was reached the 
globulin would be completely precipitated. Thus, in the prepa- ration of globulin B from 25 kilos, of corn meal, after the method 
just indicated, the dialysis of the sodium-chloride and ammonium- 
sulphate solution was continued (in running water) for two weeks, at 
the end of which time the chloride was entirely removed, and over 
42 grams of globulin had separated. The clear filtered fluid still 
contained a trace of sulphate, and as an experiment it was again 
exposed to dialysis in small parchment bags suspended in running 
water, for one week longer. Greatly to our surprise, a second 
deposit of globulin formed, which, after being washed with water, 
alcohol and ether, weighed, air-dry, 2.8 grams (Preparation B6). 
The filtrate from this product was again returned to the dialysers 
for ten days longer, when a third deposit of globulin formed, which, 
after being washed and dried, weighed 1,3 grams (Preparation B7). 
The filtrate from this product gave no further deposit even when 
dialysed in distilled water. 
These two preparations were unquestionably true globulins; 
both were wholly insoluble in water, but dissolved readily in dilute 
salt solutions. Preparation B°, dissolved in 10-per cent, solution of 
sodium chloride, coagulated as follows: the solution grew slightly 
turbid at 590 and flocked at 62°. The filtrate from this coagulum 
grew slightly turbid again at 69°, but there was no separation of flocks, 
even when the mixture was boiled, or on the addition of acetic acid. 
Evidently, therefore, the substance was precipitated at 62°. Addi- 
tion of potassium ferrocyanide to the acid fluid, howrever, gave a 
slight precipitate, indicating that the substance was not wholly 
coagulable by heat, or else that some little proteose-like substance 
had been formed in the process. 
The composition of the globulin, after drying at no° C. until 
of constant weight, was as follows; 
Analysis of Corn Globulin, Preparation 86.B6. 
I. 0.3094 gram substance gave 0.1915 gram H2Q— 6.87 per cent. H, and 
0.5911 gram C02 “ 52.10 per cent. C. 
11. 0.3516 gram substance gave 0.2121 gram II20 6,70 per cent. H, and 
0.6721 gram €O2 = 52.13 per cent. C. 
111. 0.3895 gram substance gave 47.5 cc. N at 2.50 C. and 756*° nim. 
pressure= 15.12 per cent. N. 
IV. 0.3798 gram substance gave 46.9 cc. N at 50 C. and 759-4 mnl- Pres" 
sure = 15.22 per cent. N. 
V. 0.4427 gram substance fused with KOH + KNO3 gave 0.0407 giam 
BaS04 1.26 per cent. S. 
VI. 0.3895 gram substance gave 0.0019 gram ash = 0.50 per cent. 32 
Percentage Composition of the Ash-free S7ibstance. 
c 
52-36 
52-39 
.. 
Average. 
52.38 
H 
6.90 
6.74 
6.82 
N 
• . 
. . 
15.20 15.30 
I5-25 
S 
. . 
I.26 1.26 
o 
•• 
•• 
.. 
24.29 
100.00 
Judging from the coagulation-point and composition of this 
product, it is a globulin distinctly different from the preceding 
preparations. The above data taken together dearly prove that 
it is not a mere trace of myosin, vitellin, or both, which had escaped 
separation, although a trace of these bodies may have been mixed 
with it, as is indeed suggested by the turbidity at 69°, but that it 
represents a more soluble globulin than either of the preceding, 
and one containing a much lower percentage of nitrogen. It 
resembles myosin rather than vitellin, yet differs from them both 
in composition, in the temperature at which it coagulates, and in 
its marked solubility in very dilute solutions of salts other than 
chlorides. 
The low content of nitrogen in this globulin is substantiated by 
the percentage of nitrogen in the second product (B7), which is 
undoubtedly the same as the preceding. 
I. 0.3051 gram substance gave 36.6 cc. N at 3.00 C. and 757.2 mm. pres- 
sure 14.84 per cent. N. 
11. 0.3987 gram substance gave 0.0085 gram ash —2.13 per cent. 
Percentage o£ nitrogen in the ash-free substance 15.16. 
Analysis of Corn Globulin, Preparation B\ 
Without doubt, traces of this globulin may be carried down 
with the other globulins, especially with the myosin and with the 
mixed globulin, and this may be the cause of the low coagulation- 
point occasionally observed in the other products. On the other 
hand, if the preliminary dialysis for the separation of the two more 
insoluble globulins is not continued sufficiently long, then the 
product obtained on subsequent dialysis will contain some myosin, 
and possibly vitellin. 
This is well illustrated in-the following experiment: The pro- 
teids precipitated from a sodium-chloride extract of 5 kilos, of corn 
meal (Preparation E) by ammonium sulphate, and then dissolved 33 
in water and 10-per cent, salt solution, were dialysed in running 
water for one week, when a considerable separation of globulin 
occurred. This was filtered off, and the solution, which contained an 
appreciable amount of salt, was again dialysed for one week longer, 
yielding 1.16 grams of air-dry globulin (Preparation Ec). The 
filtrate, which contained a trace of sulphate, was again dialysed for 
one week, when there resulted a small separation of globulin, 
weighing, air-dry, 0,25 gram (Preparation E7). These were dried 
at no° C. and analysed with the following results: 
Analysis of the Globulin E\ 
I. 0.3728 gram substance gave 48.1 cc. N at 3.00 C. and 768.3 mm. pres- 
sure 16.20 per cent. N. 
11. 0.4817 gram substance gave 0.0075 gram ash~ x.56 per cent. 
Analysis of the Globulin E\ 
I. 0.1700 gram substance gave 20.6 cc. N at 3.00 C. and 767.7 mm. pres- 
sure =r 15.21 per cent. N. 
Not enough substance for an ash determination. 
From this it is sufficiently evident that the first product (Ee) 
is, as might be expected, a mixture of corn myosin with the 
more soluble globulin; while the latter product (E7) corresponds 
closely, at least in its content of nitrogen, with the preceding 
products of like nature. 
A tabular arrangement of the analyses of what we may term 
pure preparations of this globulin shows a fairly close agreement 
in composition. 
c 
Prep. B6. 
52-38 
Prep. B*. 
Prep. ET. 
H 
6.82 
.. 
N 
I5-25 
15.16 
15.21 
S 
1.26 
o 
24.29 
.. 
* 
From the foregoing descriptions this body is to be considered 
as a third globulin present in small amount in the corn kernel, 
and characterised especially by a low content of nitrogen, 15.2 
per cent., and with a coagulation-point at about 62° C. 
g. Insoluble products derived from the foregoing globulins. 
As is well known, many proteid bodies undergo change by long 
contact with water or by contact with strong salt solutions, being 
converted thereby into insoluble modifications resembling albumi- 34 
nates, soluble only in dilute alkaline fluids, or, it may be, by 
longer action, into bodies closely resembling coagulated proleids. 
This is notably true of animal myosin, of freshly precipitated syn- 
tonin, and of certain vegetable globulins. We have found this 
likewise true of one or more of the corn globulins just described. 
Thus, when a perfectly clear sodium-chloride extract of corn meal 
is dialysed and the globulin thereby precipitated, a portion of the 
product is changed in the process, as evidenced by its being no 
longer soluble in solutions of sodium chloride of any strength. 
This insoluble portion, however, is dissolved by dilute solutions 
of sodium carbonate, being precipitated therefrom on neutralisa- 
tion, apparently as alkali-albuminate. Again, in precipitating the 
proteids from a sodium-chloride extract of corn meal by saturation 
of the solution with ammonium sulphate, a portion of the globulin 
precipitated thereby loses its solubility in dilute salt solutions, 
being plainly converted into the same insoluble modification. 
Further, if by chance the ammonium sulphate used in the pre- 
cipitation of the proteids is acid, the transformation is far more 
rapid and complete, apparently in proportion to the acidity. 
Naturally, there may likewise occur, in this case, some formation 
of insoluble acid compounds. 
The details of the formation and separation of some of these 
insoluble transformation products are as follows: 
I. Insoluble products formed by the action of ammonium sul- 
phate on the mixed globulin directly extracted from corn by rofer 
cent, salt solution. 
In the preparation of the mixed globulin B, 25 kilos, of finely 
ground corn were extracted with an abundance of 10-per cent, salt 
solution at the temperature of the room, and the resultant solution 
filtered through filter-paper, yielding a perfectly clear fluid. All 
of the proteids were then precipitated by saturation of the solution 
at the ordinary temperature with recrystallised and perfectly 
neutral ammonium sulphate. This precipitate was then treated 
with a small volume of water, sufficient to make with the adherent 
salt a 1- to 3-per cent, solution of ammonium sulphate, and 
then with 10-per cent, solution of sodium chloride as long as the 
latter exerted any solvent action. As a result there remained quite 
an amount of insoluble matter, wholly insoluble in water and salt 
solutions, but readily dissolved by solutions of sodium carbonate 
of sufficient strength. It was, therefore, treated with a solution of 35 
i-per cent, sodium carbonate, the fairly clear fluid filtered 
through paper, and the proteid reprecipitated by neutralisation 
with dilute hydrochloric acid. After being washed with water 
until free from salts, then with alcohol and ether, it weighed, 
air-dry, 4.7 grams (Preparation Bx). In this connection it may 
be well to recall the fact that in this extraction of corn there were 
obtained 42 grams of purified mixed globulin and 4.1 grams of the 
more soluble globulin, thus indicating that approximately one- 
tenth of the total amount of globulin was converted into this 
insoluble modification, presumably through the action of the am- 
monium sulphate. This alteration-product, after being dried at 
no° C., gave on analysis the following results : 
Analysis of Preparation Bx. 
I. 0.4400 gram substance gave 0.2781 gram H2Or 7,02 per cent. H, and 
0.8657 gram C02 zr 53.66 per cent. C. 
11. 0.3960 gram substance gave 0.2504 gram H2Q— 7.02 per cent. H, and 
0.7769 gram COoZZ 53.51 per cent. C. 
111. 0.3447 gram substance gave 44.1 cc. N at 2.00 C. and 763.2 mm. pres- 
sure zz 16.02 per cent. N. 
IV. 0,7012 gram substance fused with KOH-f-KNOs gave 0.0557 gram 
BaS04z=: 1.09 per cent. S. 
V. 0.5472 gram substance fused with KOH+KNO3 gave 0.0476 gram 
BaS04 1.19 per cent. S. 
VI. 0.5810 gram substance gave 0.0039 gram ash zz0.67 per cent. 
Average 
c 
54.02 
53-87 
.. 
53-95 
H 
7-05 
7-05 
7-o5 
N 
16.13 
16.13 
S 
•. 
1.09 
x.19 
x.14 
o 
• • 
• • 
• • 
21.73 
100.00 
Percentage Composition of the Ash-free Substance. 
Two similar preparations, each from 5 kilos, of corn meal, were 
obtained in much the same manner as the preceding, except that, 
through the inadvertent use of slightly acid ammonium sulphate 
in the precipitation of the proteids, a far larger proportion of the 
globulin was converted into this insoluble modification and, further, 
in both cases the insoluble product contained considerable matter 
which would not dissolve even in 1- to 3-per cent, sodium 
carbonate. That portion soluble in i-per cent, sodium carbonate 36 
was filtered off and in each case precipitated by neutralisation with 
dilute hydrochloric acid. 
The products were then washed free from salts with water, with 
alcohol, and thoroughly extracted with ether (Preparations M* 
and Nx). 
On analysis the following results were obtained, which show 
fairly close agreement with the preceding data: 
Analysis of Preparation Mx. 
I. 0.3874 gram substance gave 0.2403 gram H2O 2=6.89 per cent. H, and 
0.7497 gram C022=52.77 per cent. C. 
11. 0.4460 gram substance gave 0.2749 gram H2O 2=6.85 per cent. H, and 
0.8595 gram C02 2= 52.57.per cent. C. 
111. 0.4373 gram substance gave 55.5 cc. N at 7.30 C.and 765.6 mm. pres- 
sure =2 15.64 per cent. N. 
IV. 0.8302 gram substance fused with KOH -{- KNOs gave 0.0693 gram 
BaS04=2 1.14 per cent, S. 
V. 0.6978 gram substance fused with KOH + KN03 gave 0.0577 gram 
BaS04 2= 1.13 per cent. S. 
VI. 0.9290 gram substance gave 0.0176 gram ash 2= 1.89 per cent. 
Perce7itage Composition of the Ash-free Substance. 
c 
53-79 
53-59 
Average. 
53-69 
H 
7.02 
6.98 
•. 
7.00 
N 
.. 
•. 
15-94 
15.94 
S 
. . 
1.16 
1.15 1.16 
o 
•• 
•• 
22.21 
100.00 
Analysis of Preparation Nx. 
I. 0.3777 gram substance gave 0.2315 gram Ii20 6.80 per cent. H, and 
0.7174 gram C02 =r 51.79 per cent. C. 
11. 0.3437 gram substance gave 0.2101 gram H50~6.79 per cent. H, and 
0.6502 gram C02 =251.59 per cent. C. 
111. 0.4419 gram substance gave 56.6 cc. Nat 7.40 C.and 763.6 mm. pres- 
sure 2= 15.74 per cent. N. 
IV. 0.7266 gram substance fused with KOH+ KN03 gave 0.0609 gram 
BaS04=2 1.15 per cent. S. 
V. 0.8527 gram substance fused with KOH + KNO3 gave 0.0634 gram 
BaS04 2= 1.02 per cent. S. 
VI. 0.7776 gram substance gave 0.0229 gram ash 222 2.94 per cent. 37 
Percentage Composition of the Ash-free Substance. 
c 
53-39 
53-19 
Average. 
53-29 
H 
7.00 
6.99 
16.23 
. . 7-00 
N 
. • 
1.18 
16.23 
S 
. . 
• • 
1.05 1.12 
o 
•• 
.. 22.36 
100.00 
From these three analyses it is plainly manifest that these 
insoluble products formed in the manner indicated are essentially 
the same. Their composition, moreover, indicates that in the 
formation of these bodies a decided alteration occurs, if we assume 
that they are formed from the mixed globulins already described. 
In fact, none of the globulins thus far identified could yield pro- 
ducts with the high content of carbon noted here without under- 
going a change of considerable magnitude. Obviously, we do 
not know how much of this change in composition results from 
the action of the ammonium sulphate and how much from the 
action of the dilute sodium carbonate, but inasmuch as we have 
obtained from a mixed globulin an insoluble body having the 
same low content of nitrogen, without the use of the sodium 
carbonate, it seems fair to presume that the change in composition 
is independent of any action of the dilute alkaline fluid. 
2. Insoluble products formed by the action of ammonium 
sulphate on the globulin extracted from corn by water, and on the 
globulin extracted by 10-per cent, solution of salt after previous 
extraction of the corn with water. 
In the preparation of extract F, 6,5 kilos, of corn meal were 
treated with cold water—about 14 litres—in two or more portions, 
and the filtered solutions precipitated with neutral ammonium 
sulphate added to saturation. 1 his precipitate was then treated 
with water and 10-per cent, salt solution as long as anything 
was dissolved. 
The residue was then dissolved in o.s~per cent, solution of 
sodium carbonate, filtered, and the proteid precipitated by neutral- 
isation. After being washed with water, alcohol and ether, it 
weighed, air-dry, 1.35 grams (Preparation F*). 
Analysed, it gave the following results: 
I. 0.2557 gram substance gave 0.1581 gram H2Q —6.86 per cent. H, and 
0.4974 gram COsz: 53.05 per cent. C. 
Analysis of Preparation Px. 38 
11. 0.3350 gram substance gave 43.9 cc. N at 30 C. and 759.3 mm. pres- 
sure 16.27 per cent. N, 
111. 0,3800 gram substance gave 0.0028 gram ash zz. 0.73 per cent. 
Percentage Composition of the Ash-free Substance. 
c 
53-43 
Average 
, 53-43 
H 
6.90 
.. 
6.90 
N 
.. 
16.39 
i6-39 
O 
•• 
" I 
23.28 
s 
# * 
.. j 
100.00 
The residue of corn meal remaining after the above extraction 
with water, was then extracted with 10-per cent, solution of sodium 
chloride, the filtered solution precipitated with ammonium sul- 
phate, and the proteids treated exactly as described under the 
aqueous extract. As a result, 1.45 grams of insoluble proteid were 
obtained, which on analysis gave the following results; 
Analysis of Preparation Fv. 
I. 0.4967 gram substance gave 0.3086 gram HaO 6.90 per cent. H, and 
0.9537 gram €02—52.36 per cent. C. 
11. 0.3520 gram substance gave 45.1 cc. N at 30 C. and 749.1 mm. pres- 
sure 15.69 per cent. N. 
111. 0.4165 gram substance gave 0.0049 gram ash x.17 per cent. 
Percentage Co7nposition of the Ashfree Substance. 
c 
52.98 
Average. 
52.98 
H 
6.98 
6.98 
N 
. • 
15-87 
I5-87 
S 
o 
24.17 
100.00 
3. Insoluble products formed through the action of water on the 
globulins extracted from corn meal by water and by 10-per cent, 
salt sohdio7i. 
The sodium chloride and ammonium sulphate solutions of the 
globulins filtered off from the two preceding preparations, F* and 
F", were dialysed in running water until the salts were practically 
all removed and the globulins separated from their respective 
solutions. 
On attempting to dissolve these products in 10-per cent, solu- 
tion of sodium chloride, a certain proportion remained insoluble ; 39 
in other words, during the long dialysis a portion of each globulin, 
formerly soluble in the salt solution, had become insoluble. These 
insoluble products were filtered off, washed thoroughly with 10- 
per cent, salt solution, dissolved in i-per cent, sodium carbonate, 
the solution filtered and neutralised with dilute hydrochloric acid. 
The precipitated proteids, after being washed with water, alcohol 
and ether, weighed, air-dry, respectively 1.2 grams (Preparation 
F*“) and 1.73 grams (Preparation F*"1), the former having come 
from the globulin extracted by water alone, and hence presumably 
formed from the myosin-like globulin, while the latter product, 
F,ya, on similar grounds must have been formed from the vitellin- 
like globulin or from some adherent body. 
These two products, on analysis, gave the following results 
Analysis of Preparation Fxa. 
1. 0.2278 gram substance gave 29.8 cc. N at 2.50 C. and 756.9 mm. pres- 
sure rr: 16.24 per cent. N. 
11. 0.3678 gram substance gave 0.0029 gram ash 21:0.79 per cent. 
Percentage of N in the ash-free substances 16.37. 
Analysis of Preparation Fm. 
I. 0.279S gram substance gave 0.1740 gram II20 6.92 per cent. H, and 
0.5302 gram COjz: 51.74 per cent. C. 
11. 0.2844 gram substance gave 38.3 cc. N at 3.00 C. and 760.3 mm. pres- 
sure =z 16.74 Per cent. N. 
111. 0.4025 gram substance gave 0.0018 gram ash = 0.45 per cent. 
Percentage Composition of the Ashfree Substance. 
c 
Si-57 
• • 
Average. 
51-97 
H 
6-95 
6-9S 
N 
16.&2 
16.82 
S 
o 
24.26 
IOO.CO 
Arranging these several results in tabular form, it becomes easy 
to draw certain general conclusions from the data : 
Bx 
MX 
N«. 
F». 
Fw. 
Fxa. 
Ftf<». 
c 
53-95 
53-69 
53-29 
53-43 
52.98 
51-97 
H 
7.05 
7.00 
7.00 
6.90 
6.98 
6.95 
N 
16.13 
iS-94 
16.23 
i6-39 
15.87 
16.37 
16.82 
S 
o 
1.14 
21.73 
1.16 
22.21 
1.12I 
22.36/ 
23.28 
24-17 
•• 
24.26 40 
Myosin-like 
Very soluble 
Vitellin-like 
globulin. 
globulin, B6. 
globulin, F2, 
c 
52.72 
52-3S 
5i-99 
H 
7.05 
6.82 
6.81 
N 
X6.82 
I5-25 
18.02 
S 
I.32 
1.26 
0.66 
o 
22.05 
24.29 
22.52 
With the exception of the product Fl"*, all of these insoluble 
bodies have the same general composition, and are alike charac- 
terised by a high content of carbon. Taking into consideration 
the percentage of nitrogen in these bodies and the nitrogen of the 
several globulins present in the corn kernel, it is evident that the 
vitellin-like globulin cannot well be considered as an antecedent 
of these insoluble products. These latter bodies are unquestion- 
ably formed from the myosin-like, globulin and the still more 
soluble globulin with 15.25 per cent, of nitrogen. Doubtless it is 
this greater tendency of these two globulins to pass into insoluble 
modifications which facilitates the purification of the vitellin-like 
globulin. Thus the like preparations, Fx and Fw, coming as they 
do from the aqueous extract and from the sodium-chloride extract 
of the corn respectively, indicate a common origin, and it can be 
explained only on the supposition (certainly plausible enough) 
that the myosin-like globulin, not being wholly dissolved by the 
water extract, is dissolved together with the vitellin by the 10-per 
cent, salt solution and then later changed into the insoluble modi- 
fication just described. Again, the content of sulphur is of signifi- 
cance in this connection, in that the myosin-like globulin and its 
still more soluble neighbor are both characterised by a fairly high 
content of sulphur, while the vitellin-like globulin contains less 
than 1 per cent, of this element. 
In conclusion, then, we are apparently justified in making the 
statement that through the long-continued action of water and 
strong solutions of salt, as ammonium sulphate, the two globulins 
with a low content of nitrogen are gradually changed into insoluble 
modifications, which are especially characterised by a relatively 
high content of carbon. IX. Proteids Soluble both in Water and in Dilute 
Salt Solutions. 
An aqueous extract of corn meal, as well as a sodium chloride 
extract, contains in addition to the globulins already described 
more or less proteid matter soluble in water alone and having the 
general properties of ordinary albumin and proteose. These 
bodies can be detected, either in the original extract after the 
complete removal of all globulin, or better in the ammonium-sul- 
phate precipitate of the mixed proteids, after the globulins and 
their alteration-products have been entirely separated by dialysis 
and filtration. 
Of albumin-like bodies there would appear to be two present, 
both more or less coagulable by heat, but unlike in chemical com- 
position. Moreover, one is precipitable by the addition of dilute 
acetic or hydrochloric acid to a 10-per cent, sodium-chloride solu- 
tion, after the globulins have been entirely removed, while the 
other remains in solution and can be advantageously separated 
only by coagulation of the neutralised fluid. From the albumin- 
free solution, the proteose can be separated by saturation with 
sodium chloride and addition of a little acetic, or hydrochloric acid. 
An aqueous solution containing these water-soluble proteids is 
precipitated on saturation with ammonium sulphate, and on 
addition of alcohol, but not by saturation with sodium chloride. 
Addition of 0.2-per cent, hydrochloric acid or 30-per cent, acetic 
acid likewise gives no precipitate. Heated gradually, an aqueous 
solution of these proteids becomes turbid at about 46°, with sepa- 
ration of a flocculent precipitate at 6o°. Between 6o° and 70° a 
fairly large coagulum separates. The fluid again becomes turbid 
about 85°, and on boiling, a slight coagulum appears which gradu- 
ally increases as the boiling is continued. In fact, it is very diffi- 
cult to make a complete coagulation. The solution may be 
evaporated nearly to dryness, the residue taken up in water and 
filtered, and then on boiling this solution for some time an addi- 
tional coagulum may appear. Indeed, there is every evidence 
of some kind of transformation in the process of coagulation, or 
possibly it may be that we have to deal simply with a body very 
slowly, or incompletely, coagulable by heat, like some forms of 
phyt-albumose. 42 
Preparation A.—The proteids precipitated from a sodium- 
chloride extract of 5 kilos, of corn meal, by means of ammonium 
sulphate, were dissolved in water and 10-per cent, solution of 
sodium chloride, and dialysed until every trace of globulin and 
soluble salts had been removed. To the clear neutral fluid thus 
obtained, which gave no precipitate whatever on the addition 
of dilute acid or on saturation with sodium chloride, pure, crystal- 
lised salt was added in such quantity that the solution contained 
10 per cent, of sodium chloride, after which the clear fluid was 
rendered slightly acid with 0.2-per cent, hydrochloric acid. The 
precipitate thus produced was filtered off, treated with water in 
which, or in the slightly acid fluid which resulted, it was mainly 
soluble, and the solution carefully neutralised with a few drops 
of very dilute sodium carbonate. This gave rise to an abundant 
precipitate which was filtered off, washed thoroughly with water, 
alcohol and ether, weighing, when air-dry, 2.48 grains (Albumin 
A’)» This substance, when first precipitated by neutralisation, 
was very readily soluble in dilute sodium carbonate, but after 
becoming dry it was wholly insoluble in the dilute alkaline fluid. 
This description affords a very good illustration of the readiness 
with which some of these vegetable proteids undergo change by 
mere contact with dilute acids or alkalies. Evidently, the addition 
of a little 0.2-per cent, acid to the salt solution of these proteids 
gives rise to an acid compound of one of the albumins insoluble in 
salt solution (10-per cent.), but soluble in water or, rather, in the 
slightly acid fluid. Withdrawal of the acid, however, by neutral- 
isation with a few drops of very dilute sodium carbonate, yields not 
the original albumin, but apparently an insoluble acid-albumin; a 
reaction more characteristic of a globulin than of a genuine 
albumin. 
The 10-per cent, sodium-chloride solution, from which the 
above albumin was precipitated by the addition of 0.2-per cent, 
hydrochloric acid, was heated to boiling, when there resulted a 
coagulum which, after being washed with hot water until free 
from salt, and then with alcohol and ether, weighed air-dry, 1.03 
grams (Albumin A 2). 
On boiling the filtrate from this latter coagulum, no further 
precipitate could be obtained, but saturation of the slightly acid 
fluid with salt gave a considerable precipitate, readily soluble in 
water. The substance thus precipitated was therefore dissolved 43 
in water and dialysed until the sodium chloride was entirely 
removed. The salt-free solution was then evaporated to dryness 
on a water-bath. As the solution became concentrated, a portion 
of the proteid separated as a scum or skin on the surface of the 
fluid, eventually making an insoluble coagulum. On treating the 
dried residue with water, still more insoluble matter appeared, 
showing that either all of the coagulable albumins (Albumins A 1 
and A 2) had not been removed from the fluid, or else that in the 
process of treatment changes were going on by which new coagul- 
able matter was being formed. Eventually, however, by evapo- 
rating the filtered fluid to dryness several times and taking up the 
residue in water, there resulted a solution of a proteid body which 
showed no signs of coagulation, even on long-continued heating, 
and which yielded a gummy precipitate on the addition of abso- 
lute alcohol. This proteose-like body was, however, obtained 
in very small quantity—only about 0.3 gram—and might be 
considered as possibly termed by the hydrolytic action of the 
repeated evaporations, etc. 
On the other hand, it might perhaps be claimed with equal 
plausibility that the slow coagulations witnessed above were the 
result of a regressive metamorphism of a proteose normally 
present in the corn-kernel. 
The two albumins, A 1 and A 2, were dried at no° C. and 
analysed with the following results : 
Analysis of Albumin A\ 
I. 0.3828 gram substance gave 0,2355 gram H20zr6.84 per cent. H, and 
0.7398 gram CO2 52.71 per cent. C. 
IT, 0.3454 gram substance gave 43.4 cc. N at 3.00 C. and 761.7 mm. pres- 
sure" 15-64 Per cent. N. 
111. 0.3185 gram substance gave 39.4 cc. N at 2° C. and 765.5 mm. pres- 
sure zn 15.60 per cent. N. 
IV. 0.3292 gram substance gave 0.0010 gram ash 0.30 per cent. 
Percentage Composition of the Ash-free Substance. 
c 
52.86 
Average. 
52.86 
H 
6.86 
6.86 
N 
15.69 
15-65 
15,67 
S 
o 
} 
24.61 
100.00 44 
Analysis of Albumin A 2. 
I. 0.2355 gram substance gave 0.1430 gram H2O = 6.76 per cent. H, and 
0.4464 gram C02 51.72 per cent. C. 
11. 0.3498 gram substance gave 45.9 cc. N at 30 C. and 758.5 mm. pres- 
sure 16.27 per cent. N. 
111. 0.3214 gram substance gave 0.0013 gram ash = 0.40 per cent. 
Percentage Composition of the Ash-free Substa?ice. 
c 
Si-93 
Average, 
51-93 
H 
6.79 
6.79 
N 
!6-33 
16.33 
S 
o 
24-95 
100.00 
Preparation B. 
Another series of similar products was made as follows: 
6.5 kilos, of finely ground corn were extracted first with water 
and then with 10-per cent, salt solution. From these two ex- 
tracts the proteids were precipitated by saturation with ammo- 
nium sulphate, the precipitates dissolved separately, so far as 
possible, in water and 10-per cent, salt solution, and the glob- 
ulins separated by thorough dialysis. In the two filtrates, wholly 
free from globulin, the addition of an equal volume of 20-per 
cent, salt solution and a little 0.2-per cent, hydrochloric acid 
produced a flocculent precipitate, considerably heavier in the 
aqueous extract than in the sodium-chloride extract. As the two 
precipitates, however, were apparently the same, they were united 
and dissolved, so far as possible, in water. In this case, unlike the 
preceding experiment, a large proportion of the precipitate did not 
dissolve in the water added, apparently because the fluid was not 
sufficiently acid. This insoluble portion, after being washed with 
water, was therefore dissolved in a little 0.2-per cent, hydrochloric 
acid and the solution carefully neutralised with dilute sodium car- 
bonate, by which the substance was reprecipitated. After being 
washed thoroughly with water, alcohol and ether, it weighed, 
air-dry, 2.67 grams (Albumin B1). 
On analysis, after drying at no° C., it yielded the following 
results: 45 
Analysis of Albumin B\ 
I. 0.3274 gram substance gave 0.1992 gram H2O = 6.75 Per cent. H, and 
0.6369 gram CO2 = 53-05 Per cent. C. 
11. 0.3133 gram substance gave 0.1900 gram H2O = 6,74 per cent. H, and 
0.6081 gram Co2= S2-92 Per cent- c- 
111. 0.4413 gram substance gave 54.8 cc. N at 20° C. and 762.1 mm. pres- 
sure 2= 15.53 per cent. N. 
IV. 0.4758 gram substance gave 0.0048 gram ash=x.oi per cent. 
Percentage Composition of the Ashfree Substa?ice. 
c 
53-58 
53-48 
Average. 
53-53 
H 
6.82 
6.81 
, , 
6.82 
N 
15.69 
15.69 
S 
o 
::} 
23.96 
100.00 
The two original acid filtrates, from which the above albumin 
was separated, were in this preparation directly saturated with 
sodium chloride, and the precipitate of the residual albumin and 
proteose filtered off and dissolved in water. The resultant solu- 
tions were united, neutralised and dialysed until the salts were 
entirely removed. At the end of the dialysis, a small amount of 
substance was found adhering to the walls of the dialyser, not, 
however, sufficient in quantity to do anything with. On evapora- 
tion of the filtered fluid to half its volume considerable coagulum 
formed, which was washed with water, alcohol and ether, weighing, 
when air-dry, 1.32 grams (Albumin B2). On further evapora- 
tion of the mother-liquor containing the proteose an additional 
coagulum formed, which was added to the preceding. 
This preparation, dried at i io° L., gave on analysis the following 
results: 
Analysis of Albumin B\ 
I. 0.2387 gram substance gave 0.1453 gram H2O = 6.76 per cent. H, and 
0.4547 gram C02=:5i.95 per cent. C. 
II- o-3546 gram substance gave 47.6 cc. N at 30 C. and 751.8 mm. pres- 
sure zz 16.49 Per cent- N* 
111. 0.3868 gram substance gave 0.0008 gram ash = 0.21 per cent. 46 
Percentage Compositio?i of the Ash-free Substance. 
c 
52.06 
Average. 
52.06 
H 
6.77 
6.77 
N 
*6.53 
16.53 
S 
o 
' | 24.64 
100,00 
In the filtrate from which this last body was obtained there still 
remained some coagulable matter, which was finally completely 
removed by careful heating on a water-bath. The concentrated 
and perfectly neutral fluid was then precipitated with absolute 
alcohol, and the resultant proteose collected and washed with 
alcohol and ether. 
This substance, like the soluble proteoses formed from animal 
proteids, was especially characterised by extreme solubility in 
water, non-coagulability by heat, etc. It was precipitated by 
acetic acid and potassium ferrocyanide, by alcohol, phospho- 
tungstic acid, etc., and gave the usual proteid reactions with 
Millon’s reagent, and with cupric sulphate and potassium hy- 
droxide. 
Dried at no° C. until of constant weight, the product, separated 
as just described, gave on analysis the following results : 
Analysis of Proteose B. 
I. 0.3390 gram substance gave 0,1952 gram H2O 2=6.40 per cent. H, and 
0.6154 gram CQ2~ 49.51 per cent. C. 
11. 0.3409 gram substance gave o. 1993 gram H2O 2= 6.49 per cent. H, and 
0.6158 gram CQ2 49.27 per cent. C. 
111. 0.3326 gram substance gave 41.3 cc. N. at 2.50 C. and 760.9 mm. 
pressure” 15.49 per cent. N. 
IV. 0.2984 gram substance gave 37.4 cc. N at 40 C. and 758.5 mm. pres- 
sure 2= 15.49 per cent. N. 
V. 0.2784 gram substance, fused with KOH + KNOs, gave 0.0520 gram 
BaS04 =12.57 per cent. S; deducting 0.25 for S in the ash =22.32 per 
cent. S. 
VI. 0.4206 gram substance gave 0.0120 gram ash 2.42 per cent. 47 
Percentage Composition of the Ash-free Substance. 
Average. 
c 
50-74 
SO. 50 
50.62 
H 
6.56 
6.65 
6.61 
N 
bo 
00 
15.88 
15.88 
S 
2.37 
2-37 
O 
24.52 
100,00 
Preparation C. 
These same bodies were prepared again, on a somewhat larger 
scale, from 25 kilos, of finely ground corn. The process was as 
follows: The meal was extracted directly with an abundance of 
10-per cent, salt solution, and the proteids precipitated collect- 
ively by saturation of the fluid with ammonium sulphate. The 
precipitate was dissolved in water and salt solution, and the fluid 
dialysed about three weeks, until the salts were entirely removed 
and the globulins completely separated. 
The salt-free solution, containing the albumins and proteose, was 
then treated with sodium chloride until the mixture contained 10 
per cent, of salt, after which 0.2-per cent, hydrochloric acid was 
added until the precipitation was complete. The albumin so 
separated was filtered off and treated with water, in which about 
four-fifths of it dissolved. The residue, however, was dissolved 
in a little 0.2-per cent, hydrochloric acid and the two solutions 
united, and dialysed until free from chlorides. At the end of the 
dialysis the fluid was perfectly neutral, and the albumin (obviously 
altered), or a portion of it, was found precipitated in the parch- 
ment tube, the same as on neutralisation of the slightly acid fluid 
described in the previous preparations, while in the solution was 
found considerable non-coagulable proteose which must have been 
carried down with the albumin. The precipitate (Albumin C 1) 
was filtered off, washed thoroughly with water, alcohol and ether, 
and dried at no° C. for analysis. Air-dry, it weighed 3.14 grams. 
Analysis of Albumin C\ 
I. 0.3459 gram substance gave 0.2102 gram H2Q 6.75 per cent. H, and 
0.6714 gram Co2= 52.93 per cent. C, 
11. 0.3649 gram substance gave 0.2222 gram H2O 6.77 per cent. H,and 
0.7065 gram Co2= 52.81 per cent. C. 
111. 0.3546 gram substance gave 43 cc. N at 774.0 mm. pressure 15-35 
per cent. N. 48 
IV. 0.6957 gram substance fused with KOH + KNOs gave 0.0750 gram 
BaS04 x.48 per cent. S. 
V. 0.4331 gram substance gave 0.0016 gram ash —0,37 per cent. 
c 
53-12 
53.0° 
Average. 
53,06 
H 
6.78 
6.80 
6.79 
N 
iS-41 
15.41 
S 
1.48 
1.48 
o 
23.26 
100.00 
Percentage Composition of the Ash-free Substance. 
The proteose precipitated with the above albumin was sepa- 
rated from the concentrated solution, after removal of all coagu- 
lable matter, by precipitation with absolute alcohol. It weighed, 
air-dry, 2.5 grams and gave on analysis, after drying at no° C., 
the following results: 
I. 0.3131 gram substance gave 0.1903 gram 11.j0226.75 Per cent. H, and 
0.57x7 gram CO2 =249.80 per cent. C. 
Analysis of Proteose Cl. 
11. 0.3783 gram substance gave 0.2276 gram H2O 2z6.69 Per cent. H, and 
0.6873 gram C02122 49.55 per cent. C. 
111. 0.3397 gram substance gave 43.5 cc. N at 30 C. and 770.7 mm. pres- 
sure 22: 16.13 per cent. N. 
IV. 0.7246 gram substance fused with KOH + KNOs gave 0.0837 gram 
BaS04~ 1.58 per cent. S. 
V. 0.4632 gram substance gave 0.0131 gram ash 22 2.83 per cent. 
Percentage Composition of the Ashfree Substance. 
c 
51-25 
51.00 
Average. 
51- r3 
H 
6.94 
6.88 
6.91 
N 
16.59 
16.59 
S 
1.62 
1.62 
o 
23-75 
100.00 
The original filtrate from the precipitate produced by dilute 
hydrochloric acid in the presence of xo-per cent, sodium chloride 
was dialysed until nearly free from salt, and then, being perfectly 
neutral to test-papers, was concentx'ated to one-third its volume 
and filtered from the coagulum which resulted. This coagulum 49 
(Albumin C 2) was washed thoroughly with water, alcohol and 
ether, and after being dried at i io° C. was analysed. Air-dry it 
weighed 2.28 grams. 
Analysis of Albumin C", First Coagulum. 
I. 0,3226 gram substance gave 0.1927 gram HsO 6.64 per cent. H, and 
0.6092 gram Co2= 51.50 per cent. C. 
11. 0.3293 gram substance gave 0.1985 gram H2O 6.69 per cent. H, and 
0.6195 gram CO2 = 51.31 per cent. C. 
111. 0.3737 gram substance gave 46.3 cc. N at 30 C. and 774.2 mm. pres- 
sure 15.68 per cent. N. 
IV. 0.4015 gram substance gave 0.0038 gram ash = 0.94 per cent. 
Percentage Composition of the Ash-free Substance. 
S1-78 
Average. 
c 
S'-97 
51.88 
H 
6.70 
6.75 
. . 
6-73 
N 
S 
.. 
•• 
15.89 
• • 1 
15.89 
o 
• • 
• • 
25.50 
100.00 
The filtrate from the above coagulum was concentrated still 
further, without giving any appreciable coagulum, then saturated 
with salt and precipitated by the addition of 0.2-per cent, hydro- 
chloric acid. The precipitate was dissolved in water, dialysed 
until the salt had been entirely removed, and then evaporated 
to dryness on the water-bath. During this last evaporation 
a considerable coagulum formed, which was collected, washed, 
dried and analysed. It weighed 1.22 grams (Albumin C 2, second 
coagulum). 
Analysis of Albumin C\ Second Coagulum. 
I. 0.4488 gram substance gave 0.2600 gram H2Q —6.44 Per cent. H, and 
0.8224 gram 003 49.98 per cent. C, 
11. 0.3050 gram substance gave 41.8 cc. N at 30 C. and 755-8 mm. pres- 
sure 16.93 per cent. N. 
111. 0.3769 gram substance gave 0.0077 gram ash 2.04 per cent. 
Percentage Composition of the Ashfree Substance. 
c 
51.02 
Average. 
51.02 
H 
6-57 
6.57 
N 
17.28 
17.28 
S 
’• \ 25.13 
O 
.. 
100.00 50 
From the concentrated filtrate from this second coagulum, a 
small amount of proteose was separated by addition of absolute 
alcohol. Dried at i io° and analysed, it gave the following results: 
Analysis of Proteose C 2. 
I. 0.3666 gram substance gave 0.2065 gram H2O 6.26 per cent. H, and 
0.6435 gram C02 =47.87 per cent. C. 
11. 0.3333 gram substance gave 0.1901 gram H2O =6.34 per cent. H, and 
0.5934 gram C02 = 48.55 per cent, C. 
111. 0.3654 gram substance gave 46.3 cc. N at 30 C. and 768.9 mm. pres- 
sure = 15.93 per cent. N. 
IV. 0.4266 gram substance gave 0.0159 gram ash = 3.72 per cent. 
Percentage Composition of the Ashfree Substance. 
c 
49.72 
50.42 
Average. 
50.07 
H 
6.50 
6.58 
6.54 
N 
16.54 
16.54 
S 
o 
. . 
.. 
26.85 
100.00 
Preparation D. 
Another experiment after the same order as the preceding was 
carried out without, however, yielding sufficient of any one product 
for analysis, except of the albumin coagulable by heat present 
in the filtrate from the precipitate produced by 10-per cent, salt 
solution and dilute acid. 
This product (Albumin D 2) was analysed for the sake of 
additional confirmatory evidence, with the following results: 
Analysis of Albumin D 2, Coagulum. 
I. 0.4274 gram substance gave 0.2584 gram H2O = 6.71 per cent. H, and 
0.8055 gram C02 = 51.39 per cent. C. 
11. 0.3735 gram substance gave 0.2278 gram H2O = 6-77 per cent. H, and 
0.7076 gram CQ2= 51.66 per cent. C. 
111. 0.3739 gram substance gave 49.8 cc. N at 17.30 C. and 757 mm. pres- 
sure = 15.67 per cent. N. 
IV. 0.3783 gram substance gave 51.1 cc. N at 18.2° C. and 755.1 mm. pres- 
sure = 15.80 per cent N. 
V. 0.3450 gram substance gave 0.0013 gram ash = 0.38 per cent. Percentage Composition of the Ash-free Substance. 
c 
5r-5S 
51.84 
Average. 
5I-7I 
H 
6-73 
6.79 
6.76 
N 
• • 
15.72 
to 
CO 
tO 
Lri 
00 
25-75 
100.00 
The relationship in composition of these several products can 
be seen from the following table : 
Albumin Precipitated by Salt and Acid. 
A1. 
B«. 
Cl. 
C 
52.86 
53-53 
53-o6 
H 
6.86 
6.82 
6-79 
N 
15.67 
15.69 
iS-4i 
S ‘ 
O. 
} 
24.61 
23.96 
{ 
1.48 
23.26 
Albumin Coagulated by Heat. 
A2. 
B2. 
C2, 1st. 
C2, 2d. 
D2. 
c 
S*-93 
52.06 
51.88 
51.02 
5I-7I 
H 
6.79 
6.77 
6-73 
6.57 
6.76 
N 
16.33 
16.53 
15.89 
17.28 
15.78 
0} 
24.95 
24.64 
25.50 
2S-I3 
25-75 
Proteose. 
B. 
Ci. 
C2. 
c 
50.62 
5I-I3 
50.07 
H 
6.61 
6.91 
6.54 
N 
15.88 
16.59 
16.54 
n 
2-37 
1.62 
26.85 
0 / 
24.52 
23-7S 
We are so firmly convinced that these bodies are, in part at least, 
the products of changes induced by the several steps incidental to 
their separation, that we are loth to draw any very definite conclu- 
sions as to their true chemical composition. By this we do not 
mean that these three series of products are wholly artificial. 
Bodies of the general nature of the albumins here indicated unques- 
tionably exist in the corn-kernel. As already stated, an aqueous 
solution of the corn proteids, entirely freed from globulins, gives a 
coagulum between 6o° and 70°, and another one between 85° and 
ioo°, thus implying the presence of two distinct bodies. It may 52 
possibly be assumed that the albumin precipitated by salt and 
acid is simply a portion of the albumin separated later by coagu- 
lation; that this partial precipitation is simply another illustration 
of the incompleteness characteristic of the separation of these 
albumins, as witnessed, for example, in the gradual and incomplete 
coagulation of the above albumin by heat; and further, that the 
difference in chemical composition between the first and second 
series of products is simply due to their alteration by the processes 
made use of. Yet we can hardly consider this the true view. The 
peculiar heat-coagulation phenomena of the original aqueous solu- 
tion, before any separations were attempted, lend favor to the view 
that at least two coagulable albumins are present. On the other 
hand, the incompleteness of the coagulation at any one point—the 
fact that more and more coagulum continues to form as the boiling 
is continued—points clearly to gradual changes, which, doubtless, 
leave their impression upon the products separated. Thus, in 
Preparation C, the two albumins coagulated by heat, C 2 ist and 
C 3 2d, should theoretically have the same composition, the only 
tangible difference being that the second coagulum required longer 
heating to bring about its separation, and yet it differs from its 
neighbor by 1.5 per cent, of nitrogen. Observations of this 
character, and others which we will not take space to detail, 
confirm us in the opinion that these soluble bodies are exceedingly 
prone to change, and doubtless the composition here given is 
only an approximation to the true make-up of the substances as 
they exist in the corn-kernel. At the same time, it may not 
be amiss to recall the fact that in the seed itself, in its various 
stages of growth and development, kindred changes are doubtless 
taking place among its natural proteid constituents. Whether 
the proteose separated by the above methods exists as such in 
the corn-kernel, or whether it is derived from the other proteids 
by the methods of separation, is likewise uncertain. A portion of 
it is unquestionably formed by the hydrolytic changes incidental to 
the separation of the coagulable proteids, and this favors the view 
that it is wholly an artificial product. 
Further, a similar proteose-like body was directly prepared by 
heating to Bo° C. one of the preceding globulins, dissolved in 
salt-solution. Again, the somewhat variable composition of the 
proteoses analysed suggests a mixture of two or more dissimilar 
proteoses, such as might be expected in an artificial production of 
the character indicated. 53 
One point to be noted in connection with the proteose is its 
relatively low content of carbon; a point which is characteristic of 
most of the albumoses or proteoses formed by the action of the 
digestive ferments, both from animal and vegetable proteids. 
111.—Proteid Matter Soluble in Alcohol, but Insoluble 
in Water and Salt-Solutions. 
Under this head we have to deal with only one substance, viz. 
Zein or the “ maize fibrin ” of Ritthausen. The former name, 
originally given by Gorham' to this peculiar proteid found by him 
in Zea mays, seems especially appropriate, and we would suggest 
its adoption as a more fitting name than the term employed by 
Ritthausen. This body, as is well known, is especially character- 
ised by its solubility in weak alcohol, and can be obtained by 
direct extraction of corn meal with 95-per cent, alcohol, preferably 
at 40°-6o°C., or by extraction of the meal with warm alcohol after 
previous treatment with water, salt-solution, or both. 
Several preparations were made under varying conditions as 
follows: 
2.5 kilos, of finely ground corn meal were first thoroughly 
extracted with cold water, after which the residue of meal was 
treated with 95-per cent, alcohol in sufficient quantity to have the 
water retained by the meal reduce the strength to about 75 per 
cent. The mixture was warmed for some hours at about 46° C., 
and finally filtered at approximately that temperature. The filtrate 
was concentrated on the water-bath at a temperature below 65° C. 
The residue of meal was again treated with 75-per cent, alcohol 
(4 litres) at 6o°C. for several hours, and filtered while at that tem- 
perature. In all, five successive extractions were made after this 
fashion, the individual alcoholic extracts being evaporated sepa- 
rately, all at a low temperature. The last three extracts left but 
little residue on evaporation. As the first two extracts became 
concentrated the proteid separated as a tough, leathery, yellow- 
colored mass on the sides and bottom of the dish. It was cut up 
into small pieces and soaked for several days in cold absolute 
alcohol. On this first treatment of the proteid with absolute 
alcohol some little dissolved, presumably from dilution of the 
Preparation A. 
1 See Berzelius, Jahresbericht 2, (1822), p. 124. 54 
alcohol with the water contained in the substance. This alcoholic 
extract was therefore concentrated to a syrup and the proteid 
precipitated by ether. It then had the appearance of a crumbly, 
pulverisable substance, and was easily ground to a powder under 
absolute alcohol. On slightly warming the alcohol, however, the 
substance melted and finally dissolved completely in the alcoholic 
fluid. On cooling this solution, the substance separated in a very 
gummy state. 
The main portion of the proteid, which had been treated once 
with absolute alcohol, was treated with successive portions of cold 
absolute alcohol, as long as anything was dissolved by this reagent, 
after which it was treated with a mixture of one part of ether and 
two parts of absolute alcohol and, finally, was soaked in ether 
alone. After this treatment we were able to reduce it to a fine 
powder, when it was thoroughly extracted with ether for the 
complete removal of any adhering fat. 
So prepared, the substance was only partially soluble in warm 
75-per cent, alcohol, a portion of it during the foregoing process 
having been converted into an insoluble modification. This change 
from the soluble (in warm alcohol) to the insoluble form is liable 
to occur whenever the substance is heated with too watery alcohol, 
especially if the temperature approaches ioo° C.> or if the heating 
be long-continued. The soluble portion referred to above was 
therefore completely separated by repeated treatment with warm 
and with boiling dilute alcohol, and the insoluble portion ultimately 
washed with absolute alcohol and ether and dried at no° C. for 
analysis. 
The alcoholic solution of the soluble portion of the proteid 
was poured into about three volumes of water, by which the sub- 
stance was precipitated in white flocks, which, however, soon com- 
menced to unite and rise to the surface, eventually forming a dense 
cake. On heating this mass it continued to contract, squeezing out 
the water in large quantities. The substance, still containing con- 
siderable water, was next dissolved in warm 95-per cent, alcohol, 
the solution filtered, concentrated to about 400 cc. and again 
poured into a large volume of water, about 3 litres. This time 
the substance did not separate as before, but remained suspended 
in the form of a thick emulsion and was made to flock only on 
the addition of a little salt. It was then filtered off, washed with 55 
water until free from chlorides, lastly with alcohol and ether and 
dried at no° C. for analysis. 
Analysis of Zein. Preparation A.—Soluble, 
I. 0.3079 gram substance gave 0.2045 gram H2O 7.34 per cent. H, and 
0.6226 gram C02z: 55.14 per cent. C. 
11. 0.3453 gram substance gave 46.8 cc. N at 17.00 C. and 757.9 mm. pres- 
sure ~ 15.98 per cent. N. 
111. 0.5310 gram substance fused with KOH -f- KNOs gave 0.0197 gram 
BaSO4 ~ 0.51 per cent. S. 
IV. 1.0210 grams substance fused with OH + KNOs gave 0.0402 gram 
BaSQ4~ 0.54 per cent. S. 
V. 1.0600 grams substance gave 0.0024 gram ash ~ 0.22 per cent. 
VI. 1.0045 grams substance gave 0.0028 gram ash “0.27 per cent. 
Percentage Composition of the Ashfree Substance. 
c 
55-27 
Average. 
55-27 
H 
7-35 
. . 
7-35 
N 
16.01 
16.01 
S 
0.51 
0.54 
o-S3 
o 
20.84 
100.00 
Analysis of Zein. Preparation A.—Insoluble. 
I. 0.3210 gram substance gave 0.2094 gram per cent. H, and 
0,6451 gram C02 54.80 per cent. C. 
11. 0.4055 gram substance gave 0.2680 gram H20r=7,34 per cent. H, and 
0.8170 gram C02~ 54.94 per cent. C. 
111. 0.3171 gram substance gave 43.1 cc. N at 17. i° C. and 761.6 mm. pres- 
sure ~ 16.10 per cent. N. 
IV. 0.3250 gram substance gave 43.7 cc. Nat 16.80 C. and 763.5 mm. pres- 
sure =2 15.98 per cent, N. 
V. 0.5000 gram substance fused with KOH-j-KN03 gave 0.0221 gram 
BaS04 = 0.6 i per cent. S. 
VI. 0.9834 gram substance gave 0.0018 gram ash ~ 0.18 per cent. 
VII, 0.2637 gram substance gave 0.0005 gram ash “0,19 per cent. 
Percentage Composition of the Ash-free Substance. 
c 
54.90 
55-04 
Average 
54-97 
H 
7.25 
7-35 
7-30 
N 
16.01 
16.13 
16.07 
S 
0.61 
o.6x 
o 
21.05 
100.00 56 
Preparation B. 
2.5 kilos, of finely ground corn, which had been thoroughly 
extracted with 10-per cent, salt solution, were treated directly 
with warm 75-per cent, alcohol, until the alcohol ceased to 
dissolve anything further. The first alcoholic extract, on con- 
centration, yielded the proteid—doubtless on account of the large 
amount of sodium chloride it contained—as a yellow, granular 
mass which could be broken quite readily. The residues from 
the following extracts, however, were of the same general nature 
as those described under Preparation A. All of the residues 
from the several extracts were united, washed with distilled 
water until the salt was entirely removed, then dissolved in warm 
80-per cent, alcohol and the solution filtered to remove any insol- 
uble matter present. The solution was next evaporated on a 
water-bath nearly to dryness and the residue pressed as dry as pos- 
sible, yielding a yellow, elastic substance which could be pulled 
like molasses candy, but which was much more elastic and tough. 
Its elasticity was so great that it could be pulled into sheets 
several inches square and very thin. After continued kneading 
under absolute alcohol, it finally became brittle, a considerable 
portion of it passing into solution at the same time. After 
thorough extraction with absolute alcohol in this manner, it was 
soaked for some time in ether and then ground to a coarse pow- 
der. Like the preceding preparation, the proteid was now found 
only partially soluble in dilute alcohol. It was therefore separated 
into two portions by continued treatment with warm 80-per cent, 
alcohol, after first moistening the powdered product with a little 
water. The insoluble portion was washed with absolute alcohol 
and ether, and dried at 1 io° C. for analysis. 
The alcoholic solution containing the soluble portion of the 
proteid was poured into three volumes of water, by which the zein 
was precipitated in white flocks, which gradually formed into a 
cake floating on the surface of the fluid. This was redissolved in 
95-per cent, alcohol, and again precipitated as before. After a 
third precipitation in this manner, the substance was dissolved in 
95-per cent, alcohol, the solution filtered, and the filtrate evapor- 
ated on the water-bath. Towards the end, absolute alcohol was 
added several times in order to prevent the substance from separ- 
ating in consequence of the alcohol becoming too dilute. The 57 
evaporation was finally carried to dryness, and the residue cut up 
into small pieces and thoroughly extracted with ether. A portion 
of this soluble zein, dried at no° C. until of constant weight, was 
analysed with the following results ; 
Analysis of Zein. Preparaiion B.—Soluble. 
I- 0.3252 gram substance gave 0.2132 gram H2O =27.28 per cent. H, and 
0.6550 gram C02zz 54.92 per cent. C. 
11. 0.2968 gram substance gave 0.1932 gram H20=7.24 per cent. H, and 
0.5985 gram per cent. C. 
111. 0.4193 gram substance gave 57.4 cc. Nat 17.30 C. and 755.1 mm. 
pressure 16.06 per cent. N. 
IV. 0.3569 gram substance gave 49.5 cc. Nat 18.40 C. and 755.1 mm. 
pressure 16.20 per cent. N. 
V. 0.5365 gram substance gave 0.0025 gram ash=: 0.46 per cent. 
VI. 0.4590 gram substance gave 0.0020 gram ash =0.46 per cent. 
VII. 1.0270 grams substance fused with KOH + KNOs gave 0.0423 gram 
BaS04~0.57 per cent. S. 
VIII. 1.0093 grams substance fused with KOH-f-KNO3 gave 0.0385 
gram BaSQ4 0.52 per cent. S. 
Percentage Composition of the Ash-free Substance. 
c 
55-17 
55.23 
Average. 
55-20 
H 
7-31 
7.27 
7.29 
N 
16.13 16.27 
.. 16.20 
S 
0.57 
0.52 0.55 
o 
•• 
.. 20.76 
100.00 
The insoluble portion, after being dried at no° C., gave the 
following results on analysis : 
Analysis of Zein. Preparation B.—Insoluble. 
I. 0.3562 gram substance gave 0.2290 gram H2O —7.14 per cent. H, and 
0.7200 gram C02 55.12 per cent. C. 
11. 0.4284 gram substance gave 0.2772 gram HaO 7.19 per cent. H, and 
0.8660 gram C02 zz 55.12 per cent. C. 
111. 0.4203 gram substance gave 0.2708 gram H2O 7.15 per cent. H, and 
0.8504 gram C02 zz 55.17 per cent. C. 
IV. 0.3733 gram substance gave 51.2 cc. N at 17.50 C. and 762.4 mm. 
pressure 16.24 per cent. N. 
V. 0.4449 gram substance gave 60.8 cc. N at 16.9° C. and 761.8 mm. 
pressure =2 16.20 per cent. N. 
VI. 0.5238 gram substance fused with KOH-f-KNO3 gave 0.0245 gram 
BaSO4 = 0.64 per cent. S. 58 
VII. 0.7747 gram substance fused with KOH-j-KN03 gave 0.0350 gram 
BaSO* 0.62 per cent. S. 
VIII. 0.4675 gram substance gave 0.0021 gram ash~0.45 per cent. 
IX. 0.4705 gram substance gave 0.0020 gram ash~ 0.43 per cent. 
c 
55-36 
55-36 55.41 
Average 
55-37 
H 
7.17 
7.22 7.18 
7-i9 
N 
.. .. 16.31 16.27 
.. .. 16.29 
S 
0.64 0.62 0.63 
o 
20.52 
100.00 
Percentage Composition of the Ash-free Substance. 
Preparation C. 
2.5 kilos, of corn meal, previously extracted with 5-per cent, 
ammonium chloride solution, were treated several times with 
warm 75-per cent, alcohol, and the alcoholic extract concentrated at 
a low temperature. The leathery-like residues left by evapor- 
ation of the alcohol were washed somewhat with water, then dis- 
solved, so far as possible, in warm 80-per cent, alcohol, the clear 
fluid evaporated nearly to dryness, and the residue digested repeat- 
edly with absolute alcohol and, lastly, with ether. On becoming air- 
dry, the substance was pulverised and again extracted with ether. It 
was then dissolved as completely as possible in 80-per cent, alcohol, 
after having been first gently warmed with a little water. A con- 
siderable quantity of the substance remaining insoluble, this was 
removed by decantation and filtration, the residue washed first 
with dilute alcohol, lastly with alcohol and ether, and dried at 
1 io° C. for analysis. The alcoholic solution containing the soluble 
portion of the proteid was precipitated by being poured into water, 
a process which was repeated three times; after which the pre- 
cipitated zein was digested with absolute alcohol and finally with 
ether, and a portion dried at 1 io° C. for analysis. 
Analysis of Zein. Preparation C.—Soluble. 
I. 0.3728 gram substance gave 0.2396 gram H2O~ 7.14 Per cent. H, and 
0.7520 gram C02z=: 55.01 per cent. C. 
11. 0.3587 gram substance gave 0.2305 gram H2O 7.14 per cent. 11, and 
0.7236 gram C02 55.01 per cent. C. 
111. 0.4218 gram substance gave 58.0 cc. Nat 17.20 C. and 759.6 mm. pres- 
sure 16.24 per cent. N. 
IV. 0.4085 gram substance gave 55.9 cc. Nat 17.2° C. and 760.2 mm. 
pressures 16.17 per cent. N. 59 
V. i.0375 grams substance fused with KOH-f-KNOs gave 0,0460 gram 
BaS04“ 0.61 per cent. S. 
VI. 0.8267 gram substance fused with KOH -{-KNOs gave 0,0348 gram 
BaS04 22: 0.58 per cent. S. 
VII. 0.4610 gram substance gave 0.0008 gram ash 22: 0.17 per cent. 
VIII. 0.5250 gram substance gave 0.0009 gram ash 0.17 per cent. 
c 
SS-io 
SS-io 
.. 
Average. 
55-10 
H 
7-i5 
7-i5 
7-15 
N 
16.26 
x6.ig 
.. l6.22 
S 
0.61 
O.58 O.60 
o 
20.93 
100.00 
Percentage Composition of the Ash-free Substance. 
I. 0.3885 gram substance gave 0,2518 gram H2O ~ 7.20 per cent. H, and 
0.7835 gram C02 22: 54-99 per cent. C. 
Analysis of Zein, Preparation C.—lnsoluble. 
11. 0.3848 gram substance gave 0.2514 gram HzO 222 7.25 per cent. H, and 
54.94 per cent. C. 
111. 0.4964 gram substance gave 67.9 cc. Nat 17.50 C. and 763.6 mm. 
pressure ~ 16.22 per cent. N. 
IV. 0.4119 gram substance gave 57.0 cc. Nat 17.7° C. and 760.6 mm. 
pressure 16.33 per cent. N. 
V. 0.5295 gram substance fused with KOH + KNO3 gave 0.0240 gram 
BaS04 2220.62 per cent. S. 
VI. 0.5890 gram substance fused with KOH KNOs gave 0.0273 gram 
BaS04 222 0.64 per cent. S. 
VII. 0.5250 gram substance gave 0.0014 gram ash —0,27 per cent. 
VIII. 0.5737 gram substance gave 0.0013 gram ash —0.23 per cent. 
c 
55-12 
55-07 
Average, 
55.IO 
H 
7.21 
7.26 
7.23 
N 
16.26 
16,37 
16.31 
S 
o 
0.62 
O.64 0.63 
20.93 
100.00 
Percentage Composition of the Ash-free Substance. 
Preparation D. 
3.0 kilos, of freshly ground corn were extracted directly with 
about 4 litres of 75-per cent, alcohol for 24 hours, at a temperature 
varying between 40° and 6o° C, and filtered while warm. The 
clear extract was then concentrated to about 500 cc, and the proteid precipitated by pouring the solution into a large volume 
of water. The substance so separated, in the form of lumps and 
strings, was washed frequently with a large amount of water, and 
finally soaked in water for some hours. It was then collected on 
muslin, squeezed dry, treated with 95-per cent, alcohol, in which 
it dissolved readily and almost completely, and again precipitated 
by water. This process of solution in dilute alcohol and repre- 
cipitation with water was repeated several times, the product 
ultimately being divided into two fractions, which differed from 
each other simply in the number of times this process had been 
repeated. The two products, D 1 and D 2, were then treated 
repeatedly with absolute alcohol and ether, until all coloring matter 
and fat had been removed, after which they were ground to a 
fine powder and portions dried at 1 io° C. for analysis. 
The residue of meal, extracted a second time with warm dilute 
alcohol, yielded a second portion of zein, which was separated and 
purified in essentially the same manner as the preceding prepara- 
tion, the final product being wholly soluble in dilute alcohol. This 
was likewise reduced to a powder, after successive treatment with 
ether and absolute alcohol, and a portion dried at no° C. for 
analysis. (Product D 3.) 
I. 0.3028 gram substance gave 0.1991 gram H20=7.30 per cent. H, and 
0,6107 gram C02=: 54.99 per cent. C. 
Analysis of Zein. Preparation D 1 
11. 0.2455 gram substance gave 0.1590 gram H2O =27.20 per cent. H, and 
0.4952 gram C02 55.00 per cent. C. 
111. 0.3580 gram substance gave 47.8 cc. N at 16.80 C. and 766,2 mm. pres- 
sure =2 15.92 per cent. N. 
IV. 0.3557 gram substance gave 48.3 cc. N at 18.4° C. and 756.3 mm. pres- 
sure 2= 15.90 per cent. N. 
V. 0.5000 gram substance fused with KOH-fKNOj gave 0.0234 gram 
BaS042=0.64 per cent. S. 
VI. 0.5145 gram substance gave 0.0040 gram ash =0.78 per cent. 
Percentage Composition of the Ash-free Substance. 
c 
55-42 
55-43 
Average, 
55.42 
H 
7-35 
7.25 
7-3° 
N 
16.04 
16.02 
16.03 
S 
. . 
0.64 
0.64 
o 
•• 
20.61 
100.00 Analysis of Zein. Preparation D"1. 
I. 0.3401 gram substance gave 0.2220 gram H2O 33 7.25 per cent. H, and 
0.6857 gram C02 =3 54.98 per cent. C. 
11. 0.3333 gram substance gave 0.2156 gram H2O 7.19 per cent. H, and 
0.6740 gram C02 55.14 per cent. C. 
111. 0.3696 gram substance gave 50.1 cc. N at 17.00 C. and 757.4 mm. pres- 
sure 15.97 per cent. N. 
IV. 0.3731 gram substance gave 50.5 cc. N at 17.9° C. and 755.7 mm. pres- 
sure 33 15.89 per cent. N. 
V. 0.619S gram substance fused with KOH-j-KN03 gave 0.0305 gram 
BaS04 33 0.67 percent. S. 
VI. 0.5065 gram substance gave 0.0030 gram ash 33 0.59 per cent. 
VII. 0.5x31 gram substance gave 0.0035 gram ash 33 0.68 per cent. 
Percentage Composition of the Ash-free Substance. 
c 
55-33 
55-49 
Average. 
55-41 
H 
7.29 
7-23 
.. 
7.26 
N 
16.07 
x5-99 
16.03 
S 
.. 
.. 
0.67 
0.67 
o 
•• 
•• 
20.63 
100.00 
I. 0.3638 gram substance gave 0.2366 gram H2O 33 7.22 per cent. H, and 
°-73°° gram C02 33 54 72 per cent. C. 
Analysis of Zein. Preparation D*. 
11. 0.3892 gram substance gave 0.2551 gram H2O = 7.27 per cent. H, and 
0.7846 gram C02 33 54.99 per cent. C. 
111. 0.4819 gram substance gave 65.3 cc. Nat 17.90 C. and 761.2 mm. pres- 
sure 15.99 Per cent. N. 
IV. 0.4x43 gram substance gave 55.6 cc. Nat 17.0° C. and 759.7 mm. pres- 
sure 33 15.86 per cent. N. 
V. 0.5200 gram substance gave 0.0038 gram ash 33:0.73 per cent. 
VI. 0.5163 gram substance gave 0.0039 gram ash 330.75 per cent. 
VII. i.ox 17 grams substance fused with KOH -j- KN03 gave 0.0395 gram 
BaS04 33 0.54 per cent. S. 
VIII. x.0520 grams substance fused with KOH-f-KNOs gave 0.0425 gram 
BaS04 3i 0.56 per cent. S. 
Percentage Composition of the Ashfree Substance. 
c 
55-i2 
55-38 
Average. 
55-25 
H 
7.27 
7-32 
. . 
7.29 
N 
16.10 
15-97 
. . 
. . 
16.04 
S 
.. 
0.54 
0.56 
°-55 
O 
•• 
20.87 
100.00 62 
A comparison of the analyses of these several preparations of 
zein, or maize fibrin, shows a remarkable in composi- 
tion, and furthermore indicates that the insoluble modification of 
the proteid is not at all different in composition from the main 
product. This is plainly apparent from the following table: 
Soluble Zein. 
Insoluble Modification. 
A 
B 
C 
D1 
D2 
D3 
A 
B 
C 
Average. 
c 
55-2? 
55-20 
SS-io 
55-42 
55-41 
55-25 
54-97 
55-37 
SS-io 
55-23 
H 
7-35 
7.29 
7-iS 
7.30 
7.26 
7.29 
7-30 
7.19 
7-23 
7.26 
N 
l6.0I 
16.20 
16.22 
16.03 
16.03 
16.04 
16.07 
16.29 
16.31 
16.13 
S 
o-53 
o-5S 
0.60 
0.64 
0.67 
o-55 
0.61 
0.63 
0.63 
0.60 
o 
20.84 
2O.76 
20.93 
20.61 
20.63 
20 87 
21.65 
20.52 
20.73 
20.78 
100.00 
100.00 
100.00 
100.00 
100.00 
100.00 
100.00 100.00 
100.00 
100.00 
Ash 
0.24 
0.46 
0.17 
0.78 
0.63 
0.74 
0.19 
0.44 
0.25 
Maize fibrin, as prepared and analysed by Ritthausen,1 was 
found to contain 54.69 per cent. C, 7.51 per cent. H, 16.33 Per 
cent. N and 0.69 per cent. S, thus agreeing very closely with our 
data, except in carbon. This slight difference in composition obvi- 
ously cannot be explained with certainty, but considering the 
great care used in the purification of our products and their close 
agreement in composition, together with the use of unquestionable 
analytical methods, it seems probable that the above series of data 
must accurately represent the true composition of maize fibrin or 
zein. It is furthermore equally evident from the analytical data 
that this substance is a homogeneous body, and not composed of 
two or more dissimilar bodies. 
Freshly precipitated zein (precipitated from an alcoholic solu- 
tion by water) is wholly insoluble in 0.5-per cent, sodium carbon- 
ate, even when warmed with the alkaline fluid at 40° C. for 24 
hours. It is likewise equally insoluble in 0.2-per cent, hydro- 
chloric acid, but can be completely dissolved by an aqueous solu- 
tion of potassium hydroxide of 0.2-per cent, strength. So dis- 
solved it is not converted into alkali-albumin, as is indicated by 
the fact that the precipitate produced on neutralisation of the 
alkaline fluid is not soluble in excess of dilute acid, but is com- 
pletely dissolved by alcohol. This resistance of the proteid to the 
den Stickstoffgehalt der Pflanzen-Eiweisskorper.”—Pfluger’s Archiv fur die 
gesammte Physiologic 18, 246. 63 
action of alkalies is quite pronounced; thus, exposure of the sub- 
stance to the action of 0.2-per cent, caustic potash at 40° C. for 24 
hours is not followed by any conversion into alkali-albumin. This 
is likewise true even when the strength of the alkali is raised to 
2.0-per cent. KOH. Exposure to a higher temperature than 40° 
C. with this strength of alkali is, however, followed by a change 
in the proteid. 
Freshly precipitated zein is wholly insoluble in water, and on 
being boiled with water is changed into the insoluble modification 
already referred to, which is insoluble both in alcohol and dilute 
(0.2-per cent.) potassium hydroxide. Zein gives the usual proteid 
reactions with Millon’s xanthoprotein and biuret tests. 
On warming the proteid at 40° C. with pepsin-hydrochloric acid, 
the substance is gradually dissolved and converted into proteose- 
like bodies, and true peptones non-precipitable by saturation with 
ammonium sulphate. 
Boiled with dilute sulphuric acid (6 cc. concentrated acid in 300 
cc. water) zein at first melts, forming a gummy mass or lump which 
is only slowly attacked by the acid, the portion dissolved being 
mainly converted into proteoses and peptone. Heated with 
stronger sulphuric acid, it undergoes a more pronounced decom- 
position, yielding large amounts ofleucin with considerable tyrosin, 
and apparently large amounts of glutamic acid. 
Corn meal, after thorough extraction with salt-solution and warm 
dilute alcohol, yields little proteid matter to dilute solutions of 
potassium hydroxide (0.2-per cent.). 
Summary. 
1. The maize-kernel contains several distinct proteids, well 
characterised in reactions and composition. Of these, there are 
three globulins, one or more albumins, and an alcohol-soluble 
proteid. 
2, The globulin obtainable from the maize-kernel by extraction 
with 10-per cent, solution of sodium chloride and separation by the 
usual methods—as by dialysis, or by precipitation with ammonium 
sulphate, followed by dialysis—is a mixture of two or more dis- 
similar globulins, differing from each other both in composition 
and in coagulation-points. 
3. The mixed globulin can be approximately separated into its 64 
two constituents by fractional heat-coagulation, or by a process of 
“ recrystallisation” from warm dilute salt-solution. In the former 
process, there is at the same time formed a small amount of pro- 
teose-like bodies, presumably by hydrolysis of the less resistant 
globulin. 
4. The two globulins separable by the above methods from the 
mixed globulin are a myosin-like body and a vitellin-like body. 
The myosin-like globulin is characterised by containing about 
16.8 per cent, of nitrogen and 1.2 per cent, of sulphur, agreeing 
closely in its composition with animal myosin. It has, however, a 
coagulation-point (in 10-per cent, salt solution) of about 70° C. 
The vitellin-Jike globulin, on the other hand, contains about 
18.1 per cent, of nitrogen and 0.85 per cent, of sulphur, agreeing 
closely with the generally accepted composition of phyto-vitellin. 
This body, however, is almost entirely non-coagulable by heat 
when dissolved in dilute salt-solution, except in the presence of 
acetic acid. It is more soluble in warm salt-solutions than in cold, 
and when separated from the former by cooling the fluid, or on 
dialysis, almost invariably appears in the form of small spheroids. 
5. These two globulins exist as such in the corn or maize kernel, 
and are not products of a cleavage of the so-called mixed globulin. 
This is evident from the coagulation-points of a salt-solution of the 
mixed globulin; from the fact that the separation can be accom- 
plished without the aid of heat; and, lastly, since it is possible 
to directly extract the individual globulins from the kernel, by the 
use of appropriate solvents. 
6. Direct extraction of finely powdered corn meal with water 
yields a dilute salt-solution, which dissolves the myosin-like glob- 
ulin, leaving the bulk of the vitellin-like substance undissolved. 
Probably, the character of the salts present in the kernel plays an 
important part in this separation. From this solution the myosin 
can be separated in a fair degree of purity by the usual methods. 
7, Extraction of corn meal with 10-per cent, salt solution, after 
previous extraction with water, dissolves the vitellin-like globulin, 
from which solution it can be separated by the customary methods. 
So prepared, it agrees exactly with the vitellin separated by heat- 
coagulation from the mixed globulin. 
8. The third globulin present in the maize-kernel is charac- 
terised by extreme solubility in very dilute salt-solutions, especially 
phosphates and sulphates. It separates from such solutions only by prolonged dialysis—i. e. not until nearly every trace of the 
above salts has been removed. It coagulates (in a 10-per cent, 
salt solution) in the neighborhood of 62° C., and contains 15.2 per 
cent, of nitrogen and 1.26 per cent, of sulphur. 
9. Through the long-continued action of water, and also of 
strong solutions of salt, as ammonium sulphate, the myosin-like 
globulin, and the globulin with a still lower content of nitrogen, 
are changed into insoluble modifications, soluble, however, in 
0.5-per cent, sodium carbonate solution, from which they are 
precipitated on neutralisation, apparently as albuminates. So 
prepared, these insoluble modifications are characterised by a 
relatively high content of carbon. 
10. An aqueous extract of corn meal, as well as a sodium- 
chloride extract, contains, in addition to the globulins, apparently 
two albumin-like bodies, more or less coagulable by heat, but as 
prepared, unlike in chemical composition. 
11. A certain amount of proteose can be detected in the extracts 
of corn meal, after the globulins and albumins have been entirely 
removed, but apparently this is mainly, if not wholly, an artificial 
product resulting from the hydrolysis of some one or more of the 
preceding bodies. 
12. Especially noteworthy is the presence in the maize-kernel 
of a peculiar proteid body known as maize fibrin, or better as zein, 
soluble in warm dilute alcohol, but insoluble in water. 
Zein is characterised by a high content of carbon, by its resist- 
ance to the action of dilute alkalies (i. e., non-convertibility into 
alkali-albuminate), and by the ease with which it is converted into 
an insoluble modification on being warmed with water, or with 
very weak alcohol. 
Soluble zein and the insoluble modification have the same 
chemical composition. Both respond to the ordinary proteid 
reactions.