Full text of "Carnegie Institution of Washington publication"

See other formats

I 11

" ' ■ ■

llliil

MBSm

SSBSsSSjShKSK

■:••■■'.-.■■■

ifiiiiiiiiii

■Hi

■ ■■■■'*■■>■■

•■■*.-'',

{ ■'■;'. ■

'^- •■;. ■ :

■ :•;:■:::■•-■■':' ■ •

BWIi^WiiMtiiiiV'ttfflrW
nfflllKHIIIIIWWBffBff

8§£1

'V- " .
• ;■;;;-■:

.-. V .-'■ ;.

3Sg&*gg£

THE PROTEINS OF THE WHEAT KERNEL

THOMAS B. OSBORNE

WASHINGTON, D. C. :

Published by the Carnegie Institution of Washington

1907

CARNEGIE INSTITUTION OF WASHINGTON
Publication no. 84

''i^

PRESS OF JUDD & DETWEILER, INC.
WASHINGTON, D. C.

CONTENTS.

Page.

Introduction 5

Review of the Literature 6

Experimental Part. 17

The aqueous extract of wheat flour iS

The albumin of wheat flour — leucosin 20

The aqueous extract of the wheat embryo 22

Hydrolysis of leucosin 30

The proteins of wheat flour soluble in sodium-chloride solution 37

The globulin of wheat flour 37

The proteins of the wheat embryo soluble in sodium-chloride solution 40

The globulin of the wheat embryo 40

The proteose of wheat flour 45

The proteose of the wheat embryo 46

The proportion of the various protein substances of the wheat embryo 48

Digestion of the phosphorus-containing protein preparations with pepsin-
hydrochloric acid 49

Protein soluble in dilute alcohol — gliadin 53

Direct extraction with dilute alcohol 53

Extraction with dilute alcohol after extracting the flour with 10 per cent

sodium-chloride solution 58

Extraction of gluten with dilute alcohol 59

Extraction of " shorts " with dilute alcohol 60

Protein extracted by alcohol from whole-wheat flour 62

The proportion of glutaminic acid yielded by various fractions of the alcohol-
soluble protein 64

Hydrolysis of gliadin 70

Protein insoluble in water, saline solutions, and alcohol — glutenin 82

Protein extracted by dilute alkaline solutions after extracting the flour

with 10 per cent sodium-chloride brine and then with dilute alcohol. ... 82
Protein extracted by dilute alkaline solutions from gluten after extracting

the latter with dilute alcohol 84

Protein extracted by dilute alkaline solutions after complete extraction

with dilute alcohol of gluten from whole-wheat flour 88

Hydrolysis of glutenin 91

The amount of the various proteins contained in the kernel of wheat 100

The formation of gluten 103

Summary 108

Gliadin 109

Glutenin 112

Leucosin 113

The globulin 115

The proteose 116

The gluten 116

The nutritive value of the wheat proteins 117

THE PROTEINS OF THE WHEAT KERNEL.

INTRODUCTION.

Of the protein substances used as food none is of more importance than
those contained in the seeds of wheat. Although these bodies attracted
the attention of investigators more than one hundred years ago and have
since then been many times the subject of study, the published statements
respecting them are so conflicting and uncertain that it has heretofore been
impossible to know what the truth regarding them actually is. With the
purpose of clearing up the existing confusion and determining the real
value of the evidence offered, as well as extending, as far as possible, our
knowledge of these important substances, the writer some years ago under-
took an investigation of this seed which has recently been concluded by
work done under grants from the Carnegie Institution of Washington. As
the results of these investigations have been published from time to time in
a number of different papers, appearing in four different journals, it has
been thought desirable to bring all this work together in one paper. In so
doing the details have been reproduced in full, for the nature of the evidence
is such that its value largely consists in concordant results of many experi-
ments, repeated under different conditions, since it is not yet possible to
establish the chemical individuality of different protein substances by demon-
strating their possession of definite physical properties, as may be done with
the simpler organic compounds.

The experience of the writer in his endeavors to understand and repeat
the work of many of his predecessors has made him feel the importance of
these details to future workers along the same lines and is his excuse for
giving with so much minuteness the results of his own work, which to those
not familiar with the difficulties of the subject must appear to a large extent
unnecessary. In order to make this work available to those who wish sim-
ply to know the results, a comprehensive summary of this paper is given at
the end of this publication, and the details of the many operations and experi-
ments need be read only by those who wish to become familiar with these.

The account of this work is preceded by a review of the literature of the
subject, from which an idea of the unsatisfactory state of our previous

6 THE PROTEINS OF THE WHEAT KERNEL.

knowledge can be obtained. This review is interesting, also, as it shows the
slow development of the study of vegetable proteins and how the several
investigators have been influenced by the knowledge of the animal proteins
prevailing at the time the work was done. In carrying out his investiga-
tions of these proteins the writer has received the assistance of Messrs.
Voorhees, Campbell, Harris, and Clapp, for which he wishes here to make
acknowledgment ; but especially is he indebted to Prof. S. W. Johnson,
under whose direction and with whose advice and encouragement this work
was first undertaken in the laboratory of the Connecticut Agricultural
Experiment Station, where it has since been continued.

REVIEW OF THE LITERATURE.

The fact that gluten can be obtained from wheat flour by washing with
wTater appears to have been first published by Beccari.1

That alcohol extracts a protein substance from wheat flour was first stated
by Einhof,2 who considered this to be the same as the gluten.

Taddei3 found that gluten consists of two substances, one of wdiich is
soluble in alcohol, which he named " gliadin," the other insoluble in alcohol,
which he named " zymom."

De Saussure4 obtained from wheat gluten about 72 per cent of plant-
albumin in the insoluble form, about 20 per cent of plant-gelatin, or, as he
proposed to call it, "glutin," and about 1 per cent of mucin, which latter
substance he considered to be similar to the mucin described by Berzelius.

Berzelius5 thought that the alcoholic extract contained another protein
substance, which he called "mucin," and that the part of the gluten which
was insoluble in alcohol was so similar to albumin that he called this
11 plant-albumin."

Boussingault6 agreed with Einhof th.it the part of the gluten that was
soluble in alcohol was the same as the entire gluten protein.

1 Beccari. Reference to this publication has, for many years, appeared in the literature
as Comon. Bonon. I. 1, p. 122. It should be De Bononiensi Scientiarum et Artium Insti-
tuto atque Academia Commentarii, 1745, 11, part 1, p. 122. In this paper Beccari refers
to the fact that in 1728 he had orally communicated to the Academy the previously un-
published fact that wheat flour can be separated into two parts, one of vegetable, the
other of animal character. The substance of this communication was published in the
above-cited paper in which the separation of gluten from wheat flour, by washing with
water, is described.

2 Einhof, Journal der Chemie von Gehlen, 1S05, v, p. 131.

3 Taddei, Annals of Philosophy, 1820, xv, p. 390.

4De Saussure, Schweiger's Journal, 1S33, lxix, p. 188.

5 Berzelius, Lehrbuch der Chimie, Auflage 3, 1837, vi, p. 453.

e Boussingault, Annales de Chimie et de Physique, 1838, l<xv, p. 30.

REVIEW OF THE LITERATURE. 7

Iyiebig1 named the part of the gluten that was insoluble in alcohol ' ' plant-
fibrin," on account of its supposed resemblance to blood-fibrin. The sub-
stance soluble in alcohol he called ' ' plant-gelatin ' ' and considered it to be
a compound of a protein with an organic acid. In the aqueous extract of
the flour he recognized the presence of albumin.

Scherer2 prepared plant-fibrin by dissolving gluten in dilute alkali, filter-
ing, neutralizing with acetic acid, and extracting the precipitate with hot
alcohol and then with ether.

Bonchardat3 considered that wheat gluten contained a protein soluble in
extremely dilute acids, which he named " albuminose."

Dumas & Cahours4 found four protein substances in wheat flour, namely,
plant-fibrin, which remained after extracting gluten with alcohol ; a sub-
stance which they considered similar to casein, which was deposited by
cooling the alcoholic extract ; glutin, which was obtained by concentrating
and cooling the alcoholic extract, and albumin, which was present in the
aqueous washings of the gluten and was coagulated by boiling. The plant-
fibrin they considered to be identical with blood-fibrin, as both had the same
ultimate composition, and the albumin to be identical with egg-albumin for
the same reason.

Mulder5 considered the plant-gelatin, obtained by extracting gluten with
alcohol, to be a compound of sulphur with " protein," which contained the
same proportion of sulphur as blood-albumin.

Von Bibra6 recognized three proteins in gluten — plant- fibrin, which
formed 70.8 per cent; plant-gelatin, 16.2 per cent; and plant-casein, 7.1
per cent. In the water used for washing out the gluten he found 1.34 per
cent of albumin.

Giinsberg7 considered that Taddei's view that there were only two pro-
teins in wheat gluten was correct. By boiling wheat gluten with water he
obtained five preparations which separated on cooling and had the same
ultimate composition as has been established for gliadin. By treating
gliadiu in the same way he obtained a body of the same composition. The
substance which Giinsberg thus obtained was unquestionably gliadin, which
is sparingly soluble in hot water, and he appears to have been the first to
obtain correct analyses of this protein.

1 Iviebig, Annalen der Chemie und Pharmacie, 1841, xxxix, p. 129.

2 Scherer, Annalen der Chemie und Pharmacie, 1841, XI,, p. 7.

3 Bonchardat, ibid., 1842, xiji, p. 124.

* Dumas & Cahours, Journal fur praktische Chemie, 1843, xxviil, p. 398.

5 Mulder, Annalen der Chemie und Pharmacie, 1844, Lii, p. 4*9-

6 Von Bibra, Die Getreidearten und das Hrod, Nuremberg, i860.

7 Giinsberg, Journal fiir praktische Chemie, 1862, lxxxv, p. 213.

THE PROTEINS OF THE WHEAT KERNEL.

Commaille1 recognized, as protein constituents of flour, sitosin, soluble in
water and coagulable by heat ; imesin, soluble, after drying, only in water
containing o. i per cent hydrochloric acid; sitesin, soluble in o.i per cent
hydrochloric acid ; glutin, nearly insoluble in dilute acid, easily in strong acid,
forming an emulsion with alcohol, which is separated by much water ; and
mucin, which dissolves easily, even after drying, in water and in cold 80
per cent alcohol.

Ritthausen2 next published the results of his extensive investigations in a
volume in which he discussed at length the proteins of the wheat kernel.
The composition, properties, and the proportions in which they occur in the
seed were given in detail, and also the evidence which he considered showed
the individuality of each. He recognized five proteins, namely, gluten-casein,
gluten-fibrin, plant-gelatin or gliadin, and mucedin, as constituents of the
gluten, and also albumin, which he found in the aqueous extracts of the
seed.

The gluten-casein was not soluble in water, very slightly soluble in dilute
alcohol, and readily soluble in very dilute acids and alkalis. When decom-
posed by boiling with sulphuric acid it yielded tyrosine, leucine, 5.3 per
cent of glutaminic acid, and 0.33 per cent of aspartic acid. In the dry gluten
he found from 26 to 31.4 per cent of gluten-casein, which he considered to
be minimal quantities.

Table giving results as ascertained by Ritthausen.

Gluten-
casein.

Gluten-
fibrin.

Plant-
gelatin.

Mucedin.

Albumin.

Hydrogen

P.ct.

52.94
7.04

17.14

0.96

21.92

P.ct.

54-31
7.18

16.89

1. 01

20.61

P.ct.
52.76

7,10
18.01

0.85
21.37

P.ct.

54.li
6.90

16.63
0.88

21.48

P.ct.
53-12

7.18
17.60

i-55
20.55

100.00

The gluten-fibrin formed that fraction of the alcohol- soluble proteins
which was soluble in the strongest alcohol and separated from a hot con-
centrated solution in 50 to 60 per cent alcohol on cooling. Owing to the
difficulty encountered in separating gluten-fibrin from the other alcohol-
soluble proteins, it was impossible to determine its amount. Usually from

1 Commaille, Journal de Pharmacie, 1866 (4), iv, p. 108.

2 Ritthausen, Die Eiweisskorper, etc., Bonn, 1872.

REVIEW OF THE LITERATURE. 9

2 to 3 per cent of the gluten was obtained, which corresponds to 0.25 to 0.35
per cent of the flour. The actual quantity he considered to be much more,
and that in different varieties of wheat the proportion of gluten-fibrin varied
greatly.

Ritthausen described gluten-fibrin as insoluble in water, but by boiling
with water it was decomposed and rendered insoluble in alcohol. In
alcohol of 30 to 70 per cent it dissolved readily when heated, and sepa-
rated again on cooling, more completely from the more dilute alcohol.
From dilute solutions on concentration and from concentrated solutions on
cooling the gluten-fibrin separated on the surface of the liquid as a thick,
soft skin, which was renewed as often as it was removed, which property
Ritthausen considered distinguished it from mucedin and plant- gelatin. In
cold alcohol of 80 to 90 per cent the gluten-fibrin was soluble to a consider-
able degree. Dilute acids and alkalis dissolved this protein freely, yielding
solutions from which it was precipitated on neutralizing to a slight acid
reaction.

The composition of gluten-fibrin is shown in the table on page 8.

Plant-gelatin or gliadin formed the fraction of the alcohol-soluble protein
that dissolved freely in alcohol of 60 to 70 per cent. The solubility of this
protein decreased rapidly when the proportion of alcohol to water fell below
or above this strength. It was very slightly soluble in cold water, more
so in hot water. By boiling with water it was gradually rendered insol-
uble in alcohol. Extremely dilute acids and alkalis dissolved plant-gelatin
readily.

The amount of plant-gelatin which different wheats contain was not deter-
mined, owing to the impossibility of separating it from the other proteins.
The composition of this protein is shown in the preceding table.

Mucedin formed the fraction of the alcohol-soluble proteins which was
soluble in the most dilute alcohol. Except for its greater solubility in water
and in very dilute alcohol, mucedin does not appear to differ greatly in its
properties from plant-gelatin. Its composition is given in the table on
page 8.

Mucedin yielded 25 per cent of glutaminic acid when boiled with sul-
phuric acid, but other decomposition products were not determined. Only
a very small quantity of mucedin was obtained in a pure state, and no
estimate of its total amount was made. The relative proportion, however,
Ritthausen considered to vary greatly in different sorts of wheat.

Albumin was obtained by heating the acidified wash- waters of the gluten.
This, however, he considered as possibly derived from the soluble part of
the gluten-proteins. The composition of this albumin is given in the table
on page 8.

IO TIIK PROTEINS OF THE WHEAT KERNEL.

Weyl1 was the first to recognize the presence of globulin in wheat flour,
and states that besides vegetable-vitellin, vegetable-myosin, which coagu-
lated at 550 to 6o°, was also present.

Weyl & Bischoff2 considered the protein matter of wheat to consist chiefly
of a myosin-like globulin which they called vegetable-myosin, and that, if
so, this must be the substance from which gluten is derived, for other pro-
teins are present only in small quantity. Extraction with 15 per cent salt
solution left a residue from which they obtained no gluten. They therefore
considered it probable that the gluten forms from the myosin in consequence
of a ferment action similarly to the formation of fibrin from fibrinogen.
No ferment, however, could be detected. They also found that large
amounts of sodium chloride, sodium sulphate, and magnesium sulphate
hindered the formation of gluten in the same way that sodium and mag-
nesium sulphates hinder the formation of fibrin. As no gluten was obtained
from flour extracted with alcohol, they concluded that the myosin had been
coagulated. By warming flour 48 to 96 hours below 6o°, the coagulation
point of myosin, no gluten was obtained from the meal after adding a little
unwarmed flour, showing that the gluten-forming substance had been
coagulated.

Balland3 found that nearly the same amount of gluten was formed with
water at 20, 150, and 6o°, and therefore concluded that no ferment action
took part in its formation.

According to Martin,* alcohol extracts from gluten but one protein sub-
stance. This is soluble in hot water, but insoluble in cold ; hence is insoluble
phytalbumose. The residue remaining after treatment with alcohol is
uncoagulated protein, soluble in dilute acids and alkalis. This he called
"gluten-fibrin." The insoluble phytalbumose is not present, as such, in
flour, since direct extraction of the meal with 75 per cent alcohol removes
no protein. Extraction with water yields less globulin and soluble albumose
than extraction with sodium chloride somtion of 10 to 15 per cent. Martin
therefore concluded that the insoluble phytalbumose is formed from the
soluble by the action of water, the gluten-fibrin being formed by a similar
action of water on the globulin ; that is, conversion into an albuminate.
This albuminate and the insoluble phytalbumose together constitute gluten.

W. Johannsen5 believed that there was no ferment action in the formation
of gluten. Dough was obtained by grinding dried gluten and mixing with
starch, and also by mixing moist gluten with starch.

1 Weyl, Zeitschrift fur physiologische Chemie, 1877, 1, p. 72.

' Weyl & Bischoff, Berichte der deutschen chemischen Gesellschaft, 1880, xni, p. 367.

3 Balland, Comptes rendus de l'Academie des Sciences, 18S3, cxv, p. 202.

4 Martin, British Medical Journal, 1886, 11, p. 104.

5 W. Johannsen, Annales Agronomiques, 1S88, xiv, p. 420.

REVIEW OF THE LITERATURE. II

Chittenden & Smith1 prepared many samples of gluten-casein according to
Ritthausen's method. As an average of eight analyses they found the fol-
lowing composition :

p. ct.

Carbon 52. 87

Hydrogen 6.99

Nitrogen 15.86

Sulphur 1. 17

Oxygen 23. 1 1

100.00

They also prepared and analyzed the various products of peptic digestion
of this protein.

Osborne & Voorhees2 investigated the number and character of the proteins
of wheat, but as their results are given in detail in the body of this paper
they need not here be further mentioned.

O'Brien* recognized globulin, proteose, and the gluten-proteins in extracts
of wheat flour. The protein-leucosin, which Osborne & Voorhees considered
to be an albumin, was regarded as a globulin by O'Brien, since it is precip-
itated by saturating its solutions with magnesium sulphate.

About 1 per cent of the flour consists of proteins soluble in saline solu-
tions and coagulating on boiling. Neither ferment action nor globulin take
part in gluten formation. Gluten consists of zymom, insoluble in alcohol,
and glian, soluble therein. Glian is formed by hydration of the protein of
the flour and zymom from glian by further hydration. Glian yields myxon,
glutine, and mucine, which are not constituents of glian, but derived from it.

Frankfurt4 estimated the proportion of various constituents of the embryo
of wheat, and found globulin 21.62 per cent and albumose 13.62 per cent.

O'Brien5 stated that the proteins of the wheat embryo consist of globulins
of the myosin type, coagulating at 550, soluble in dilute solutions of sodium
chloride or magnesium sulphate and precipitated by excess of these salts ;
globulins of the vitellin type, coagulating at 750 to 780, and soluble in dilute
solution of sodium chloride, but not precipitated by an excess ; proteose
and albumin, not coagulating below 8o°, soluble in sodium chloride solution,
but not precipitated by an excess, nor by dialysis, nor by carbonic acid.

Kjeldahl6 found that all of a number of preparations of the alcohol-soluble
protein made from wheat flour showed an almost constant content of about
52 per cent of carbon and 17.25 per cent of nitrogen, and when dissolved in

1 Chittenden & Smith, Journal of Physiology, 1890, xi, p. 419.

2 Osborne & Voorhees, American Chemical Journal, 1893, xv, p. 392.
3 O'Brien, Annals of Botany, 1895, IX, p. 171.

4 Frankfurt, Versuchs-Stationen, 1S95, xivVii, p. 449.

5 O'Brien, Annals of Botany, 1895, ix, p. 543.

6 Kjeldahl, Agricultur chemischen Centralblatt, 1896, xxv, p. 197.

12 THE PROTEINS OF THE WHEAT KERNEL.

75 per cent alcohol a specific rotation of — 920. This rotation was so con-
stant, not only for the protein from different sorts of wheat from different
regions, but also for those from crops of four different years, that it seemed
to Kjeldahl that wheat flour contained only one single protein substance
soluble in alcohol.

Fleurent1 held the view that only one protein substance soluble in alcohol
was present in wheat flour, and proposed a method for determining the
amount of gliadin and glutenin. By this method he found that gluten con-
tained from 60 to 80 per cent of gliadin and 18 to 25 per cent of glutenin,
according to the variety of the wheat from which it was obtained.

Guthrie2 concluded that the water-absorbing power of wheat flour was
greater when the proportion of glutenin to gliadin was greater, strong flours
being those relatively rich in glutenin.

Teller3 devised methods for determining the relative quantities of the
different proteins in wheat flour and applied them to flours of different
origin and to various mill products.

Teller* also concluded that the proteose found by Osborne & Voorhees
was gliadin that had been dissolved in small quantity in the aqueous extract.
Osborne5 showed that this was erroneous and gave the reasons why he had
not mistaken gliadin for proteose.

Morishima's6 investigations led him to believe that wheat gluten contained
but a single protein, and that glutenin and gliadin were derivatives of one
and the same substance, which he named artolin.

Teller7 determined the proportion of the several proteins present in the
wheat kernel on many consecutive days during the ripening of the grain
and found a large increase of gliadin nitrogen during this period, together
with a smaller though marked decrease of the glutenin nitrogen when consid-
ered in proportion to the whole amount of nitrogen present. The changes
in the proportion of leucosin and globulin nitrogen were less marked and
more irregular.

Ritthausen8 again asserted his belief in the existence of three distinct
proteins in wheat that were soluble in alcohol, but offered no new evidence
of their existence.

1 Fleurent, Comptes rendus de l'Academie des Sciences, 1S96, cxxni, p. 755.
7 Guthrie, Agricultural Gazette of New South Wales, September, 1896.

3 Teller, Arkansas Agr. Exp. Sta. Bull. 42, part 2, p. 81. 1896.

4 Teller, American Chemical Journal, 1S97, xix, p. 65.
6 Osborne, ibid., 1S97, xix, p. 263.

6 Morishima, Archiv fur experim. Pathologie und Pharmakologie, 1898, xu, p. 34S.

7 Teller, Arkansas Agr. Exp. Sta. , Bull. 53, p. 53. 1898.

8 Ritthausen, Journal fur praktische Chemie, 1899, Lix, p. 474.

REVIEW OF THE LITERATURE. 1 3

Snyder1 determined the amount of the different proteins in various flours
and mill products. He found 73.9 per cent of the total protein to be
gliadin in patent flour from soft winter wheat and 63.7 per cent in that from
hard winter wheat. He concluded that the protein in the gluten of a flour
good for bread-making consists of 65 per cent of gliadin and 35 per cent of
glutenin. The ratio of gliadin to glutenin in different grades of flour varies
between 1 to 4 and nearly 2 to 1 . While the lower grades of flour contain
more protein than the higher, the proportion of gliadin to glutenin is not
such as to produce bread of the best physical properties.

Osborne & Campbell2 found that the leucosin, globulin, and proteose,
obtained in very small quantity from the entire wheat kernel, together con-
stitute nearly the whole of the protein of the embryo, and that gliadin and
glutenin, which are the principal proteins of the endosperm, could not be
obtained from the embryo. The details of this investigation are given in
full in subsequent pages of this paper.

Kossel & Kutscher,3 following Ritthausen's directions, prepared the pro-
teins of wheat gluten and determined the proportion of basic products which
they yielded on decomposition with acids. They found that glutenin was
sharply distinguished from the protein soluble in alcohol by the fact that it
yields a notable quantity of lysine, whereas all their products derived from
the alcoholic extract of gluten yielded none of this diamino-acid. They
held the view, advanced by Ritthausen, that in gluten there are three pro-
tein substances soluble in alcohol. Of these mucedin yielded 3.13, gliadin
2.75, and gluten-fibrin 3.05 per cent of arginine and 0.43, 1.20, and 1.53
per cent respectively of histidine ; but in view of the methods employed
for the determinations of these bases they consider these differences too
small to justify the conclusion that these are distinct protein substances.

Dennstedt4 decomposed " wheat fibrin " by boiling with baryta and found
that one-third of the nitrogen was split off as ammonia and one-fifth of the
sulphur as sulphide and sulphate. After removing the barium and treating
the solution with lead acetate he separated proteoses, which he analyzed.

Osborne5 made careful determinations of total sulphur in four samples of
thoroughly purified gliadin and found an average of 1.027 percent, of which
0.619 per cent was split off as sulphide by boiling with caustic alkali.

1 Snyder, Minnesota Agr. Exp. Sta., Bull. 63. 1899.

2 Osborne & Campbell. Journal American Chemical Society, 1899, xxi, p. 486.

3 Kossel & Kutscher, Zeitschrift fur physiologische Chemie, 1901, xxxi, p. 165.
4Dennstedt, Chemiker Zeitung, 1901, p. 5.

5 Osborne, Journal American Chemical Society, 1902, xxiv, p. 140.

14 THE PROTEINS OF Till; WHEAT KF.RXEL.

Osborne & Harris,1 in a study of the different forms of binding of nitrogen
in proteins, found that leucosin of wheat yielded 1.16 per cent of nitrogen
as ammonia and 3.50 per cent of nitrogen in basic compounds, precipitable
by phosphotungstic acid ; that the globulin yielded 1.42 per cent of nitrogen
as ammonia and 6.83 per cent of basic nitrogen ; that gliadin yielded 4.3
per cent of nitrogen as ammonia and 1.09 per cent of basic nitrogen, and
that gluteuin yielded 3.31 percent of nitrogen as ammonia and 2.05 per cent
of basic nitrogen. They also found2 that, while none of the wheat proteins
yielded any furfurol on distillation with hydrochloric acid, glutenin gave a
moderately strong, gliadin a strong, and leucosin a very strong reaction with
the Molisch test. This test is commonly regarded as giving evidence of a
carbohydrate complex in the protein molecule, but they decided that other
evidence is necessary before such a conclusion is justified.

Osborne & Harris3 found the specific rotation of gliadin dissolved in
alcohol of 80 per cent by volume to be — 91.90 and — 92.50. In comparing
the tryptophane reaction of many proteins these same authors found4 that
the globulin of wheat gave only a slight reaction, gliadin and glutenin one
of medium intensity, and leucosin the strongest reaction of all the proteins
examined, thus indicating the relative amounts of tryptophane or indol-
amino-propionic acid which these proteins yield on decomposition with acid.

Kutscher5 determined the amount of tyrosine and glutamiuic acid 3'ielded
by the proteins of wheat gluten when decomposed by boiling with sulphuric
acid. In glutenin he found 2.75, in gluten-fibrin 4.43, in gliadin 2.09, and
in mucedin 2.35 per cent of tyrosine ; in glutenin 9, in gluten-fibrin 13.07,
in gliadin 18.54, aud in mucedin 19.81 per cent of glutaminic acid.

Naysmith6 found that gluten contained 0.12 per cent of phosphorus.
Gliadin extracted by 70 per cent alcohol from gluten and separated by
evaporating to dryness contained 0.29 per cent of phosphorus, but when
precipitated from the alcoholic extract by dilute sodium chloride solution it
contained only 0.19 per cent.

Glutenin also contained 0.21 per cent of phosphorus. Both these proteins
contained iron. Phosphorus and iron are not constituents of the molecules
of these proteins, but are derived from the cell nuclei.

Although gliadin and glutenin contain phosphorus, they are not nucleo-
proteids. Naysmith also concluded that no ferment action occurred in the
formation of gluten.

1 Osborne & Harris, Journal American Chemical Society, 1903, xxv, p. 323.
2 Ibid., p. 474.

3 Ibid. , p. 844.

4 Ibid., p. 854.

5 Kutscher, Zeitschrift fur physiologische Chemie, 1903, xxxvur, p. III.

6 Naysmith, Transactions of the Canadian Institute, 1903, vn.

REVIEW OF THE LITERATURE. 1 5

The composition of gliadin and glutenin as prepared by Naysmith were
as follows :

Composition of gliaditi and glutenin as prepared by Naysmith.

Carbon ....
Hydrogen .
Nitrogen . .
Sulphur . . .
Oxygen . . .
Phosphorus
Iron

Gliadin.

P. d.

52.39
6.84

1747
1.12

21.89
0.267
0.034

100.000

Glutenin.

P.ct.

52.75
7.22

16.15
1.06

22.58
0.215
0.026

100.000

Snyder1 proposed a method for determining gliadin in wheat flour which
was based on the optical rotation of the alcoholic extract of a definite
quantity of the flour. The results of this method agreed closely with those
obtained by determining nitrogen in the alcoholic extract. The specific
rotation of gliadin calculated from the mean of several determinations by
the above method is («)D = — 900, approximately.

Chamberlain''' studied the method of determining gliadin and glutenin in
wheat flour which had been proposed by Fleurent and modified by Manget,
and found that the results for gliadin were too high and for glutenin too
low. He therefore proposed a method based on the work of Snyder and of
Osborne & Voorhees.

Konig & Rintelen3 published an account of their investigation of the pro-
teins of wheat gluten and conclude with Ritthausen that there are three
soluble in alcohol. Their analyses of the preparations representing these
three proteins agreed closely with those of Ritthausen for gliadin, but
differed considerably in carbon for those representing gluten-fibrin and
mucedin.

Osborne & Harris4 reviewed the work that had been done on the alcohol-
soluble proteins of wheat published since 1893 anc^ gave their reasons for
adhering to the views of Osborne & Voorhees, that only one such protein

1 Snyder, Journal American Chemical Society, 1904, xxvi, p. 263.

2 Chamberlain, U. S. Dept. of Agriculture, Bureau of Chemistry, 1904, Bulletin 81.
'Kpnig & Rintelen, Zeitschrift fur Untersuchuug der Nahrungs und Genussmittel,

1904, viii, p. 401.
4 Osborne & Harris, American Journal of Physiology, 1905, XIII, p. 35.

THE PROTEINS OF THE WHEAT KERNEL.

existed, namely, gliadin, the gluten-fibrin and mucedin being, in their
opinion, impure preparations of gliadin. This opinion was supported by
further experimental evidence and by quantitative determinations of the
glutamiuic acid yielded by hydrolyzing different fractional preparations of
gliadin, some of which, according to Ritthausen's statements, should have
contained the gluten-fibrin. The amount of glutaminic acid found in the
different fractions was essentially the same.

The glutaminic acid thus obtained from gliadin was 37.3 per cent, which
is more than that from any protein substance yet examined.

Abderhalden & Samuely1 determined the amount of the various primary
decomposition products yielded by gliadin and found —

P.ct.

Glycocoll 0.68

Alanine 2.66

Amino valerianic acid 0.33

a-proline 2.40

Leucine 6.00

Glutaminic acid 27.60

P.ct.

Aspartic acid 1.24

Phenylalanine 2.60

Serine o. 12

Tyrosine 2.37

Tryptophane, about 1.00

The gliadin used for this analysis yielded 12 per cent of humus, which,
together with moisture and ash, were deducted in calculating the above
percentages.

Mathewson2 determined the specific rotation of gliadin in various organic

solvents with the following results

40°

Methyl alcohol, 70 per cent (a) - — — 95.65

Ethyl alcohol, 70 " " " —9195

Ethyl alcohol, 60 " " " —96.66

Ethyl alcohol, 50 " " " —98.45

Propyl alcohol, 60 " " " — 101.10

Phenol, 70 " " " — 123.15

Phenol, anhydrous — L3I-77

Paracresol — 121.00

Benzyl alcohol — 53. 10

Glacial acetic acid — 78. 60

Osborne & Harris3 described the preparation of large quantities of the
wheat proteins to be used for the quantitative determination of the products
of hydrolysis. These determinations were made by Osborne & Clapp.4 The
results described in these papers are given in full later in this publication.

1 Abderhalden & Samuely, Zeitschrift fur physiologische Chemie, 1905, xi,vi, p. 193.

2 Mathewson, Journal American Chemical Society, 1906, xxvin, p. 1482.
1 Osborne & Harris, American Journal of Physiology, 1906, xvn, p. 223.
* Osborne & Clapp, ibid., p. 231.

EXPERIMENTAL. \J

EXPERIMENTAL PART.

Although positive evidence of the chemical individuality of protein sub-
stances can not yet be obtained, there is no question that protein preparations
can be isolated from seeds and animal tissues which, beyond doubt, represent
distinctly different substances. Thus five unquestionably different forms
of protein, differing in composition, solubility, and physical characters, can
be isolated from the wheat kernel. Whether each of these is itself a chem-
ical individual or a mixture of two or more very similar substances can not
at present be asserted. All that can be said is that it has not yet been pos-
sible to separate them into fractions, the properties of which indicate a
mixture.

Owing to the extreme sensitiveness of proteins to the action of acids,
alkalis, and salts, the minor differences in solubility are not to be depended
upon as a basis for characterizing individual proteins. Thus the protein
edestin, which in pure water is entirely insoluble, in the presence of a slight
amount of acid is freely soluble therein. The addition of a small quantity
of a neutral salt throws the edestin out of this acid solution, while a larger
quantity of salt at once redissolves it.

Such differences in solubility have nothing to do with the protein mole-
cule proper, but depend on the formation of protein salts, the solubility of
which is different from that of the free protein itself. As the formation of
such protein salts depends on conditions that in most cases can not be taken
into account, such differences in solubility can not be made a basis for char-
acterizing the different individual proteins.

We are therefore limited, in dealing with such problems, to the more marked
differences in solubility, such as that in alcohol, strong saline solutions, or
alkalis, and to constant ultimate composition of successive fractional precip-
itations. Thus, when proteins have been separated into fractions which
have the same composition, general solubility, and physical properties, we
are not justified in concluding that we have in hand a single individual pro-
tein. All we can conclude is that we have reached the limit of separation
attainable with the means now available, and that for the present we must
accept such products as the simplest units with which we can now deal and
which for the present must serve as our basis for further study. If, on the
other hand, protein preparations, characterized in the manner above de-
scribed, show distinct and constant differences from one another, we are
justified in considering them to be different substances.

That the wheat kernel contains at least five such distinct protein sub-
stances will be shown in the following pages.

l8 THE PROTEINS OF Till'. WHEAT KERNEL.

THE AQUEOUS EXTRACT OF WHEAT FLOUR.

The water-extract of wheat flour is of a straw-yellow color, becoming red-
brown on standing, and has a very slight acid reaction towards litmus.

Saturated with ammonium sulphate, a bulky precipitate forms, which on
standing contracts, showing the solution to contain but a small amount of
protein matter. After 24 hours this precipitate can be completely dissolved
in water, giving no evidence of the formation of insoluble derivatives. Sat-
uration with sodium chloride gives a small precipitate. Acetic acid in the
cold solution gives no precipitate until sodium chloride is added.

On slowly heating, the solution becomes turbid at 480 and yields a floccu-
lent coagulum at 520. After heating to65° for some time and filtering, the
solution becomes turbid again at 730, flocks forming in very small amount
at 82 °. No more separation occurs on further heating the extract even to
boiling. The addition of a little acetic acid and sodium chloride gives a
small precipitate. The body coagulating at 520 forms the greater part of
the protein in solution. The complete coagulation of this protein is accom-
plished with difficulty, prolonged heating at 65 ° being necessary to cause it
to separate completely. The addition of sodium chloride greatly facilitates
the final coagulation. The temperature at which the flocculent coagulum
separates depends upon the rate of heating. Unless the solution is heated
very slowly, the point at which flocculation occurs is much above 52 °.

When the sodium-chloride extract of the wheat flour is saturated with
ammonium sulphate and the precipitate redissolved, and its solution dialyzed
until all of the globulin has separated, the solution, when slowly heated in
a double water-bath, becomes turbid at 480 and flocks separate at 550. After
heating for some time at 650 and filtering off the coagulum, the solution,
when again heated, becomes turbid at 700 and a very minute amount of
flocculent coagulum forms at 8o°. Boiling the solution after filtering gives
no more precipitate, and nothing is obtained by adding a little salt and acetic
acid. If the amount of salt is increased and acetic acid added, a precipitate
results. Equal volumes of a solution so prepared were treated with 20 per
cent of sodium chloride and a little acetic acid. To the first the salt and
acid were added directly, to the second after heating to 65 ° and filtering off
the coagulum, and to the third after heating to 950 and likewise filtering.
The first portion gave the most precipitate, the last the least, showing that the
coagulable proteins are thus precipitated by salt and acid. The filtrate from
the first portion when neutralized and boiled gave no precipitate, indicating
that the separation of the albumin was complete.

The above solution, freed from globulins by dialysis, gave a precipitate
on saturation with sodium chloride, the filtrate from which became turbid

EXPERIMENTAL,. 19

when heated to 430, flocculent at 560, and no more precipitation on further
heating, showing that either a higher-coagulating protein was thus removed
or that coagulation of the albumin was more complete in the strong saline
solution at the lower temperature. This dialyzed solution likewise gave
a considerable precipitate with nitric acid. On heating, a part remained
insoluble, and on filtering this off, the filtrate gave a precipitate on cooling,
which dissolved again on heating and reappeared as often as the solution
was cooled. The filtrate from the salt and acid precipitate did not give
this reaction, but the solution of the precipitate in water gave it very
strongly.

This reaction is characteristic of some proteoses, and shows that the salt
and acid precipitate contains a proteose together with the albumins. This
proteose is likewise precipitated by saturating the extract with salt, for
on dissolving the precipitate so produced and separating the albumin con-
tained in it by coagulation the filtrate gave a strong red biuret reaction, and
a precipitate with nitric acid, which dissolved on warming and precipitated
again on cooling. The filtrate from the precipitate caused by saturation
with salt gives no reaction with nitric acid, showing that the proteose is thus
completely precipitated.

In order to be sure that the coagulable protein, which was apparently an
albumin, was not a globulin held in solution by the small amount of salts
contained in the river- water used for dialysis, as was suggested by its partial
precipitation by saturation with sodium chloride, the following experiment
was tried : 250 cc. of a strong aqueous extract of winter-wheat meal was
dialyzed in running distilled water for 48 hours. A small precipitate was
then filtered off, the clear solution returned to the dialyzer, and the process
continued for five days longer. No more substance separated. The entire
solution, which was still found to coagulate at 540, was then evaporated to
dryness, the considerable protein residue burned off, and the total mineral
matter found to weigh only 0.0008 gram, thus proving the protein to be an
albumin.

We are able, then, to recognize two distinct protein substances soluble in
pure water, namely, a coagulable albumin and a proteose. As it was found
that the protein removed from the flour by treatment with alcohol was to a
slight extent soluble in pure water, it might be thought that one of these
bodies was identical with this alcohol-soluble protein. Its identity with the
albumin is excluded by the fact that the latter is precipitated by heat, and
with the proteose by the fact that the alcohol-soluble proteose gives a pre-
cipitate with hydrochloric acid when it is dissolved in distilled water and also
with a little sodium chloride, which the proteose does not.

THE PROTEINS OF THE WHEAT KERNEL.

THE ALBUMIN OP WHEAT FLOUR — LEUCOSIN.

A quantity of wheat flour was extracted with 10 per cent sodium-chloride
solution, the extract saturated with ammonium sulphate, the precipitated
protein dissolved in sodium-chloride brine, and the clear solution dialyzed
until all the globulin had been precipitated. The filtered solution was then
heated to 6o° in a water-bath kept between 6o° and 650. After an hour the
precipitate was filtered off, washed thoroughly with water, alcohol, absolute
alcohol and ether, and dried. Before drying, the coagulum was a white,
voluminous, semisolid mass, which, when completely dried over sulphuric
acid, became dense and horny. This preparation, 1, after drying at 1100,
was found to have the composition shown in the following table :

Preparations 1 and

Preparation 1.

Preparation 2.

II.

Aver-
age.

Ash-
free.

II.

Aver-
age.

Ash-
free.

Hydrogen ....

P.ct.

53-04
6.74

16.86
1.27

P.ct.
53-28

6.89
16.95

P.ct.

53-16
6.82

16.91
1.27

P.ct.

53-27
6.83

16.95

1.27

21.68

P.ct.

52.91
6.86

16.93
1.28

P.ct.

52.93

6-74

17.OI

I.3I

P.ct.

52.95
6.80

16.97
1.30

P.ct.
53-o6

6.82
17.01

1.30
21.81

Ash

0.22

100.00

Another lot of 10,000 grams of floui was extracted with 10 per cent
sodium chloride solution and treated in the same way as the extract last
described. The albumin obtained weighed 6.4 grams and had the composi-
tion given in the above table, preparation 2.

The filtrate from preparation 2 was then heated to 75 ° and 0.65 gram
of coagulum obtained, which on analysis gave the results shown in the table
on page 21, preparation 3.

Another preparation was made from the same flour by extracting 10,000
grams with 10 per cent sodium chloride solution, filtering the extract, and
dialyzing at once. In this case the precipitation of the proteins with ammo-
nium sulphate was omitted. After complete dialysis the solution was filtered
from the globulin which had separated, and the solution heated at once to
900 — a temperature high enough to precipitate all of the albumin.

Dried over sulphuric acid, this preparation, 4, which in all respects resem-
bled 1 and 2, weighed 16.5 grams and when analyzed gave the results shown
in the table at the top of page 21.

EXPERIMENTAL.
Preparations j, 4, and 5.

Preparation 3.

Preparation 4.

Preparation 5.

Ash-free.

II.

Average.

Ash-free.

P. ct.

P.ct.

P.ct.
52-86

6.85
16.21

I.20

P. ct.

16.20

P.ct.
52.86

6.85
16.21

1.20

P.ct.

53-02

6.87

16.26

1.20

22.65

P. ct.
52.36
6.80
16.62

1-34

0.67

P.ct.

52.71
685

16.73

1-34

22.37

Sulphur

16.91

16.94

Ash

0.18

O.32

0.32

100 00

100.00

Another preparation of albumin was made by extracting with 10 per cent
sodium chloride solution 2000 grams of so-called ' ' shorts ' ' from the spring-
wheat flour. This substance consisted chiefly of particles of the outer coats
of the seed to which more or less of the adjacent embryo and endosperm
adhered. After three hours the extract was strained through a coarse cloth
and squeezed out from the residue in a screw-press. After the starch had
settled, the nearly clear extract, which had a deep red-brown color, was
siphoned off and saturated with ammonium sulphate. The precipitate thus
produced was filtered off and dissolved in 10 per cent sodium-chloride brine.
The resulting solution, filtered from all the insoluble matter, was then
dialyzed free from chlorides, the precipitated globulins filtered off, and the
albumin contained in the solution separated by heating to 65 °. This
preparation, 5, had the composition shown in the preceding table.

Table i. — Summary of analyses of coagulated wheat albumin.

Carbon . . .
Hydrogen
Nitrogen .
Sulphur . .
Oxygen. . .

Average.

P. ct.

P.ct.

P. ct.

P.ct.

53-27
6.83

16.95

53-o6

6.82

17.01

16.94

5302

6.87

16.26

52.71

6.85

16.83

53-Q2

6.84

16.80

1.27
21.68

1.30
21.81

1.20

22.65

1-34
22.27

1.28
22.06

100.00

The agreement of the figures in table 1 is satisfactory, with the exception
of the nitrogen in 4. The accuracy of this analysis in this respect, however,
can not be doubted, as four determinations of this element were made, all
of which agreed closely. As this preparation was separated at a higher
temperature than any of the others, it is possible that it had in consequence
lost some of its nitrogen.

22 THE PROTEINS OF THE WHEAT KERNEL.

THE AQUEOUS EXTRACT OF THE WHEAT EMBRYO.

The embryo flour, when treated with water, yields a gummy mass, from
which a clear extract is secured with difficulty. From 500 grams of meal
an extract was obtained with 2000 cc. of water, of which 1400 cc. could be
filtered clear. This extract was neutral to litmus, alkaline to lacmoid, and
so acid to phenolphthalein that 19 cc. of decinormal alkali were required to
neutralize 100 cc. of it to this indicator.

When a freshly prepared dilute aqueous extract of the recently ground
wheat germs is heated in a water-bath, no coagulation occurs, the solution
becoming slightly opalescent. If a more concentrated extract, such as may
be obtained by treating one part of meal with five parts of water, is thus
heated, the entire solution solidifies to a firm, opaque jelly, free from visible
particles. If to either of these solutions a very little hydrochloric acid
is added previous to heating, an abundant flocculent coagulum separates on
heating.

After standing a while the aqueous extract becomes gradually acid to
litmus, so that when heated slowly it becomes turbid at about 500 and a
large flocculent coagulum separates at 550. Heated to 650 for some time
and filtered, a second coagulum may be obtained on raising the heat from
65 ° to ioo°. The amount of this second coagulum is about one-third that
of the first.

The coagulated protein is dissolved by 0.5 per cent potassium-hydroxide
solution, but not perceptibly by 0.4 per cent hydrochloric acid solution,
unless the latter is heated, when a clear transparent jelly is formed.

Freed from coagulable protein, the aqueous extract still contains a rela-
tively large amount of substance which has the reactions of proteose.

When the concentrated aqueous extract is poured into a large volume of
distilled water, a turbidity forms at first, which mostly disappears after
shaking, indicating the absence of a notable quantity of globulin held in
solution by the salts dissolved from the meal.

Saturation of the extracts with sodium chloride gives a considerable pre-
cipitate, only a small part of which can be redissolved in dilute sodium-
chloride solution. When this dissolved part is precipitated by again satu-
rating with sodium chloride, it also is converted, to a large extent, into an
insoluble form ; the part still remaining in solution is precipitated, like a
globulin, by dialysis.

When the solution, saturated with sodium chloride, is filtered and the
diluted filtrate saturated with ammonium sulphate, a part of the precipitate
produced, when redissolved in water, is thrown out of solution by saturating
with sodium chloride, though before precipitation with ammonium sulphate
it was soluble in saturated sodium-chloride solution.

EXPERIMENTAL. 23

These reactions show that changes occur which involve the albumin coag-
ulating at 55°, for after freeing the extract from all protein precipitable by-
saturating with sodium chloride or by dialysis there remains in solution only
a small proportion of this albumin.

Thus an aqueous extract corresponding to 666 grams of germ meal, when
heated to 65 °, yielded 62 grams of coagulum, or 9.3 per cent ; a similar ex-
tract on dialysis deposited 9.2 per cent ; only 0.87 per cent of coagulable and
2.0 per cent of uncoagulable protein remaining in solution. The precipitate,
produced by dialysis, was but slightly soluble in sodium chloride solution,
having become largely coagulated. From these facts it is clear that one and
the same protein substance gives rise to these apparently different protein
bodies, and consequently the substance which O'Brien considered to be a
globulin of the myosin type and an albumin, coagulating at 8o°, are in fact
derivatives of the albumin, which coagulates mostly at 650.

These changes are due to a slow development of acid in the extract, which
not only brings about hydroly tic changes in the protein, but may also lead to
the formation of different compounds between the protein and the various
acids contained in the extract, and so give rise to chemically different sub-
stances. Such a development of acid takes place rapidly in muscle plasma,
under the influence of which quite similar changes in the proteins there
present can be observed.

Why Frankfurt overlooked albumin, present in such large proportion in
the aqueous extract, is not easily understood, unless, before heating his solu-
tions, he either added no acid or so much that he converted this substance
into an uncoagulable acid compound.

Hydrochloric acid added to the extract in very small quantity causes a
flocculent coagulum to separate on heating, while a slightly larger quantity,
added before heating, entirely prevents the formation of this coagulum.
Acetic acid and nitric acid give precipitates in the extracts which are not
soluble in a reasonable excess of either of these acids.

The Albumin of the Wheat Embryo.

In order to determine definitely the relations of these variously obtained
substances, a large number of fractional precipitations have been made under
quite different conditions, an account of which is now given ■

An extract was made by treating 700 grams of germ meal with 5 liters of
water, straining through bolting-cloth and filtering the fluid perfectly clear.
A portion of it was at once heated for 1 hour in a water-bath at 6o°, and the
large coagulum produced gave 24 grams of preparation 6.

Another preparation was made by heating in a water-bath at 65 ° 2000 cc.
of a clear aqueous extract, obtained by treating 3000 grams of the germ meal

24 THE PROTEIN'S OF THE WHEAT KERNEL.

with 9 liters of water. Thecoagulum produced, weighing 62 grams, formed
more than 9 per cent of the oil-free germ meal. This is preparation 7.

Another aqueous extract was heated at 650 until all the protein coagu-
lable at this temperature had separated. The coagulum produced, when
washed with hot water and alcohol, was dried over sulphuric acid and
found to weigh 16.68 grams. The filtrate from this coagulum, heated in a
boiling water-bath, yielded a second coagulum which formed preparation 8,
weighing 4.9 grams.

A portion of the extract which yielded preparation 6 was saturated with
ammonium sulphate; the resulting precipitate was dissolved as far as possi-
ble in water, its solution filtered clear and dialyzed for 4 days. During
this time a considerable precipitate formed that, when filtered out, was
found to be insoluble in salt solution. The solution, filtered from that sub-
stance and dialyzed in running water until nothing more separated, was
filtered and heated at 6o°, which caused a coagulum. This coagulum
weighed 7.1 grams and made preparation 9.

When 2000 cc. of an extract of 650 grams of wheat-germ meal was dia-
lyzed 4 days, a dense turbidity formed, due to a globulin, since it dissolved
on adding sodium chloride. Passing carbon dioxide gas through the dialyz-
ing solution seemed to increase the turbidity, but effected no definite separa-
tion. As it was found that 10 cc. of decinormal hydrochloric acid per 100 cc.
of the extract caused a separable precipitate, this proportion was added and
the resulting flocculent precipitate brought into solution again by adding
sodium chloride. The clear solution was then dialyzed for 2 days in run-
ning water and filtered from an amorphous precipitate, which was treated as
later described on page 44.

The filtrate from this precipitate was further dialyzed for 3 days more in
running water and then, as nothing separated, for 4 days more in alcohol.
The precipitate which resulted was dried over sulphuric acid, exhausted
with water, in order to remove all uncoagulated proteins, as well as other
soluble substances, dehydrated with absolute alcohol, again dried and
weighed, yielding 12 grams of preparation 10.

Another aqueous extract was saturated with pure sodium chloride, the
abundant precipitate filtered out, treated with dilute brine, and the resulting
solution filtered from a relatively considerable quantity of insoluble matter.
This filtrate was saturated with sodium chloride, a second precipitate filtered
out, and likewise treated with dilute sodium-chloride solution. The insol-
uble portion was removed by filtration and the clear filtrate dialyzed. The
small precipitate separated by dialysis when washed and dried, weighed 4.8
grams and formed preparation 11.

EXPERI MENTAL. 25

The filtrate from the first precipitation of the substance of preparation 11,
caused by saturating its solution with sodium chloride, as described above,
was diluted with water and saturated with ammonium sulphate. The pre-
cipitate which resulted was dissolved in water and its solution precipitated
by saturating with sodium chloride. Although this substance had previ-
ously been soluble in saturated brine, after precipitation with ammonium
sulphate it was found to be nearly all insoluble therein, so that almost com-
plete precipitation resulted on again saturating with sodium chloride. The
precipitate so produced was filtered out, dissolved in dilute sodium chloride
solution, and reprecipitated by dialysis. We thus secured 7.6 grams of
preparation 12.

By saturating another aqueous extract of germ meal with sodium chloride
a very large quantity of protein was separated, which was filtered out, ex-
hausted with dilute sodium chloride solution, and the insoluble part washed
thoroughly with water and alcohol. This preparation, 13, weighed 17
grams.

The filtrate and saline washings from preparation 13 were united and again
saturated with sodium chloride and yielded a small precipitate, which, when
dissolved in brine and precipitated by dialysis, gave preparation 14, weighing
2.8 grams. As the salt-saturated solution from which this preparation had
separated contained so little protein, it appears that nearly all the protein
precipitated from the aqueous extract by saturating with sodium chloride had
been converted into the insoluble substance forming preparation 13.

The filtrate from the salt-saturated precipitate produced in the aqueous
extract was dialyzed in water for several days, and the still clear solution
then dialyzed in alcohol for 24 hours. The protein thereby precipitated in
a coagulated state yielded 12.4 grams of preparation 15.

Another aqueous extract was saturated with sodium chloride, and the
precipitate, treated in the same way as preparation 13, yielded 18 grams
of preparation 16.

The saline washings of the last preparation were dialyzed free from chlo-
rides and gave a precipitate weighing 2.86 grams, which formed preparation
17, having the properties of a globulin, dissolving readily on adding sodium
chloride, and being precipitated from such solution by water.

The filtrate from the final precipitation of 17, when heated in a boiling
water-bath, gave a coagulum, which formed preparation 18, weighing 1.64
grams.

The salt-saturated filtrate from the first precipitation of 16, as already
described, was heated to boiling, and the coagulum produced was filtered out,
giving preparation 19, weighing 5.47 grams.

Since analysis showed that most of the preparations already described
contained phosphorus, some even in large amount, we made an attempt to

26 THE TROTEINS OE THE WHEAT KERNEL.

separate the phosphorus from our extract in order to determine, if possible,
the relation of the preparations free from phosphorus to those which con-
tained much phosphorus.

2000 grams of meal were treated with 6 liters of distilled water, and the
extract (4 liters) was squeezed out as completely as possible in a press.

As a preliminary experiment 100 cc. of this clear, filtered extract were
made faintly alkaline to pheuolphthalein, with about 40 cc. of decinormal
potassium-hydroxide solution. To insure a sufficient quantity 20 cc. more
of alkali were added, and thereupon a little calcium chloride, which gave a
precipitate that seemed to partly dissolve on adding sodium chloride. The
undissolved part, when washed with dilute sodium-chloride solution, water,
and alcohol, and dried, formed 1.7 per cent of the meal, contained about 55
per cent of organic matter, and left 45 per cent of ash, consisting of tricalcium
phosphate.

To 2000 cc. of the original extract were then added 1350 cc. of a solution
containing alkali, equivalent to 1560 cc. decinormal solution, with sodium
chloride enough to form 6.5 per cent of the total liquid. To this a solution
of calcium chloride was added as long as a precipitate formed, and after
standing over night the solution was decanted from the precipitate and filtered
clear on a pulp filter. Of the clear filtrate 2200 cc. were made as neutral as
possible to litmus by adding 180 cc. of decinormal hydrochloric acid solution.
Of the solution thus neutralized 1000 cc, when gradually heated in a
water-bath, became turbid at 520 and a considerable coagulum separated

at 53°-

After the temperature had been slowly raised to 65 ° and kept at this point
sometime, the coagulum was filtered out, washed, and dried as usual, giving
preparation 20, weighing 6.4 grams. Another portion of this extract, filtered
from the calcium-chloride precipitate, was saturated with ammonium sulphate
while still slightly alkaline to litmus, the resulting precipitate filtered out,
dissolved in water, its solution filtered clear, and dialyzed. A slight precip-
itate formed on dialysis, which was removed by filtering and the solution
heated in a boiling water-bath. The protein thus coagulated weighed 3.07
grams, preparation 21.

To determine what effect the removal of the phosphorized substance thrown
out by calcium chloride had upon the precipitation with sodium chloride, we
made neutral to litmus a liter of the filtrate from the calcium-chloride precip-
itate and then saturated it with sodium chloride. The large precipitate
which formed was washed by decantation with water, in which it gradually
dissolved, until only an insignificant quantity remained. The similarly ob-
tained precipitate from the simple aqueous extract we have shown to be nearly
all insoluble in water.

EXPERIMENTAL.

To separate globulin from the aqueous extract 1200 cc. of clear, filtered
extract were obtained from 200 grams of the germ meal treated with 2000 cc.
of water. 1000 cc. of this extract were dialyzed in running water for 6
days, and the large precipitate resulting gave preparation 22, weighing 9.17
grams.

These preparations, thus variously obtained from the aqueous extract,
were dried to constant weight at no° and analyzed with the results given
in table 2, most of the figures being the average of closely agreeing duplicate
determinations.

Table 2. — Composition of preparations of protein from the water extract of

the wheat embryo.

Carbon. . . .
Hydrogen .
Nitrogen . .
Sulphur . . .
Phosphorus
Ash

P2Os in ash

10.

11.

12.

13-

P. ct.

Pet.

P. ct.

Pet.

P.ct.

51-13

6.85

16.28

50.52

6.81

16.47

50.17

7.01

16.66

52.39
6.83

16.20

51.77

6.81

16.11

52.13

7.04

16.48

52.73

7.H

16.00

43-59

5-77

15.16

1.18

0.72

1. 17
0.97

1. 00
0.91

I.32
trace

1.30

0.17

1.49

0.06

i-53
none

0.90
3-38

2.73

1.83

2.90
2.09

3-03
1.91

o.35
trace

i-39
0.47

0.43

trace

o-39
none

13.04
6-73

14.

P. ct.

52.28

6.97

16.38

i-39
0.07

0.44

trace

Carbon

Hydrogen .
Nitrogen . . .
Sulphur . . .
Phosphorus

Ash

P2Oj in ash.

15.

16.

17-

18.

19-

20.

21.

P. ct.

P.ct.

P. ct.

P.ct.

P. ct.

51.21

46.67

51-87

51-91

51-65

52.02

6.85

6.19

6.89

6.86

6.66

7.00

16.18

15.89

16.65

16.31

16.08

16.02

16.45

1. 10

0.93

1. 19

1-35

1.60

i-i3

1.24

0.46

2.53

trace

none

2.19

8.17

0.38

o.45

0.32

1.09

0.56

I. II

5-71

trace

none

22.

P. ct.

49-59
6.68

16.34
0.91

1.85
2.50
1.79

Assuming that those of the foregoing preparations which contain phos-
phorus are compounds of protein with the nucleic acid which has been
separated from the aqueous extract of wheat germs,1 and also assuming that
all the phosphorus of these preparations is a part of the nucleic acid, the
composition of these preparations was calculated free from nucleic acid.
The analyses were further calculated ash-free by subtracting the P205
contained in the ash from the total ash, which seems permissible, since
the ash consisted almost wholly of metaphosphates of potassium and sodium,

1 Osborne & Campbell, Journal American Chemical Society, 1900, xxii, p. 379 ; also
Osborne & Harris, Report Connecticut Agricultural Experiment Station for 1901, p. 365.

26 THE TROTEINS OF THE WHEAT KERNEL.

separate the phosphorus from our extract in order to determine, if possible,
the relation of the preparations free from phosphorus to those which con-
tained much phosphorus.

2000 grams of meal were treated with 6 liters of distilled water, and the
extract (4 liters) was squeezed out as completely as possible in a press.

at 53°-

EXPERIMENTAL.

Tabi,e 2. — Composition of preparations of protein from the water extract of

the wheat embryo.

Carbon

Hydrogen . .
Nitrogen . . .
Sulphur
Phosphorus .

Ash

P205 in ash .

10.

11.

12.

13-

P. ct.

P.ct.

P. ct.

P.ct.

51-13

6.85

16.28

50.52

6.81

16.47

50.17

7.01

16.66

52.39

6.83

16.20

51.77
6.81

16.11

52.13

7.04

16.48

52.73

7.11

16.00

43-59

5-77

15-16

1. 18

0.72

1. 17
0.97

1. 00
0.91

1.32

trace

1.30

0.17

1.49

0.06

i-53
none

0.90
3-38

2.73

1.88

2.90
2.09

3-03
1. 91

o.35
trace

i-39
0.47

0.43

trace

o.39
none

13.04
6.73

14.

P.ct.

52.28

6.97

16.38

1-39

0.07

0.44

trace

Carbon

Hydrogen .
Nitrogen . . .
Sulphur . . .
Phosphorus

Ash

P2Os in ash .

15-

16.

17-

18.

19-

20.

21.

P. ct.

P.ct.

P. ct.

P.ct.

P. ct.

51.21

46.67

51-87

51-91

51-65

52.02

6.85

6.19

6.89

6.86

6.66

7.00

16.18

15-89

16.65

16.31

16.08

16.02

16.45

I. IO

0.93

1. 19

i-35

1.60

I-I3

1.24

0.46

2.53

trace

none

2.19

8.17

0.38

o.45

0.32

1.09

0.56

I. II

5-71

trace

none

22.

P. ct.

49-59
6.68

16.34
0.91

1.85
2.50
1.79

1 Osborne & Campbell, Journal American Chemical Society, 1900, xxii, p. 379 ; also
Osborne & Harris, Report Connecticut Agricultural Experiment Station for 1901, p. 365.

THE PROTEINS OF THE WHEAT KERNEL.

strongly indicating that the P205 was derived from the nucleic acid. These
calculations gave the results shown in table 3.

Table 3. — Composition of lencosin contained in the preparations from water

extracts of the ivheat embryo.

Carbon . . .
Hydrogen
Nitrogen .
Sulphur.. .
Oxygen. . .

10.

11.

12.

13-

P.ct.

P. ct.

P.ct.

52.93

52.75

52.41

52.57

5257

52.47

52.93

53-23

7.12

7.16

7.33

6.85

6.91

7.08

7.13

7.09

16.45

16.68

16.94

16 26

16.27

16.55

16 06

16.30

1.29

1.32

113

I.32

1-34

1.50

1-53

I.60

22.21

22.09

22.14

23.00

22.91

22.40

22.35

21.78

100.00

IOO.OO

14.

P.ct.

52.64

7.02
16.46

1.41
22.47

100.00

Carbon . .
Hydrogen
Nitrogen.
Sulphur .
Oxygen . .

15-

16.

17-

18.

19.

20.

21.

P.ct.

P. ct.

P.ct.

52.63

7.06
16.40

52.44

7.10
16.26

52.06

6.92

16.71

16.38

52.11

6.88
16.13

52.1b

6-73
16.20

52.30

7.04

16.54

1. 17
22.74

i-34
22.86

I.19
23.12

1.35

1.60
23.28

I.14
23-77

1.24

22.88

100.00

22.

P.ct.

53-45
7-30

16.57
1. 16

21.52

100.00

Of these preparations, 6, 7, 8, 9, 18, 19, 20, and 21 were obtained by coagu-
lation with heat, 10 and 15 by coagulation with alcohol, 13 and 16 by satu-
ration with sodium chloride, 11, 12, 14, and 17 by dialyzing salt solutions
in water, and 22 by direct dialysis of the aqueous extract. Since some of
these preparations formed a large part of the protein contained in the extract,
while others represented fractions of it, it is evident that all contain pro-
tein of the same composition, mostly combined with various proportions of
nucleic acid.

Eliminating the nucleic acid, it thus appears that the composition of the
protein part of those preparations which contain phosphorus is the same as
that of the phosphorus-free protein preparations, although the former con-
tain from very little up to more than 37 per cent of nucleic acid.

Most of these preparations might, in accordance with custom, be called
nucleoproteids, while 13 and 16 are, both in properties and composition, very
much like nuclein. It is probable that these nucleoproteids and nucleins are
nucleic acid compounds of one and the same protein.

It is to be noted that these preparations show very diverse properties.
Some are like albumin; some like globulin; some are precipitated by satura-

EXPERIMENTAL. 29

tion with salt, while others are not. As we have shown, these different prop-
erties are the result of changes caused by varying the conditions under which
the protein exists in the extract, and depend chiefly on the degree of acidity
of the extract, whereby the numbers and kinds of acid molecules that com-
bine with the protein molecule are altered.

Whatever may be the true cause of these changes, it is evident from the
results here described that the distinctions heretofore made between globulin
and albumin, myosin, and vitellin, etc., have very little value as a basis
for classifying protein substances. This explains the difference between
O'Brien's classification of leucosin as a myosin-like globulin, to which refer-
ence was made at the beginning of this paper, and our designation of it as
an albumin, because of the ready solubility in water and coagulability by heat
of the preparations which we had made.

Thus preparation 22, weighing 9. 17 grams, was insoluble in water and in
salt solution, and was not a precipitate of globulin, since in the filtrate from
which it had separated on dialysis only 0.87 gram of coagulable albumin
was found instead of 9.5 grams, as usually found by direct coagulation of
the aqueous extracts ; moreover, the analysis indicates that it is a compound
of leucosin, with 20 per cent of nucleic acid.

On the preceding pages it was shown that a small part of the precipitate,
produced by saturating the aqueous extract with sodium chloride, is soluble
in dilute salt solution, and can be precipitated from this solution by dialysis,
as a globulin-like substance, readily soluble again in salt solution. This
globulin-like substance contains little or no nucleic acid, and has very nearly
the same elementary composition as leucosin, of which it is probably a com-
pound with a small proportion of some body of low molecular weight.

It is plain from these facts that O'Brien's myosin contains the same pro-
tein substance as my leucosin.

O'Brien's " albumin," coagulating at 750 to 8o°, is probably more of this
same leucosin, as shown by preparation 8, which formed about 25 per cent
of the total coagulable protein. The experience of the writer has been that
complete coagulation, especially in a solution nearly free from salts, can be
effected, if at all, only by heating the solution much above the lower coagu-
lation temperature of the protein to be separated.

From the whole seed leucosin was obtained with the same composition and
general properties as from the embryo, but preparations from the whole seed
were free from phosphorus. This was probably because the proportion of
nucleic acid to protein matter was smaller in the whole seed than in the embryo,
so that on extracting with water the nucleic acid did not form soluble com-
pounds with the leucosin, but remained undissolved in combination with the
other proteins. In table 4, on the following page, is given the average of
analyses of albumin from the cereals.

3°

THE PROTEINS OF THE WHEAT KERNEL.
Table 4. — Composition of albumin prepared from various cereals.

Wheat
embryo.

Wheat
kernel.

Rye

kernel.

Barley
kernel.

Barley
malt.

Carbon

P. Ct.

5265
7.04

1643
1.32

22.56

P.ct.
53-02

6.84
16.80

1.28
22.06

P. ct.
52-97
6.79
16.66

i-35
22.23

P. ct.
52.81
6.78
1662

1.47
22.32

P. ct.

53-Q7

6.72

16.71

} 23.50

Sulphur

Oxvgen

100 00

100.00

In an earlier paper on the "Chemical Nature of Diastase" ' I pointed out
that diastatic action appeared to be always associated with leucosin. Since
extracts of wheat embryo were so rich in leucosin, the diastatic power of the
germ meal was determined by extracting with four times its weight of water,
and, under the conditions of L,itner's test, o. 10 cc. of the extract so made,
when added to 10 cc. of a 2 per cent solution of soluble starch, formed within
1 hour, at 200, enough sugar to reduce 5 cc. of Fehling's solution. The
o. 10 cc. of extract corresponds to 25 mg. of the germs, from which it is seen
that this meal possesses high diastatic power, though it is inferior in this
respect to active malt.

HYDROLYSIS OF LEUCOSIN.

In order to determine the proportion of the different products which are
formed by boiling proteins with strong acids, it is necessary to use relatively
large amounts of the protein. Commercial wheat-germ meal was therefore
used in order to obtain a sufficient quantity of leucosin for this purpose.
The freshly ground meal was extracted with water, and, as the gummy
solution could not be filtered within a reasonable time, an equal volume of
saturated ammonium sulphate solution was added. The precipitate thus
produced was filtered out, dissolved in water, the solution filtered perfectly
clear, and the leucosin coagulated by heating the dilute solution to 650 in a
water-bath at 700. As the only other protein substance present in this solu-
tion was a relatively insignificant quantity of proteose, the product obtained
was practically free from any other protein. This coagulum was thoroughly
washed with hot water, in order to remove any admixed proteose, and dehy-
drated with absolute alcohol. The preparation formed a light white powder.

Owing to the difficulty of preparing large quantities of this protein, which
occurs in very small quantity in the wheat kernel, we were limited in this

1 Osborne, Journal American Chemical Society, 1895, xvn, p. 587.

EXPERIMENTAL.

hydrolysis to 257.8 grams of water and ash-free leucosin. This was hydro-
lyzed and the glutaminic acid separated as hydrochloride. When dried in
vacuo over sulphuric acid, 12.39 grams of the hydrochloride were obtained,
which melted at 1980 with effervescence.

Nitrogen : 0.5482 gram substance gave NH3 = 4.i7 cc. HC1 (1 cc. HCl = o.oi gram N).

Chlorine : 0.2528 gram substance gave 0.1989 gram AgCl.

Calculated for C5H10O4NCl, N 7.64, CI 19.35 p. ct.; found, N 7.61, CI 19.45 P- ct.

The filtrate from the glutaminic acid hydrochloride was concentrated to a
sirup under reduced pressure, the residue taken up in alcohol and saturated
with dry hydrochloric acid gas. The solution was then evaporated to a
thick sirup under reduced pressure, the residue again esterified with alcohol
and hydrochloric acid, and the solution concentrated as before. The esteri-
fication was again repeated, the final concentration being made at a pressure
of 10 mm. from a bath, the temperature of which did not rise above 400.
The free esters of the amino-acids were then liberated from the residue,
extracted with ether, and dried with potassium carbonate and anhydrous
sodium sulphate in the usual way. The aqueous layer was then made
strongly acid with hydrochloric acid and the salts removed by concentra-
tion and treatment with alcoholic hydrochloric acid. The alcoholic extracts
containing the hydrochlorides of the amino-acids were evaporated to a thick
sirup under reduced pressure and the residue esterified as above described.
The free esters were then liberated and their ether solution dried as before.
After distilling off the ether on the water-bath, at atmospheric pressure, the
residue was distilled with the following results :

Fraction.

Temperature
of bath
up to —

Pressure.

Weight.

III

75
80

125

200

m m .
I2.0
10.0

0.8

Grains.
21.76
25.86
49.60

49-39

146.61

Fraction I. Temperature of bath up to 750
Pressure, 12 mm. Weight, 21.76 grams.

This fraction was saponified directly after collection by evaporating on the
water- bath with concentrated hydrochloric acid. The residue was esterified
with alcohol and hydrochloric acid and allowed to stand for several days
on ice. The glycocoll ester hydrochloride which separated weighed 1.73
grams and melted at 145°.

32 THE PROTEINS OF THE WHEAT KERNEL.

Nitrogen: 0.2851 gram substance, dried in vacuo, gave NHj=2.88 cc. HC1 (1 cc.

HCl = o.oi gram N).
Chlorine: 0.2843 gram substance, dried in vacuo, gave 0.2889 gram AgCl.
Calculated for C4H10O2 NCI, N 10.05, CI 25.40 p. ct.; found, N 10.12, CI 25.12 p. ct.

In the filtrate from the glycocoll ester hydrochloride the free amino-acids
were regenerated and subjected to fractional crystallization in water and
alcohol. There were obtained 3.06 grams of nearly pure alanine.

Carbon and hydrogen: 0.3682 gram substance gave 0.5460 gram C02 and 0.2649

gram H20.
Nitrogen: o. 250S gram substance gave NH3 = 3.92 cc. HC1 (1 cc. HCl = o.oi gram N).
Calculated for C3H702N, C 40.40, H 7.93, N 15.75 P- ct. ; found, C 40.44, H 7.99,

N 1563 p. ct.

The alanine decomposed at 2900.

f Fraction II. Temperature of bath up to 8o°. )
{ Pressure, 10 mm. Weight, 25.86 grams. j

After saponifying with boiling water, the solution of the amino-acids was
evaporated to dryness under reduced pressure. The dried residue, which
weighed 18.78 grams, was extracted with boiling alcohol, whereby 1.68
grams went into solution. From the part insoluble in alcohol there were
isolated, by fractional crystallization, 5.53 grams of leucine, 5.79 grams
alanine, and 0.47 gram of substance which had the percentage composition
of amino- valerianic acid.

Carbon and hydrogen: 0.2094 gram substance gave 0.3913 gram C02 and 0.1793

gram H20.
Calculated for C5Hu02N, C 51.22, H 9.48 p. ct. ; found, C 50.96, H 9.51 p. ct.

From the more soluble part of this fraction glycocoll was isolated as the
hydrochloride of the ester. This weighed 2.78 grams, equivalent to 1.50
grams of glycocoll, and melted at 1440 to 1450.

j Fraction III. Temperature of bath up to 1250.
{ Pressure, 0.8 mm. Weight, 49.6 grams.

Fraction III was saponified by boiling with 10 parts of water for 10 hours,
and the solution evaporated to dryness under reduced pressure. After
extracting the proline with boiling alcohol the insoluble part was fractionally
crystallized. There were obtained 23.72 grams of leucine and 2.62 grams
of alanine. The isolated leucine decomposed at 298 °.

Carbon and hydrogen : o. 1S35 gram substance dried at no0 gave 0.3670 gram C02 and

0.1670 gram H20.
Nitrogen : 0.30S6 gram substance gave NHS = 3.34 cc. HC1 (1 cc. HCl = o.oi gram N).
Calculated for C6H1302N, C 54.89, H 10.01, N 10.70 p. ct. ; found C 54.63, H io.n,

N 10.82 p. ct.

EXPERIMENTAL. 33

The combined alcoholic solutions from fractions II and III, which con-
tained the proline, were evaporated to dryness under reduced pressure, and
the residue extracted with boiling alcohol, in which 2.3 grams did not dis-
solve. The solution filtered from this was again evaporated to dryness
under reduced pressure, the residue dissolved in water, and the copper salts
prepared by boiling for an hour with an excess of copper hydroxide. The
deep blue solution was evaporated to dryness under reduced pressure, and
the residue boiled with absolute alcohol, which dissolved the /-proline copper
salt. The residue of racemic copper salt, insoluble in alcohol, was dissolved
in water and the solution concentrated. Of racemic proline copper 2.41
grams were obtained, equivalent to 1.69 grams of ^-proline.

Water: 0.3190 gram substance, air-dried, lost 0.0353 gram H20 at 110°.
Copper: 0.2827 gram substance, dried at no0, gave 0.0766 gram CuO.
Calculated for C10H16O4N2Cu. 2 H20, H20 11.00 p. ct. ; found, H20 11.07 P- ct.
Calculated for C]0H16O4N2Cu, Cu 21.79 P- ct- > found, Cu 21.65 P- ct.

The alcoholic solution of the copper salt of the /-proline was evaporated
to dryness under reduced pressure. The dried residue weighed 8.24 grams,
equivalent to 6.5 grams of /-proline.

For identification a small portion was freed from copper with hydrogen
sulphide, and the free proline converted into the phenylhydantoin, accord-
ing to the directions of Emil Fischer.1

The hydantoin melted sharply at 1430 and gave the following analysis :

Carbon and hydrogen : 0.2334 gm. substance gave 0.5676 gm. C02 and 0.1204 gm. H20.
Nitrogen : 0.1373 gram substance gave NH3 = 1.78 cc. HC1 (1 cc. HC1 = 0.01 gram N).
Calculated for C12H1202N2, C 66.60, H 5.61, N 12.99 p. ct. ; found, C 66.32, H 5.73, N
12.96 p. ct.

J Fraction IV. Temperature of bath up to 2000.
( Pressure, 0.8 mm. Weight, 49.39 grams.

From fraction IV the ester of phenylalanine was removed in the usual
manner by shaking out with ether, and after freeing from ether the residual
ester was saponified by dissolving in concentrated hydrochloric acid and
evaporating on the water-bath. Of phenylalanine hydrochloride 12.12
grams were obtained, which is equivalent to 9.93 grams of phenylalanine.
For identification the hydrochloride was recrystallized from strong hydro-
chloric acid and converted into the free acid by evaporation with excess of
ammonia. When once recrystallized from water, it melted at 2630 t0 265°.

Carbon and hydrogen : 0.2962 gram substance, dried at no°, gave 0.7089 gram C02 and

o. 180 1 gram H20.
Nitrogen : 0.1924 gram substance gave NH3 = 1.65 cc. HC1 (1 cc. HC1 = 0.01 gram N).
Calculated for C9Hn02N, C 65.39, H 6-73. N 8-5° P- ct- \ found, C 65.27, H 6.76, N

8.57 p. ct.

^iscber, E., Zeitschrift fur pbysiologische Cbetnie, 1901, xxxnr, p. 251.
3

34 THE PROTEINS OF THE WHEAT KERNEL.

The aqueous layer was saponified by warming with an excess of barium
hydroxide on the water-bath for 7 hours. After standing for some time
the crystals of racemic barium aspartate were filtered out and decomposed
with an equivalent quantity of sulphuric acid. The filtrate from the barium
sulphate gave, on concentration, 5.06 grams of aspartic acid, which, when
recrystallized from water, was analyzed.

Carbon and hydrogen : 0.24S3 gram substance, dried at no0, gave 0.3278 gram CO, and

0.1 2 15 gram H20.
Nitrogen : 0.2769 gram substance gave NH3 = 2.93 cc. HC1 (1 cc. HCl = o.oi gram N).
Calculated for C4H.04N, C 36.06, H 5.31, N 10.55 P- ct. ; found, C 36.00, H 5.44, N

10.59 P- ct.

The filtrate from barium aspartate was freed from barium quantitatively
with sulphuric acid and the filtrate from the barium sulphate concentrated to
small volume and saturated with hydrochloric acid gas. After long standing
on ice, 2.15 grams of glutamiuic acid hydrochloride separated.

The filtrate from this was evaporated under reduced pressure, the residue
taken up in water, and the chlorine removed with silver sulphate.

After removing the sulphuric acid with an equivalent quantity of barium
hydroxide, the solution was boiled with an excess of copper hydroxide. The
filtered solution, on standing, separated tyrosine-like needles of copper laevo-
aspartate which weighed 7.41 grams, equivalent to 3.57 grams of aspartic
acid.

Nitrogen : 0.1853 gram substance, air-dried, gave NH3 = o.93 cc. HC1 (1 cc. HC1 = 0.01

gram N).
Copper : 0.1677 gram substance, air-dried, gave 0.0491 gram CuO.
Calculated for C4H504N, Cu ■ $% H20, N 5.09, Cu 23.06 p. ct.; found, N5.02, Cu 23.37

p. ct.

From the copper salt the free acid was regenerated and analyzed.

Carbon and hydrogen: 0.2041 gram substance gave 0.2715 gram C02 and 0.1054 gram

H20.
Calculated for C4H704N, C 36.06, H 5.31 p. ct.; found, C 36.29, H 5.74 p. ct.

Specific rotation. — Dissolved in 20 per cent hydrochloric acid,

(a) ^ = +23.8°

Fischer & Dorpinghaus1 found

/ n 2°° 1 O

(«)-5-= + 22.0°

An effort to isolate serine in the filtrate from the copper aspartate failed.
Fischer & Dorpinghaus, Zeitschrift fur physiologische Chemie, 1902, xsxvi, p. 462.

EXPERIMENTAL. 25

The Residue after Distillation.

The residue remaining after distillation of the esters weighed 57 grams.
This was dissolved in boiling alcohol, and after cooling 1.98 grams of needle
crystals were filtered out. The filtrate was evaporated to a sirup under
reduced pressure, saponified by heating with an excess of barium hydroxide,
and, after removing the barium, evaporated to small volume under reduced
pressure. The solution was then saturated with hydrochloric acid, and,
after standing on ice for a long time, yielded 7.39 grams of glutaminic acid
hydrochloride. The free acid prepared by evaporating with an exactly
equivalent quantity of potassium hydroxide, when recrystallized from water,
melted at 202 ° to 2030 with effervescence.

Carbon and hydrogen : 0.3646 gram substance, dried at no°, gave 0.5432 gram C02 and

0.2019 gram H20.
Nitrogen: 0.3696 gram substance gave NH3=3.55cc. HC1 (1 cc. HCl = o.oi gram N).
Calculated for C3H;,04N, C 40.82, H 6.12, N 9.52 p. ct.; found, C 40.63, H 6.15, N 9.60

p. ct.

The total glutaminic acid obtained from leucosin was 17.5 grams, or 6.73
per cent. This result is higher than that recently recorded in this labora-
tory, namely, 5.72.1

This protein is one from which the glutaminic acid hydrochloride can be
directly obtained only with great difficulty. In the former paper attention
was directed to this fact, and the statement made that it is possible that the
result given was too low.

Tyrosine.

Forty grams of leucosin, equal to 34.96 grams dried at no°, were boiled
for 12 hours with a mixture of 120 grams of sulphuric acid and 240 grams
of water. After removing the sulphuric acid with an equivalent quantity of
barium hydroxide, the solution was evaporated with an excess of barium
carbonate in order to remove ammonia. After removing the barium, the
solution was concentrated to a small volume on the water-bath and allowed
to stand for some time. The substance which separated was washed with
cold water, dissolved in ammonia, the solution treated with bone-black, and
evaporated. On cooling, 1.0360 grams tyrosine separated in colorless needles.
The filtration from this, on further concentration, yielded 0.13 gram more
tyrosine, making a total of 1. 1660 grams, or 3.33 per cent. This was recrys-
talized and analyzed.

Carbon and hydrogen : 0.4573 gram substance, dried at no0, gave 0.9994 gram C02 and

0.2813 gram H20.
Calculated for C9Hu03N, C 59.62, H 6.13 p. ct. ; found, C 59.60, H 6. 11 p. ct.

1 Osborne & Gilbert, American Journal of Physiology, 1906, xv, p. 333.

3^ THE) PROTEINS OF THE WHEAT KERNEL.

Arginine, Histidine, and Lysine.

The filtrate from the tyrosine, by Kossel's method, yielded a solution in
which the nitrogen found corresponded to 0.99 gram of histidine, or 2.83
per cent.

The identity of the histidine could not be established owing to its very
small amount.

The solution of the arginine contained nitrogen equal to 0.6720 gram,
which is equal to 2.08 grams of arginine, or 5.94 per cent. A part was con-
verted into the nitrate and this latter into the copper salt by boiling with an
excess of copper hydroxide and the copper salt recrystallized from water.

Water: 0.19S4 gram substance, air-dried, lost 0.019 1 gram H20 at no0.
Copper : 0.1766 gram substance, dried at no0, gave 0.0262 gram CuO.
Calculated for C12H2804N8Cu (N03)2 ' 3 H20, H20 9.15 p. ct.; found, H20 9.62 p. ct.
Calculated for C12H2804N8Cu (N03)2, Cu 11.85 P- ct.; found, Cu 11.83 P- ct.

The filtrate from the copper salt was freed from copper and the solution
evaporated with an excess of sulphuric acid under reduced pressure. The
sulphuric acid was removed with an excess of barium hydroxide, and the
barium with carbonic acid. The filtrate from the barium carbonate was evap-
orated to dryness and the arginine converted into the picrolonate, according
to the directions of Steudel. This melted at 2260 to 2270; Steudel gives 2250.

Nitrogen: 0.0516 gram substance, dried at no0, gave 11. 8 cc. moist N3 at 250 and

765 mm.
Calculated for C6Hu02N4 • C10H8O5N4, N 25.62 p. ct. ; found, N 25.71 p. ct.

The filtrate from the silver precipitate which contained the arginine and
histidine was freed from silver and barium, and the lysine precipitated with
phosphotungstic acid and then converted into the picrate, of which 2.47
grams, equal to 0.9616 gram of lysine, or 2.75 per cent, was obtained.
This was recrystallized from water and analyzed.

Nitrogen: 0.2288 gram substance gave 38.6 cc. moist N2 at 25. 50 and 759 mm.
Calculated for C6Hu02N2 ■ C6H307N3, N 1S.70 p. ct. ; found, N 18.77 p. ct.

The results of this hydrolysis are given in table 5.

Table 5. — Leucosin.

p. ct.

Glycocoll 0.94

Alanine 4.45

Aminovalerianic acid 0.18

Leucine n-34

a-proline 3. 18

Phenylalanine 3-83

Aspartic acid 3.35

Glutaminic acid 6.73

P. ct.

Tyrosine 3.34

Lysine 2 75

Histidine 2.83

Arginine 5.94

Ammonia 1.41

Tryptophane present

5032

EXPERIMENTAL. 37

PROTEINS OF WHEAT FLOUR SOLUBLE IN SODIUM-CHLORIDE SOLUTION.

The Globulin of Wheat Flour.

Beside the proteins soluble in water, 10 per cent sodium-chloride brine
extracts from ground wheat kernels a globulin which is present in the seed
in small quantity. Ten kilograms of ' ' straight flour ' ' were extracted with
34 liters of 10 per cent sodium-chloride solution by suspending the flour in
the liquid, stirring frequently, and then allowing the whole to stand at
rest over night. The extract, separated from the flour and filtered as clear
as possible, had a very slight acid reaction, was of a pink color, very viscid
consistence, and formed about one-half of the solution added to the flour.
This was saturated with ammonium sulphate, and the resulting precipitate
filtered off and dissolved as far as possible in 4 liters of 10 per cent sodium-
chloride solution. The exceedingly viscid solution was filtered with diffi-
culty, placed in a dialyzer, and left in a stream of running water until the
chlorides were removed. As the salts dialyzed out the globulin gradually
separated in minute particles, the larger of these being evidently spheroidal
in form. This precipitate weighed 5.8 grams. This protein, dissolved in
a 10 per cent sodium-chloride solution, when heated slowly, gave a very
slight turbidity at 870, which increased slightly up to 990. On boiling,
some coagulum developed, and on adding acid to the solution filtered from
this coagulum a very considerable precipitate formed.

Dilution of the solution of the globulin in 10 per cent sodium-chloride
brine precipitated the protein. Saturation with sodium chloride gave no
precipitate. Saturation with magnesium sulphate, and also with ammonium
sulphate, completely precipitated the globulin. When dried at no°, this
preparation, 23, gave, when analyzed, the results which are shown in the
table on page 38.

Another preparation, 24, was made in the same way as the preceding, ex-
cept that the precipitation with ammonium sulphate was omitted, the filtered
extract being placed at once in dialyzers. Like the preceding solution, this
was at first very viscid, but after the removal of the chloride the viscid
property was entirely lost. This viscidity can hardly be due to the globulin,
for solutions of the precipitated globulin showed no trace of it. The aqueous
extract of the flour had no such property, and it is difficult to say to what
this was due unless to the presence of carbohydrate. After complete
removal of the chlorides the solution was filtered from the precipitate, the
latter dissolved in 10 per cent sodium-chloride solution, and the insoluble
matter filtered off. The residue so removed consisted chiefly of an insoluble
form of the globulin. This was dissolved in o. 2 per cent potassium-hydroxide
water, the solution filtered clear, and precipitated by neutralization with

THE PROTEINS OF THE WHEAT KERNEL.

0.2 per cent hydrochloric acid. The precipitate weighed 2 grams. The
solution of the globulin was dialyzed till free from chlorides and the sepa-
rated globulin found to weigh 5 grams. The yield in this extraction was
therefore 7 grams, or nearly the same as in the preceding. The composi-
tion of preparation 24 is given by the analysis shown in the following table :

Preparations 23 and 24.

Preparation 23.

Preparation 24.

II.

Average.

Ash-
free.

II.

Average.

Ash-
free.

Hydrogen

Oxygen

P.ct.

50.87

6.74

18.13

0.97

P.ct.

50.79

6.67

18.25

P.ct.
50.83

6.71
18.19

o.97

P.ct.
51-07

6-75
18.27

0.97
22.94

P. Ct.

5o.77
7-03

18.43
0.71

P.ct.

50-63

6.S4

18.32

P. ct.
50.70

6-93

18.38

0.71

P.ct.

51 01

6.97

18.48

0.71

22.83

Ash

0.48

0.62

100.00

The globulin contained in the " shorts" from the spring wheat was next
extracted by treating 2000 grams with 10 per cent sodium-chloride solution
for 3 hours, with frequent stirring, and then squeezing out the extract
in a screw-press. After the suspended starch had settled, the extract was
decanted and saturated with ammonium sulphate. The precipitate was dis-
solved in 10 per cent sodium-chloride brine, and the solution filtered from
the insoluble matter. The clear extract was then dialyzed till free from
chlorides, and the precipitated globulin found to consist of well-formed
spheroids and masses of confluent spheroids. The globulin was strongly
colored, since much coloring matter was extracted from the shorts, render-
ing the extract a deep red-brown in color. The precipitate was then filtered
off, again dissolved in the salt solution and placed in a dialyzer. When free
from chlorides, the precipitated globulin was filtered off and found to be still
much colored. After pa'rtly washing with water it began to dissolve,1 and
was therefore next washed with dilute alcohol and finally with absolute
alcohol and with ether. Dried over sulphuric acid, it weighed 2.22 grams,
and had the composition shown under the heading " Preparation 25 " in the
table on page 39.

JThis solubility of a globulin is due to the formation of acid compounds which are
soluble in water, but insoluble in a dilute saline solution. Cf. Osborne, Journal Amer-
ican Chemical Society, 1902, xxiv, p. 39.

EXPERIMENTAL.

The filtrate and washings obtained from this preparation after the second
precipitation by dialysis were precipitated by adding a few drops of sodium-
chloride solution. The precipitate produced, which was nearly free from
coloring matter, weighed 1.25 gram. When analyzed it gave the results
shown under the heading " Preparation 26" in the accompanying table.

Preparations 25, 26, and 2j.

Preparation 25.

Preparation 26.

Preparation 27.

Ash-
free.

II.

Average.

Hydrogen . . .
Nitrogen ....

Oxveren

P.ct.

50.79
6.80

18.19
0.66

P. ct.
51.00

6.83
18.26

0.66
23-25

P. ct.

P.ct.

P. ct.

P.ct.

18.56

18.64

18.15

18.43

18.29

Ash

0.40

0.47

IOO.00

The solution filtered from this preparation was next heated to boiling
and the coagulum obtained, which weighed 1 gram, when analyzed was found
to contain the same amount of nitrogen as the globulin. It was considered
to be a coagulated globulin which had been held in solution in water by
acid. Its nitrogen content is shown under the heading " Preparation 27."

The total quantity of globulin obtained from the shorts was in this case
4.47 grams, being nearly twice as much as was similarly obtained from a
like quantity of the flour.1

Table 6. — Summary of analyses of wheat globulin.

23-

24.

25-

26.

27.

Average.

Hydrogen ....

P. ct.
GI-07
6-75
18.27

} 23.91

P.ct.

51.01
6.97

18.48
f 0.71
I 22.83

P. ct.

51.00

6.83

18.26

0.66

23-25

P.ct.

51-03

6.85

13.39

0.69

23.04

18.64

18.29

100.00

1 Teller (Bull. 42, Ark. Agr. Exp. Sta. , 1896) found a much larger quantity of leucosin
and globulin nitrogen in the samples of bran which he examined than he found in the
flours.

4-0 THE PROTEINS OE THE WHEAT KERNEL.

THE PROTBINS OF THE WHEAT EMBRYO SOEUBEE IN SODIUM-
CHLORIDE SOLUTION.

The Globulin of the Wheat Embryo.

Wheat-germ meal treated with 10 per cent sodium-chloride brine forms a
dense jelly-like mass, from which it is nearly impossible to separate the
solution.

With 3 per cent brine a manageable extract can be made by using from
six to ten times as much solvent as meal. Thus ioo grams of the meal
treated with 600 cc. of 3 per cent sodium chloride yielded in 15 hours 400 cc.
of clear filtrate. As has just been shown, the aqueous extract on dialysis,
in consequence of a change which affects leucosin, deposits a large amount
of protein, chiefly in the coagulated form. In order to obtain preparations
of the protein substance soluble in salt solutions, but insoluble in water,
which should be free from this coagulable albumin, 2000 grams of germ
meal were treated with 20 liters of 3 per cent sodium-chloride solution
heated to 700, whereby the leucosin was coagulated and the salt-soluble
globulin brought into solution. The extract, neutral to litmus, was filtered
clear, at once saturated with ammonium sulphate, and the proteins thus
precipitated collected on a filter, dissolved in water, and the clear solution
dialyzed in running water.

Protein matter separated on dialysis in spheroids, which, like many
other plant-globulins, united to a plastic mass on the bottom of the dia-
lyzer. This precipitate was dissolved in sodium-chloride solution and, after
filtering absolutely clear, dialyzed for 48 hours, the large precipitate which
separated allowed to settle, and the solution, which was nearly free from
protein, decanted.

A portion of the precipitate was washed first with water, which rendered
it opaque and dense, then with dilute and finally absolute alcohol, and dried
over sulphuric acid. This weighed 5.22 grams, and is preparation 28. The
rest of the precipitate was completely dissolved in 125 cc. of 10 per cent
sodium-chloride solution. To this, water was added until its volume was
425 cc, thus making a sodium-chloride solution of nearly 3 per cent. From
this diluted solution a gummy deposit separated, from which the fluid was
soon completely decanted. The latter was further diluted with 325 cc. of
water and the precipitate which resulted allowed to settle to a viscid trans-
parent deposit. From this precipitate the solution was again decanted and
dialyzed for 48 hours, but not more than a trace of globulin was deposited.
The two precipitates produced by dilution formed preparations 29 and 30,
weighing respectively 11.4 grams and 8.15 grams. A part of each of these
preparations was set aside for analysis, and the rest, dissolved together in 10
per cent sodium-chloride solution, allowed to stand over night at 40. The

EXPERIMENTAL. 41

solution was then decanted from a slight sediment, filtered clear, and heated
to 8o°, in order to coagulate any leucosin which might be present, and after
2 hours filtered from a very small coagulum which had gradually formed.
This filtrate was dialyzed in water for 4 days, and the globulin which sepa-
rated gave preparation 31. The solution filtered from the first dialysis
precipitates, which yielded preparations 28, 29, and 30, was further dialyzed ;
a little globulin, which separated, was filtered out and the filtrate dialyzed
iii alcohol for 4 days. A precipitate was produced which, when washed
with absolute alcohol and dried, weighed 25 grams. This substance con-
sisted of protein which is described on page 47.

Another series of fractional precipitations of this globulin-like protein
was made by extracting 4 kilograms of the oil-free germ meal with 27
liters of 3 per cent sodium-chloride solution heated to 67 ° at the time
it was applied to the meal. The mixture was thoroughly stirred and
thrown on filters. A clear filtrate of about 12 liters was finally obtained,
which was saturated with ammonium sulphate. The precipitate produced
was dissolved in water and its solution dialyzed for 48 hours ; where-
upon a large quantity of spheroids separated, which on settling united
to a coherent mass. This precipitate was washed by decantation with
water, dissolved in brine, and its solution made faintly alkaline to litmus by
cautiously adding decinormal potassium-hydroxide solution. In order to
separate phosphoric acid, a little calcium-chloride solution was then added
to this very slightly alkaline liquid, and the latter, though apparently free
from any precipitate of calcium phosphate, was filtered, whereby a little
suspended matter was removed. The solution was made exactly neutral to
litmus by adding 56 cc. decinormal hydrochloric acid and dialyzed for 18
hours. A gummy precipitate (A) adhering to the bottom of the dialyzer
then separated, from which the solution (B) was decanted almost completely.

The precipitate (A) was dissolved in about 200 cc. of 5 per cent sodium-
chloride solution and the liquid was poured into 800 cc. of water. The
resulting flocculent precipitate settled rapidly to a coherent deposit, from
which the solution was decanted. The deposit was repeatedly washed by
decantation with water, which caused it to lose its gummy character and
become opaque, white, and granular. It weighed 15.5 grams and was
preparation 32. The solution marked B was further dialyzed for 48 hours,
when a second precipitate formed, which, like 32, completely dissolved
in sodium-chloride solution to a solution perfectly neutral to litmus. This
precipitate was washed by decantation with water, but the finer part
settled so slowly that it was necessary to decant it together with the water.
The sediment, after exhausting with absolute alcohol and drying, weighed
23.5 grams, and formed preparation 33. On long standing the decanted
washings deposited the finely divided matter, which was then collected on a

THE TROTEINS OF THE WIII-AT KERNEL.

filter, dissolved in sodium-chloride solution, and precipitated by water, giving
15.4 grams of preparation 34.

To determine the quantity of globulin contained in the oil-free germ meal,
200 grams of the meal were treated with 2000 cc. of 3 per cent sodium-
chloride solution heated to 65 ° and the extract filtered perfectly clear. Of
this, 1000 cc. were dialyzed until free from chlorides, when the precipitate of
spheroids was filtered out. This preparation, 35, formed 5.05 per cent of
the oil- free meal.

To obtain a quantity of this globulin for digestion with pepsin, a quantity
of germ meal was extracted with 3 per cent sodium-chloride solution heated
to 700; the extract was filtered clear and saturated with ammonium sul-
phate. The precipitate produced was dissolved in water and the resulting
gummy and somewhat turbid solution filtered clear. The filtrate was dia-
lyzed until the solution gave no turbidity on pouring into distilled water.
The protein, which had then separated in spheroids, weighed 27.3 grams,
preparation 36.

A part of the extract from which 36 had been prepared was mixed with an
equal volume of decinormal potassium hydroxide solution — about twice the
quantity necessary to neutralize the extract to phenolphthalein. The solu-
tion was then dialyzed in distilled water frequently renewed, and in this way a
considerable quantity of phosphorus was separated in the alkaline dialysate.
When all, or nearly all, which it was possible to separate in this way had
been removed, the solution in the dialyzer was neutralized with hydrochloric
acid until it no longer reacted alkaline to litmus. This caused a turbidity.
The acid was then further added until an acid reaction with litmus was
obtained, producing a precipitate from which, after settling, the solution was
decanted. The precipitate was then dissolved in sodium-chloride solution,
its solution filtered clear and dialyzed, whereby a substance was precipitated
in spheroids, which formed preparation 37, weighing 3.0 grams. These
preparations had the composition shown in table 7.

Table 7. — Composition of preparations extracted by sodium-chloride solution

from the wheat embryo.

Carbon

Hydrogen. . .
Nitrogen

Sulphur

Phosphorus .
Ash

2Os in ash.

28.

P.ct.

18.14
0.49

1.15
2.29
1.66

29.

P.ct.

IS.2I
0.56

1.03
1.86
1-34

30-

P.ct.

48.77
6.44

18.12

0.51

i-35
2.25

1.68

3i-

P.ct.

50-03
7.04

18.39

0.60
0.76

I.V>
0.84

32.

P. ct.

50.23

6.89

IS.23

0.53
0.56

1.22
O.80

33-

P.ct.

48.17

6.54
IS.06

0.55
1.41

3-85

2.00

34-

35-

P.ct.

49-39
6.78

17.95
0.4S

1. 17
2.60
1.82

P.ct.

48.75

6.52

18.16

0.63

1. 41

2.66
2.00

*6.

P.ct.

49-79
6.76

18.01
0.61
1. 11
1. 11
0.68

37-

P.ct.
48.67
6.56

17-97
0.61

1-55
2-94
2.30

EXPERIMENTAL.

These analyses, when calculated free from nucleic acid and ash, as was
done for the albumin preparations, in the manner described on page 27, gave
.the results set out in table 8.

Table 8. — Composition of the globulin contained in the preparations extracted
from the wheat embryo by sodium-chloride solution.

28.

29.

3°-

3i-

32.

33-

34-

35-

36.

37-

P.ct.

P. ct.

P.ct.

51-37
6.83

1S.62
0.60

22.58

P.ct.

51-58

7-31

18.70

0.66

21-75

P. ct.

51.40
7.08

18.45

0.57

22.50

IOO.CO

P. ct.
5I-56

7.07
18.85

0.67
2185

P.ct.

51-86
7.19

18.41

o.55
21.99

P.ct.
51.40
6.94
18.71

o.75
22.20

100.00

P.ct.

51-98

7.12

18.37

0.70

21.83

P. ct.

51-70
7-05

18.53

0.75
21.97

Nitrogen . . .

Sulphur

Oxygen.. . .

18.59
0.57

18.59

0.63

roo.oo

IOO.CO

100.00

These figures plainly indicate that these globulin preparations are mixtures
of nucleates of protein substance of the same ultimate composition, and con-
tain from 5 to 15 per cent of nucleic acid. This protein has very nearly the
same composition as the globulin occurring in the kernel of wheat, rye,
barley, and maize. In the entire kernel so little of this globulin is present
that it is difficult to prepare it pure therefrom. For this reason the analyses
given in table 9 do not agree as closely as they might otherwise be expected
to. From the whole seed this globulin is obtained entirely free from phos-
phorus, which is probably due to the much greater proportion of protein
matter to nucleic acid in the entire seed compared with that existing in the
embryo.

Table 9. — Composition of the globulin contained in various cereals.

Wheat
embryo.

Wheat
kernel.

Rye1
kernel.

Maize2
kernel.

Barley3
kernel.

Carbon

P. ct.

51-57
7.07

18.60
0.65

22.11

P. Ct.

5I-03

6.S5

18.39

0.65

23.08

P. Ct.

51-19

6-74

18.19

} 23.88

P.ct.

51-99
6.81

18.02
f 0.66
I 22.52

P.ct.
5088
6.65
18.10

} 24.37

Oxygen

100.00

1 Osborne, Journal American Chemical Society, 1895, xvn, p. 429.

2 Chittenden & Osborne, American Chemical Journal, 1891, xin, pp. 327, 385,

and 1892, xiv, p. 20.

3 Osborne, Journal American Chemical Society, 1895, xvn, p. 539.

THE PROTEINS OF THE WHEAT KERNEL.

Having determined the composition of this globulin-like protein and also
that of the albumin, it became clear that several preparations obtained from
the aqueous extract were mixtures of these two substances, thus showing
the globulin to be present to some extent in the aqueous extract.

As noted on page 24, when 2000 cc. of an aqueous extract of about 650
grams of the meal were dialyzed in running water for 4 days a dense tur-
bidity was formed, which could not be removed by filtration. This, however,
on adding a little hydrochloric acid, was converted into a precipitate, which
was readily dissolved by adding sodium chloride sufficient to make a 3 per
cent solution, and was precipitated from this solution by dialysis.

Preparation 38, weighing 9 grams, was thus obtained, which, dried at 1 io°,
had a composition which corresponds pretty nearly with that of a mixture of
60 per cent of the globulin with 40 per cent of leucosin, except that the
amount of sulphur found was somewhat greater than that calculated. The
analysis is shown in the following table :

Preparations 38 and jg.

Preparation 38.

Preparation 39.

II.

Arer-
age.

Corrected
for ash
and nu-
cleic acid.

Calculated
for 60 per
cent globu-
lin and 40
per cent
leucosin.

Corrected
for ash
and nu-
cleic acid.

Calculated
for 40 per

cent globu-
lin aud 60
per cent
leucosin.

Phosphorus ....

P. ct.

48.30
6.49

17.40
0.83
1. 91

P.ct.
47.92

6.41
17.24

0.85

P.ct.
48.11

6.45

17.32

0.84

1. 91

P. ct.
5I-70
7.07

17-74
1.08

P.ct.

51-95

7.07

17-74
0.91

P.ct.

49-49

6.81

16.87

0-93
0.89

4.00
2.01

P.ct.
5I-80

7.14
17.32

1. 14

P. ct.
52.13

7-03
17.30

I.05

22.51

22.53

22.6o

22.49

Ash

3-95
2-95

P205 in ash

100.00

IOO.OO

After heating another portion of the same aqueous extract to 65 ° for some
time and filtering off the coagulum, the filtrate was dialyzed for 5 days into
alcohol and the precipitate thereby produced filtered out and exhausted with
water. The residue of protein matter coagulated by alcohol, weighing 6.7
grams and marked preparation 39, was then dried at no° and analyzed with
the results shown in the preceding table.

This analysis corresponds quite nearly with that of a mixture of 40 per
cent of the globulin with 60 per cent of leucosin.

EXPERIMENTAL. 45

No earlier analyses of the wheat globulin are on record. Weyl1 was the
first to call attention to the presence of globulin in wheat, and says that
" besides vegetable-vitellin I detected in the 10 per cent sodium-chloride
extract of the pulverized seeds of wheat, peas, oats, white mustard, and
sweet almonds a second globulin substance. ' ' This he calls vegetable- myosin
and gives its coagulation-point at 55 ° to 6o°. It is probable that Weyl in
some way mistook for a globulin the albumin already described.

Later, Weyl & Bischoff 2 state :

On investigating the proteins of wheat, one of us found chiefly an albuminous sub-
stance which, on account of its resemblance to myosin, was named vegetable-myosin.
This vegetable-myosin must be the mother-substance of the gluten, since in wheat meal,
together with it, other nitrogenous substances exist, at the most, only in very small
amount.

On what experimental evidence this statement rests the writer has been
unable to discover, and in view of his experience he is at a loss to under-
stand it.

Martin 3 considers wheat flour to contain a large amount of globulin of the
myosin type, coagulating between 55 ° and 6o°, precipitated by saturation
with sodium chloride and ammonium sulphate. Here again the small
quantity of albumin contained in the flour has evidently been mistaken
for a large quantity of vegetable-myosin. This perhaps is not surprising,
as the precipitates obtained by saturating sodium-chloride extracts with
ammonium sulphate appear very bulky, and in the absence of an actual
determination of the weight of these precipitates misleading conclusions
might easily be reached. The only globulin found by the writer in extracts
of wheat meal, either winter or spring wheat, is the one just described,
which in properties and composition closely resembles those globulins which
have been found in other seeds.

THE PROTEOSE OF WHEAT FLOUR.

As already stated in describing the reaction of the extract freed from
globulin by dialysis, there was found in it one or more proteoses, besides
the albumin just described. These were almost wholly precipitated by
saturation with sodium chloride or by adding 20 per cent of this salt to the
solution, together with a little acetic acid.

If the albumin is completely removed by heat and the filtered solution
then concentrated, a coagulum gradually develops. This substance must
be derived from the proteose-like protein, as this forms nearly if not quite
all the protein substance remaining in solution before concentration. If

1 Weyl, Zeitschrift fur physiologische Chemie, 1877, 1, p. 72.

2 Weyl & Bischoff, Berichte der deutschen chemischen Gesellschaft, 1880, xiii, p. 367.

3 Martin, British Medical Journal, 18S6, 11, p. 104.

the; proteins of the wheat kernel.

the coagulum is removed b)' concentration and long-continued heating and
subsequent filtration, wholly uncoagulable proteose-like substances are
found in solution. The amount of proteose is extremely small, and no
preparations were made for analysis. The coagula obtained by concen-
trating the solution filtered from preparations 3 and 4, respectively, were
analyzed with the results shown in the accompanying table under the head
" Preparation 40."

Another coagulum similarly obtained from the solution filtered from prep-
aration 4 was also analyzed and the following figures obtained :

Preparations 40 and 41.

Preparation 40.

Preparation 41.

II.

Average.

Ash-free.

II.

Average.

Ash-free.

Carbon

Hydrogen . . .

P.ct.

51.35

P.ct.

51.60

P. ct.

51.48

P.ct.
51.62

P.ct.

51-73

6.82

17.29

I....

P. ct.

17.26

P. ct.

51-73
6. 82

17.28

P.ct.
51-86
6.82
17.32

24.00

Nitrogen ....
Sulphur

16.79

16.58

16.69

16.73

Oxygen

J
0.25

0.25

Ash

0.27

100.00

the proteose op the wheat embryo.

In making the preparations from the embryo already described consider-
able quantities of crude proteose were obtained from both the aqueous and
sodium-chloride extracts. After the leucosiu and the globulin had been
separated as completely as possible, the solutions containing the proteoses
were dialyzed in alcohol, and the precipitates produced were washed and
dried over sulphuric acid.

A mixture weighing 15.4 grams was made by uniting several such prep-
arations that had been obtained from aqueous extracts from which most of
the other proteins had been separated, without heat, by saturating with
sodium chloride and dialysis in alcohol. The mixture contained much
matter made insoluble in water by the final treatment with alcohol. This
was filtered out, washed thoroughly with water and with alcohol, and when
dried weighed 4.18 grams, and was marked preparation 42. The filtrate
from this was saturated with ammonium sulphate, the precipitate redissolved?
and again precipitated in the same way. The solution of the second pre-
cipitate was dialyzed in cold distilled water until free from sulphate, and then
for several days in alcohol ; the precipitate thus produced was dissolved in
water, a little insoluble matter filtered out, and its clear solution saturated

EXPERIMENTAL. 47

with sodium chloride, which produced a small precipitate. This was filtered
out, dissolved, and its solution dialyzed in water. The salt-saturated filtrate
was likewise dialyzed, and when both solutions were free from chlorine the
dialyzers were transferred to alcohol and the proteose thereby precipitated.
The proteose separating on saturation with salt gave o. 6 gram of prepara-
tion 43 ; that from the salt-saturated solution 0.97 gram of preparation 44.
This small yield of proteose indicates that the greater part had diffused
through the parchment paper during the long dialysis to which the solutions
had been subjected.

Another crude product was obtained by dialyzing an aqueous extract in
alcohol after separating the leucosin which had been coagulated by heat.
This, weighing 35 grams, was dissolved in water and the insoluble matter
filtered out, washed and dried, giving preparation 45, weighing 7.26 grams.

The filtered solution was saturated with ammonium sulphate, the pre-
cipitate dissolved in water, and the clear solution dialyzed in distilled water
until free from sulphates, and then in alcohol. The substance thus sep-
arated was again dissolved in water and its solution saturated with salt ; the
precipitate thus produced was dissolved in water, and its solution, as well as
the salt-saturated filtrate, were dialyzed in water. When free from chlorine,
these solutions were dialyzed in alcohol and yielded, respectively, prepara-
tions 46, weighing 4 grams, and 47, weighing 1.84 grams.

Another preparation of crude proteose was obtained by extracting the
meal as described on page 41 with 3 per cent sodium-chloride solution heated
to 700, dialyzing the extract in water, coagulating the leucosin by heat, and
precipitating the proteose by dialysis in alcohol. A mixture of such prep-
arations, weighing 31.6 grams, was treated with water, the insoluble matter
filtered out, washed, and dried, giving 5.16 grams of preparation 48.

The filtered solution was saturated with ammonium sulphate, the precipi-
tate dissolved in water, the solution dialyzed in distilled water till free from
sulphate, and then in alcohol. The separated proteose was redissolved in
water and its solution saturated with sodium chloride. The precipitate
which resulted was filtered out, dissolved in water, and its solution, as well
as the salt-saturated filtrate, were dialyzed in water till free from chlorine,
and finally in alcohol.

The products thus obtained formed, respectively, preparation 49, weigh-
ing 0.75 gram, and 50, weighing 1.35 grams. One other proteose prepara-
tion was made from the aqueous extract described on page 26, from which
the phosphorus was largely separated by making it slightly alkaline and
adding calcium chloride. After heating the extract to boiling and filtering
out the coagulum, the filtrate was dialyzed in alcohol, the resulting pre-
cipitate dehydrated with absolute alcohol, dried over sulphuric acid, redis-
solved in water, and precipitated by saturating with ammonium sulphate.

THE PROTEINS OF THE WHEAT KERNEL.

The gumm)' precipitate, having the general appearance and properties of
similar precipitates of the proteoses obtained by the action of pepsin, was
dissolved in water, dialyzed free from sulphates, and then precipitated by
dialysis in alcohol, giving 2 grams of preparation 51. These preparations
were dried at no0 and analyzed with the results shown in table 10.

Tabi^e 10.-

—Composition

of alcohol coagula and of proteose prepared from
the wheat embryo.

Residues of other pro-
teins coagulated by
alcohol.

Proteose precipitated
by sodium chloride.

Proteose soluble in saturated
NaCl solution.

42.

45-

48.

43-

46. 49-

44-

47-

5°-

5i-

Carbon

Hydrogen

Nitrogen

Ash

P. ct.

52.36

6.98

16.01

I.85
22.80

P.ct.
49-44

6.85
16.00

4.08
23-63

P. ct.

5i 93
6.87

16.30
1.30

23.60

100.00
o.95

P.ct.
16.79

P.ct.

49-94
6.80

17.08
1.24

24.94

P.ct.
16.26

P.ct.
48.46
6.70
16.91

} 27.93

P.ct.
48.70

6-73
16.76

27.81

P.ct.

48.44

6.71

16.16

28.6c

P. ct.

48.99

6.85

16.89

f 1. 10

I 26.17

IOO.00
O.Sl

100.00
14-13

100.00

0. t,o\ 0.77

IOO.OO
i-i3

100.00
1. 00

100.00
0.74

100.00
1.27

From these analyses it is seen that the matter insoluble in water, forming
preparations 42, 45, and 48, consists of coagulated protein apparently mostly
derived from leucosin. The high proportion of sulphur in 42 and 45 is due
to calcium sulphate precipitated by alcohol from the aqueous extract. The
remaining preparations have the low percentage of carbon characteristic of
proteoses made by pepsin digestion.

THE PROPORTIONS OF THE VARIOUS PROTEIN SUBSTANCES OF

THE WHEAT EMBRYO.

Twenty grams of fresh germ meal, from which the ether-soluble constit-
uents had not been separated, were treated with 500 cc. of water, and after
shaking for some time the extract was filtered clear. Two portions of 100
cc. each were treated with a few drops of very dilute hydrochloric acid and
heated in a boiling water-bath. The coagulum which separated was col-
lected on a filter and its nitrogen determined. To the filtrate from one
coagulum tannin was added, and nitrogen was determined both in the pre-
cipitate and in the filtrate. Another lot of 20 grams was treated in the same
way and nitrogen determined in the heat-coagulum formed in each of two
portions of 100 cc. The amount of nitrogen corresponding to 1 gram of
germ meal was found in the four coagula to be 0.0163 gram, 0.0156 gram,
0.0159 gram, and 0.0162 gram, in the tannin precipitate 0.0062 gram, and in
the solution filtered from the latter 0.0062 gram.

EXPERIMENTAL. 49

Twenty grams of germ meal were extracted with 500 cc. of 3 per cent
sodium-chloride solution heated to 700, whereby the leucosin was coagu-
lated and the globulin and proteose dissolved. Of the clear filtered extract
100 cc. yielded with tannin a precipitate containing 0.0166 gram nitrogen
per gram of meal extracted.

Two portions of the meal, each of 1 gram, were exhausted with 3 per
cent sodium-chloride solution heated to yo° and nitrogen determined in the
residues. The 0.0331 gram and 0.0309 gram of nitrogen found in the resi-
dues were from the leucosin and insoluble nitrogenous bodies, so that the
nitrogen belonging to the latter equaled 0.0171 gram and 0.0149 gram.
From the average of these figures we find the following amounts of the
different forms of nitrogen in 1 gram of the wheat-germ meal :

N, grains.

Insoluble in water and salt solution 0160

Insoluble in water, but soluble in salt solution (globulin nitrogen) . . .0100

Soluble in water and coagulable by heat (albumin nitrogen) 0160

Soluble in water, uncoagulable by heat, precipitable by tannin (pro-
teose nitrogen) 0050

Not precipitable by tannin (non-protein nitrogen) 0060

Total 0530

Found by direct nitrogen determination 0531

It has been shown that the coagulated leucosin preparations contain about
10 per cent of nucleic acid, the globulin about 15 per cent, while those of
the proteose contain none. Deducting these quantities from the nitrogen
given above, it is found that 9.5 percent of the embryo is leucosin, 4.84 per
cent globulin, and 3.03 per cent proteose.

The bodies which are represented by the insoluble nitrogen could not be
separated from the embryo. The residue, after extraction with hot-salt
solution, contained 0.0076 gram of phosphorus. In view of the large
proportion of nucleic acid found in the extracts of the embryo, it is not
improbable that this phosphorus mostly belongs to nucleic acid, and that the
insoluble nitrogen largely belongs to compounds of protein with relatively
much nucleic acid.

DIGKSTION OF THE PHOSPHORUS-CONTAINING PROTEIN PREPARATIONS
WITH PEPSIN-HYDROCHEORIC ACID.

IvEucosin Nucleate.

Ten grams of the coagulated albumin, preparation S, were suspended in
400 cc. of water and dissolved by adding 100 cc. of decinormal potassium-
hydroxide solution. To the nearly clear solution which resulted an equal
volume of 0.4 per cent hydrochloric acid was added, together with some
pepsin, and the mixture digested at 37 °. In a short time the solution
4

50 THE PROTEINS OF THE WHEAT KERNEL.

became perfectly clear, but later deposited a large coherent precipitate, which
gradually contracted, but at the same time retained the form of the lower
part of the beaker. From this the clear solution was decanted, the precip-
itate thoroughly washed by decantatiou, suspended in water and dissolved
by adding 28 cc. of decinormal potassium-hydroxide solution, an amount of
alkali just sufficient to dissolve all the substance, and at the same time make
the solution neutral to litmus. When to this solution decinormal acid was
gradually added, no precipitate appeared until nearly one-half the quantity
of acid required for complete neutralization had been added, but with 28 cc.
the solution was neutralized and the nuclein completely precipitated, the
addition of 2 cc. more acid giving no turbidity in the filtered solution. This
precipitate formed preparation 52, weighing 1.54 grams.

To precipitate this substance a quantity of acid was added exceeding that
of the alkali employed for solution by just 2 cc. The filtrate from the pre-
cipitate, however, required not 2 cc. of alkali, but 8.5 cc. for neutralization
to phenolphthalein, showing 6.5 cc. of alkali to have been neutralized by
the acid of the nuclein originally dissolved. The neutralized filtrate left on
evaporation 0.3975 gram of substance, the aqueous solution of which was
precipitated by hydrochloric or nitric acid, but not by ammonium molybdate
solution until after boiling with acid for some little time, when yellow phos-
phomolybdate was precipitated. These facts indicate the presence in this
filtrate of a nucleic acid.

More nuclein was made from the same preparation, 7, by suspending 30
grams in 0.2 per cent hydrochloric acid containing pepsin, which, even at
200, caused within 2 hours complete solution of the coagulated protein.
The solution was digested at 37 ° for 48 hours, during which time much
nuclein separated, having the appearance and properties of the preparation
just described.

After decanting the clear solution and thoroughly washing the residual
nuclein, the latter was suspended in water and dissolved in 72 cc. decinor-
mal potassium-hydroxide solution. The solution thus obtained was made
neutral to litmus by adding 11 cc. of decinormal hydrochloric acid, but no
precipitate appeared till 1.5 cc more of acid were added. To the solution
72 cc. decinormal hydrochloric acid were added, giving a precipitate, prepa-
ration 53, which weighed 3.4 grams. The filtrate from this precipitate, as
in the former case, was strongly acid, requiring 12 cc. of decinormal potas-
sium hydroxide to neutralize it to phenolphthalein. Two other preparations
of nuclein were made from 8.493 grams of 13 and 9.804 grams of 16, both
being substances precipitated from the aqueous extract by saturating with
sodium chloride. Each portion was suspended in about 300 cc. of 0.2 per
cent hydrochloric acid, containing 0.1 gram of pepsin, and, with frequent

EXPERIMENTAL. 51

stirring, digested at 400 for 24 hours. Throughout the digestion a large
part of the substance remained undissolved. An equal volume of 0.2 per
cent hydrochloric acid, containing o. 1 gram of pepsin, was again added to
each, and the digestion continued for 24 hours longer. The insoluble matter
which remained was not coherent like the two former nuclein products, but
consisted of a white, very finely divided substance, which was easily filtered
out and washed. From 13, 4.04 grams of preparation 54 were obtained,
and from 16, 4.16 grams of 55.

Globulin Nucleate.

Fifteen grams of a mixture of nearly equal parts of the globulin prepa-
rations 32 and 33 were next suspended in 0.2 per cent hydrochloric acid
containing 0.2 gram of pepsin, which, within a short time, almost completely
dissolved the protein matter. From this solution, on further digestion, the
nuclein separated, forming a coherent deposit. After 72 hours' digestion
the clear solution was decanted, the deposit dissolved in a little ammonia, and
its solution filtered perfectly clear from a very slight gelatinous residue.
The solution was then treated with acetic acid added in excess of the
amount necessary to neutralize it to litmus. Since, even on standing, the
precipitate so produced separated imperfectly, an equal volume of alcohol
was added. The substance, which then separated well, gave 2.38 grams of
preparation 56, or about 16 per cent of the original substance.

The filtrate from the acetic acid precipitate, on adding hydrochloric acid,
gave a further slight precipitate, which had properties characteristic of
nucleic acid.

Still another preparation of nuclein was made from the globulin by sus-
pending 10 grams of 36 in water and adding 50 cc. of decinormal potassium-
hydroxide solution. This solution was neutralized and an equal volume of
0.4 per cent hydrochloric acid at once added, producing a turbid solution,
which, however, contained no visible particles. To this pepsin was added
and the mixture digested for 40 hours, during which time a coherent deposit
of nuclein formed on the bottom of the beaker. From this the clear solution
was decanted. The deposit was then thoroughly washed with water and
dissolved in 43 cc. of decinormal potassium-hydroxide solution. To this
clear solution 43 cc. of decinormal hydrochloric acid were added, causing a
gummy precipitate, which could not be filtered until 15 cc. more acid had
been added, when the precipitate rapidly settled as a coherent deposit, from
which the solution was soon decanted. This solution required for neutrali-
zation to litmus 16 cc. of decinormal potassium-hydroxide solution, and to
phenolphthalein 18 cc. The precipitate when washed and dried gave 2.2
grams of preparation 57.

THE TROTEINS OF THE WHEAT KERNEL.

These six preparations were all dried at i io° and analyzed with the results
shown in table n.

Table ii. — Composition of nuclein from the proteins of the wheat embryo.

Carbon

Hydrogen.. .
Nitrogen.. . .

Sulphur

Phosphorus .

Ash

P205 in ash. .
Bases in ash.

52.

53-

54-

55-

56.

57-

P. ct.

P.ct.

P. ct.

P.ct.

44.87

44-35

42.68

43-35

39-42

41.92

5.82

5-77

5-45

5-47

5-03

5-25

16.04

16.64

16.12

16.01

16.05

17.00

0.97

1.03

0.65

0.85

o-53

0.46

4.58

5-07

5-32

4.88

5-27

5.63

0.60

0.78

1.72

17.42

1. 17

0.29

o.55

1.24

0.94

10.56

0.69

0.31

0.23

0.48

0.78

6.86

0.48

These analyses of nuclein were calculated free from the bases of the ash
and from nucleic acid, in the way previously described, with the results given
in table 12.

Table 12. — Composition of protein matter contained in the nuclein.

Carbon . . .
Hydrogen
Nitrogen. .
Sulphur.. .
Oxygen. . .

52.

53-

54-

55-

56.

57-

P.ct.

P. ct.

P.ct.

P. ct.

53.65

54-77

5l-8o

52.36

52 30

51.64

7.23

7.46

6.S5

6.73

6.91

6.60

16.68

17-56

16.31

19.31

18.93

1.98

2-37

1. 61

1.89

1-53

125

20.46

17.84

23-43

22.71

19-95

21.58

100.00

The composition of the protein matter in 54 and 55 is very nearly that of
leucosin, except as regards sulphur, the amount of which is decidedly greater.
On the other hand, 52 and 53, which also were derived from preparations
whose protein matter was leucosin, differ in composition very decidedly from
that substance. This is probably because on pepsin digestion the substance
of preparations 54 and 55 remained throughout undissolved, whereas 52 and
53 separated on pepsin digestion from nearly clear solutions, and therefore
doubtless their protein matter had been to some degree altered by the pepsin
before separating as an insoluble compound with nucleic acid. The two
nucleins, 56 and 57, from the globulin, which also had separated from
solution, show similar differences in composition when compared with the
unaltered globulin, carbon and nitrogen being higher and sulphur very much
higher than in the globulin.

EXPERIMENTAL.

PROTEIN SOLUBLE IN DILUTE ALCOHOL — GLIADIN.

As already stated, wheat flour yields to dilute alcohol a considerable
amount of protein matter. Treatment of the residue remaining after ex-
tracting the flour with ioper cent sodium-chloride brine likewise removes a
large amount of protein, as does also extraction of the gluten obtained by
washing the dough with water. Extracts were made with alcohol under all
these conditions, and the protein extracted subjected to repeated fractional

precipitations.

Direct Extraction with Dilute Alcohol.

5000 grams of the straight flour were extracted with 10 liters of alcohol,
0.90 sp. gr., and allowed to soak over night. The next morning the mixture
was stirred, and, after settling, the clear solution poured off. Three liters
more of alcohol, 0.90 sp. gr., were then added, and after standing some time
the supernatant liquid was decanted and the residue squeezed nearly dry.
The solution so obtained was designated "extract 1." The residue was
again treated with 4 liters of 0.90 sp. gr. alcohol and pressed nearly dry.
This formed extract 2. The same process twice repeated gave two extracts
which, when united, formed extract 3. Each of these three extracts, after
filtering perfectly clear, was separately concentrated to one-third its volume,
and, after cooling, decanted from the very glutinous, viscid mass which had
separated. 1 and 3 yielded much more substance than 2. On stirring with
a glass rod the precipitated mass formed a very thick, viscid liquid. This
substance was in each case dissolved in a small amount of hot alcohol of 0.90
sp. gr. , in which it was very soluble, and the solution was allowed to cool
over night. Most of the substance separated on cooling and the liquid was
decanted from it. The solution decanted from the second and third extracts
was treated with a quantity of distilled water and a little sodium chloride
added. This threw down a small precipitate, which on standing collected
on the bottom of the vessel as a clear semi-fluid mass. This was treated
with water, absolute alcohol, and ether, and yielded 7.27 grams of prepara-
tion 58 from extract 2, and 10.7 grams of the preparation 59 from extract 3.
These had the following compositions :

Preparations 58 and 50.

Preparation 58.

Preparation 59.

Ash-free.

Nitrogen

P. Ct.

17-05
0.76

P. ct.
17.18

P. ct.

17-15
0.65

P. ct.
17.26

Ash

the; proteins op the; wheat kernel.

On examination both were found to contain some fat which could not be
wholly removed, as the substance had dried in a dense, horny form. The
residues which had separated from the solutions just described were next
washed by thoroughly intermixing with distilled water. The water was
found to dissolve some of the protein, which was subsequently precipitated
by the addition of a little sodium-chloride solution. After standing over
night this precipitate settled to the bottom of the vessel in a transparent
layer, from which the solution could be completely decanted. After treating
this substance with absolute alcohol, it formed a voluminous white, porous
mass, which was digested for some time with ether. From extract i
preparation 60 was obtained, weighing 12.4 grams ; from extract 2, united
with that from 3, preparation 61, weighing 8.6 grams.

These preparations were found on analysis to have the following compo-
sitions :

Preparations 60 and 61.

Preparation 60.

Preparation 61.

II.

Average.

Ash-free.

Carbon
Hydrogen . .
Nitrogen . . .
Sulphur ....

P. ct.
52.40

6.77
I7.52

1.05

P.ct.

52.58
6.78

17.73

I. II

P. Ct.

52.49
6. 78

17-63
1.08

P.ct.

52.52
6.78

17.64
1.08

21.98

P.ct.

52.69

6.77

17.74

1.26

0.15

P. ct.

52-17
6.78

17.77
1.26

21.42

100.00

Ash

0.06

100.00

The residues remaining after washing with distilled water were then
digested with alcohol of 0.820 sp. gr. , which dissolved much of the
substance. After standing some time, the strong alcoholic solutions were
decanted from the residues and found to consist of milky liquids. The
addition of a few drops of 10 per cent sodium-chloride solution immediately
produced in each a very large, curdy precipitate, the liquids from which
they separated being left perfectly clear. From the solution from the first
extract, which was about one liter in volume, 32.26 grams of substance
were obtained after dehydration with absolute alcohol and digestion with
ether. This was marked preparation 62. From the second extract was
similarly obtained preparation 63, weighing 5.34 grams, and from the third
extract preparation 64, weighing 17.43 grams. The filtrates from 62 and
63 were found to be almost wholly free from protein, but that from 64 left
on evaporation a residue which, when freed from fat, weighed 7.53 grams,
preparation 65.

EXPERIMENTAL.

These preparations were found to have the following compositions :

Preparations 62 and 63.

Preparation 62.

Preparation 63.

Ash-free.

II.

Average.

Ash-free.

Hydrogen . .
Nitrogen . . .
Sulphur , , ,
Oxygen ....

P. ct.

52.59
6.70

17.64

1.22

P.ct.
52.67

6.71
17.66

1.22
21.74

P. ct.

52.28

6.87

17.90

1. 21

P.ct.

52.54
6.79

P. Ct.

52.41
6.83

17.90
1. 21

P. Ct.

52.55
6.85

17.94
1. 21

21.45

Ash

O.I5

O.27

100.00

Preparations 64 and 65.

Preparation 64.

Preparation 65.

II.

Average.

Ash-free.

II.

Average.

Ash-free.

Carbon

Hydrogen . .
Nitrogen . . .
Sulphur ....
Oxygen ....

P. ct.
52.52

6.72
17.60

1.23

P. ct.

52.82
6.79

P.ct.
52.67

6.76
17.60

1.23

P. ct.

52.74
6.77

17.62
1.23

21.64

P. ct.
52.13

6-97
17-35

i-35

P. ct.

52.42

6.85

17.19
1.41

P.ct.
52.28

6.91
17.27

1.38

P. ct.
52.39

6-93
I7.3I

1.38
21.99

Ash

0.14

0.22

100.00

The residues which remained after treatment with alcohol of 0.820 sp. gr.
were then dehydrated with absolute alcohol and digested with ether. From
extract 1 preparation 66 was obtained, weighing 63 grams ; from extract
2 preparation 67, weighing 2.1 grams, and from extract 3 preparation 68,
weighing 41.2 grams.

Preparation 66, which constituted the principal fraction of the protein
extracted, was further treated in the following manner : 20 grams were
dissolved in 250 cc. of 0.90 sp. gr. alcohol and found to yield a clear solu-
tion, which was then poured into 800 cc. of absolute alcohol, whereby a
considerable precipitate was at once separated, leaving the solution milky.
This substance was dehydrated with absolute alcohol and digested with
ether, yielding preparation 69. The filtrate was then treated with a few
drops of 10 per cent sodium-chloride solution, causing a heavy precipitate,
which on stirring rapidly agglutinated and adhered as a mass to the stirring-

Tin: PROTEINS OE THE WHEAT Ki'.RXEE.

rod. This was removed, treated in the usual manner, and marked prepara-
tion 70. The mother- liquor from which this separated, after standing over
night, deposited a further small amount of protein, which, after treatment
with absolute alcohol and ether, gave preparation 71.

These six substances were analyzed and found to have the following
compositions :

Preparations 66 to 7/.

Preparation 66.

Preparation 67.

Ash-free.

II.

Average.

Ash-free.

Carbon

Hydrogen . . .
Nitrogen ....

Sulphur

Oxygen

P. Ct.

52.81
6.81

17.66
1. 11

P. ct.

52.S4

6.S1

17.67

1. 11

21-57

P. ct.

Pet.

P. ct.

15-42

15-28

15-35

15-42

Ash

0.06

0.42

100.00

Preparation 68.

Preparation 69.

II.

Average.

Ash-free.

Carbon

Hydrogen. . .
Nitrogen ....

Sulphur

Oxvsren

P. ct.

52.90
6.74

17.24
1.05

P. ct.

52.99
6.82

17.36

P. ct.

52.95

6.78

17.30

1.05

Pet.
53-02
6.79

17-32

1.05

21. S2

P. ct.

17.67

17.69

Ash

0.15

0. 10

100.00

Preparation 70.

Preparation 71.

II.

Average.

Ash-free.

Carbon

Hydrogen . . .
Nitrogen . .
Sulphur

Ash

P. Ct.

52.15

6-93

I7-52

P. ct.

52.35
6.87

17.84

P. Ct.

52.25

6.90

17.68

P. ct.

52.33

6.91

17.70

23.06

P. ct.
52.2S
7.12
17.79

0.19

Pet.

52.33

7-13
17.82

22.67

0.15

I CO. 00

100.00

EXPERIMENTAL.

If the preceding analyses are brought together as in table 13, the effect
of the various fractional solutions and precipitations may be seen at a glance.

Tabi^e 13. — Gliadin extracted by direct treatment of the flour with alcohol.

From 0.9 sp. gr. al-
coholic solution.

From water wash-
ings.

From 0.82 sp. gr. alcoholic
solution.

53.

59-

60.

61.

62.

63.

64.

P. ct.

P. ct.
52.52

6.78
17.64

I.08
21.98

IOO.OO

12.40

P. ct.

52.77
6.78

17.77
1.26

21.42

P. ct.
52.67
6.70
17.66
1.22
21-75

P. ct.

52.55
6.S5

17.94

I. 21
21-45

P. ct.

52.74
6.77

17.62

1.23

21.64

Nitrogen

17. iS

17.26

Weight of sub-
stance in grams.

100.00

8.60

IOO.OO
32.26

IOO.OO
5-34

100.00

1743

7.27

10.70

From

nitrate
from 64.

Residue after extraction with
0.820 sp. gr. alcohol.

Fractional reprecipitations
of preparation 66.

65.

66.

67.

68.

69.

70.

7i-

Carbon

P. ct.
52.39

5-93
I7.3I

1.38
21.99

P. ct.
52.82

6.8l
17.67

I. II
21.57

Pet.
15.42

Pet.
53-o2

6.79
17-32

1.05
21.82

P. ct.
17.69

P. Ct.

52.33

6.91

17.70
1 23.06

P. ct.

52.38

7.13
17.82

22.67

Hydrogen

Oxveen

Weight of sub-
stance in grams.

IOO.OO
7-53

IOO.OO
63.O

2.IO

100.00
41.20

100.00

It is evident that 58 and 59 contain less nitrogen than the great bulk of
the protein extracted. This is due to fat which they were found to contain
and which could not be wholly removed by extraction with ether, since in
drying these preparations were converted into the horny condition which
rendered penetration with ether impossible.

Preparation 67 was evidently impure, as might be expected, since it con-
tained all of the insoluble particles of the entire extract which had escaped
nitration, and owing to its small amount these impurities produced a marked
effect on its percentage composition. The same is true of 68, but as the
quantity of this preparation is so much greater this contamination has pro-
duced much less effect on its composition. Preparation 65 was obtained by
evaporating the mother-liquor from 64 nearly to dryness and then extracting
with absolute alcohol and ether. It would hardly be expected that under

the; proteins of the wheat kernel.

such circumstances it would be entirely pure. The analyses of the other
preparations are in good agreement, and it is evident that no fractional
separation of the extracted protein is indicated. The preparations obtained
from solution in pure water have the same composition as those from solu-
tions in alcohol of 0.820 sp. gr., and also the same composition as the
residue remaining after treatment with each of these reagents.

The total amount of protein contained in these several preparations is
207.83 grams, being equal to 4.16 per cent of the flour.

Table 14. — Gliadin extracted from flour zvitk dilute alcohol after extraction with

10 per cent sodium-chloride solution.

Preparation 72.

Preparation 73.

II.

Average.

Ash-free.

II.

Average.

Ash-free.

P. ct.
52.72

6.86
17.89

o-95
21.58

Hydrogen . .
Nitrogen.. . .
Sulphur ....
Oxveren ....

P.ct.
52.61

6.82
17.62

1. 00

P.ct.

17.78
I.04

P.ct.
52.61

6.82
17.70

1.02

P. Ct.

52.69
6.84

17.73

1.02

21.72

P.ct.
52.65

6.85
17.87

o-95

P.ct.
0.94

P.ct.
52.65

6.85
17.87

o.95

Ash

0.16

0.13

100.00

Preparation 74.

Preparation 75.

Preparation 76.

II.

Average.

Ash-free.

Hydrogen . .
Nitrogen.. . .
Sulphur ....

P.ct.
52.52

6.82
17.72

1. 10

P.ct.

52.67
6.76

P. ct.
52.60

6.79
17.72

1. 10

P. ct.

52.71
6.81

17-75

1. 10

21.63

P. ct.

P. ct.
52.62

6.83
17.78

1.08

P.ct.

52.65

6. S3

17.79

1.08

21.65

16.93

17.08

Ash

0.21

0.91

0.05

100.00

Extraction with Dilute Alcohol After Extracting the Flour
with 10 per cent Sodium- Chloride Solution.

Four kilograms of "straight flour" were extracted with 10 per cent
sodium-chloride solution as long as anything was removed. After squeez-
ing as dry as possible in a screw-press, the residue was treated with alcohol
so as to yield with the water retained by the meal as nearly as possible a

EXPERIMENTAL. 59

solution containing 75 per cent of alcohol. After digesting 2 days with the
solvent, the extract was squeezed out in a press and the process repeated
three times. Four extracts were thus obtained. These were each concen-
trated to small volume, cooled, and the solution decanted from the precipi-
tated mass. This was then washed with distilled water. After removing
the salts, the substance from extracts 1 and 2 dissolved to some extent ;
that from extract 3 dissolved completely to a turbid solution. By adding
sodium chloride the dissolved protein was precipitated.

The residues remaining after washing with water were treated with abso-
lute alcohol and digested with ether. The precipitates obtained from the
water washings by adding salt were treated in the same way. From extract
1 preparation 72 was obtained, weighing 82 grams ; extract 2, preparation
73, weighing 57 grams ; from extract 3, after dissolving in water and
precipitating with sodium chloride, preparation 74, weighing 11.3 grams;
from extract 4 preparation 75, weighing only 1.35 grams, and from the
united water washings of 72 and 73 preparation 76, weighing 5.8 grams.
The total weight of these preparations was 157.45 grams, equal to 3.94 per
cent of the flour taken. Their composition is shown by the analyses given
in table 14 on the preceding page.

Extraction of Gluten with Dilute Alcohol.

Two kilograms of "straight spring- wheat flour " were made into a dough
with distilled water of 200, and then washed in a stream of river water of
50. After washing until nearly all the starch was removed, the gluten was
chopped up fine and digested with alcohol of 0.90 sp. gr. at a tempera-
ture of about 200. This extraction was continued with repeatedly re-
newed portions of alcohol of the same strength as long as anything was
removed. The extracts were united, filtered perfectly clear, and concen-
trated to about one-fourth their original volume. The residual solution
was then cooled and allowed to stand over night to deposit the separated
gliadin. The supernatant solution was poured off and the large amount
of protein which had separated was then dehydrated by treatment with
absolute alcohol. The decanted mother-liquor from which this protein had
separated, and also the strong alcoholic solution which resulted from dehy-
drating the precipitated mass, were each precipitated by adding a little
sodium-chloride solution. The three products thus obtained were united,
digested with fresh quantities of absolute alcohol in order to complete the
dehydration, and then extracted with absolute ether. Dried over sulphuric
acid, the preparation weighed 82 grams, and formed, therefore, 4.10 per cent
of the flour taken. Dried at no°, this substance had the composition
shown under the head " Preparation 77 " in the table on page 60.

THE PROTEINS OF THE WHEAT KERNEL-

Thirty grams of preparation 77 were then dissolved in alcohol of o. 90 sp. gr. ,
and the clear solution evaporated to small volume, cooled, and, as no protein
separated, strong alcohol was added until a considerable precipitate resulted,
equal to about one-half the dissolved protein. This precipitate, which, if
the extracted protein were a mixture, as stated by Ritthausen, would con-
tain the bulk of the substances insoluble in strong alcohol, weighed 12 grams.
This was marked preparation 78.

The solution from which this substance had separated must have con-
tained the chief part of the protein called by Ritthausen gluten-fibrin. It
was then concentrated to small volume, cooled, water added until a con-
siderable precipitate resulted, the solution then heated until all dissolved,
and, after cooling, the mother-liquor was decanted from the separated
protein. This process was repeated four times, and the precipitate finally
obtained dehydrated with absolute alcohol and digested with ether. This
preparation, 79, weighed 1.6 grams.

Preparations 77, 78, and 79.

Preparation 77.

Preparation 78.

Preparation 79.

Ash-free.

Carbon

Hydrogen . .
Nitrogen . . .
Sulphur ....
Oxygen ....

P.ct.

52.33
6.63

17.57
1.08

P.ct.
52.58
6.67

I7-65

1.08

22.02

P. ct.
52.82

6-77

17.62

1.09

0.19

P.ct.
52.68
6.78

17.65

1.09

21.80

P.ct.
52.82
7.18
17.57
1

0.04

P.ct.
52.84
7.18
17-57
22.41

100.00

Ash

0.50

100.00

It is clear from these analyses that no separation into proteins of differing
composition had thus been effected.

Extraction of "Shorts" with Dilute Alcohol.

Two kilograms of "shorts" from the spring-wheat flour were extracted
with alcohol of 0.90 sp. gr. and the extract squeezed out with a screw- press.
The extract, which was a deep red-brown in color, was filtered perfectly
clear, and then concentrated by distillation to about one-third. On cooling,
the protein separated, leaving the mother-liquor as a deep coffee-brown
liquid. This was decanted, the precipitate dissolved in alcohol of 0.90
sp. gr., and again precipitated by concentration and cooling; the strongly

EXPERIMENTAL.

colored mother-liquor was decanted and this process repeated. The pre-
cipitated protein was then again dissolved in a little dilute alcohol, and the
resulting solution poured into absolute alcohol, thereby precipitating the
greater part of the protein and leaving the alcohol strongly colored. The
precipitate was thus freed from a very considerable part of its coloring
matter. After digestion with absolute alcohol, and finally with ether, this
preparation, 80, was dried and analyzed with the result shown in the
table below :

Owing to the fact that this preparation, 80, was still contaminated with
coloring matter, and also showed slight differences in composition from the
protein extracted by similar treatment from the flour, it was subjected to
further treatment with a view to its more complete purification.

Apart of the preparation was dissolved in 150 cc. of alcohol of 0.90 sp. gr.
and the solution poured into 1000 cc. of absolute alcohol. This produced a
turbid liquid, which, on adding a drop or two of sodium-chloride solution,
gave a heavy precipitate that rapidly settled, leaving the alcohol colored
yellow. This precipitate was again dissolved in diluted alcohol, and pre-
cipitated by pouring into ether in order to remove anything soluble in this
liquid. No coloring matter was thus removed. The precipitate was then
digested with absolute alcohol, yielding preparation 81.

The strong alcoholic solution from which this preparation had separated
on longer standing deposited a small amount of substance which, when
dehydrated, yielded preparation 82. Analysis showed these two substances
to have the composition shown in the following table :

Preparations 80, 81, and 82.

Preparation 80.

Preparation 8

Preparation 82.

Ash-
free.

II.

Aver-
age.

Ash-
free.

II.

Aver-
age.

Ash-
free.

Carbon. . . .
Hydrogen .
Nitrogen . .
Sulphur. . .
Oxveren . . ,

P. ct.

52.75
6.96

17.22
I.36

P. ct.

53-25
7.02

17-38

1-37
20.98

P. Ct.

52.57
6.74

17.39

p. a.

52.52

6.80

P.ct.

52.55
6.77

!7-39

P. Ct.

52.85
6.81

17.48

22.S6

P. Ct.

52.43
6.79

17-59

P.ct.

52.56

6.88

P. Ct.

52.50
6.84

17.59

P. Ct.

52.74

6.87

17-67

22.72

J
o.54

0.46

Ash

0-95

100.00

These figures show that the protein extracted from the " shorts " has the
same composition as that similarly obtained from the flour.

62 THE PROTEINS OF THE WHEAT KERNEL.

Protein Extracted by Alcohol from Whole-wheat Flour.

As Ritthausen and probably others used whole-wheat flour in extracting
the various proteins described by them as soluble in dilute alcohol, it was
thought best to carry out some extractions with meal obtained by grinding
the entire wheat kernel in the laboratory.

Preparations 83-88.

Preparation S3.

Preparation 84.

II.

Aver-
age.

Ash-
free.

II.

Aver-
age.

Ash-
free.

Carbon

Hydrogen...
Nitrogen. . .
Sulphur. . . .
Oxygen ....

P. ct.
52.88

7.00
17-47

i-43

P.ct.

52.68
6.97

17.48

P.ct.
52.78

6.98
17.48

1-43

P.ct.
52.90

6.99

I7.52

i-43
21.16

P.ct.

52.59
6.87

17.92
0.92

P.ct.

52-73
6.80

18.06

P.ct.
52.66

6.84
17.99

0.92

P.ct.
52.89

6.S7
18.06

0.92
21.26

Ash

0.23

o.45

IOO.OO

100.00

Preparation 85.

Preparation 86.

II.

Average.

Ash-free.

P.ct.
51-03

6-59

16.98

0.92

P.ct.

50.93

6.50

17.08

P.ct.

50.98

6.55

17.03

0.92

P.ct.

53-i6

6. S3

17-75

0.96

21.30

P.ct.

52.64

6.S6
17-49

o.35

P.ct.
52.82

6.88
17-55
22.75

100.00

Hydrogen . .
Nitrogen . . .

Sulphur

Oxygen

Ash

4.11

4-cj

4.07

100.00

Preparation 87.

Preparation 88.

Ash- free.

II.

\verage.

Ash-free.

Carbon

P.ct.

52.66

6.80

17.62

P.ct.

52.68

6.81

17-63

22.88

P.ct.

52-03
6.66

17.48
f 1.0S

P.ct.

51-88

6.69

P.ct.

51-96
6.68

17.48
1.0S

P. ct.

52.24

6.71

17-57

1.0S

22.40

Hydrogen

Nitrogen ....

Sulphur

Oxygen ....

0.04

Ash ,

0.52

100.00

EXPERIMENTAL. 63

One kilogram of whole spring- wheat flour, freshly ground, was made into a
dough, and the gluten obtained from this by washing with water was then
chopped fine and thoroughly extracted with alcohol of 0.90 sp. gr., the
yellow extract concentrated, and the protein separated by cooling. The
deposit thus produced was dissolved as far as possible in dilute alcohol, and
the insoluble substance, which was coagulated protein, was washed with
dilute alcohol, absolute alcohol, and ether. This was preparation 83.

The solution filtered from 83 was poured into absolute alcohol and a small
amount of protein separated ; this was treated with absolute alcohol and
ether in the usual way, yielding preparation 84. The filtrate from 84 was
concentrated to small volume and poured into absolute alcohol, whereby
nearly all the protein was precipitated. This substance was dehydrated with
absolute alcohol and digested with ether, giving preparation 85. These three
bodies had the composition shown by the figures for preparations 83, 84,
and 85 in the table on page 62.

In a similar manner an extract was made of winter-wheat meal obtained
by grinding the entire wheat kernel in the laboratory, the alcoholic extract
concentrated to about one-third of its volume, cooled, and the solution
decanted from the deposit. This was then dissolved in alcohol of 0.90
specific gravity and the coagulated protein filtered off, washed with dilute
alcohol, digested with absolute alcohol and then with ether, giving prepara-
tion 86.

The solution filtered from this preparation was concentrated to small
volume, cooled, and the protein separated was digested with absolute alcohol
and with ether, yielding preparation 87. (See table on p. 62. ) The complete
extraction of this protein from the gluten is very difficult, a little generally
remaining in the insoluble residue after extracting with dilute alcohol. In
one case the residue thus remaining was dissolved in 0.2 per cent potassium-
hydroxide solution, and the resulting solution, after standing some time to
deposit suspended impurities, was decanted and precipitated with dilute
hydrochloric acid. This precipitate was washed by decantation with water
and then digested for some time with dilute alcohol. The alcoholic solution
was then filtered and concentrated to small volume and cooled. The protein
separated was then digested with absolute alcohol and with ether and dried
at 1 io° for analysis.

From this analysis it is seen that the protein soluble in dilute alcohol is
not changed in composition by solution with potassium hydroxide, nor is its
solubility altered, so far as could be learned.

In order to facilitate a comparison of these analyses they have been
brought together in table 15.

THE PROTEINS OF THE WHEAT KERNEL.
Table IS. — Composition of protein extracted by dilute alcohol.

By direct extrac-
tion

After extraction
with 10 per cent
sodium - chlo-
ride solution .

From gluten
spring - wheat
flour

From gluten
whole spring
wheat flour.

From gluten
whole winter
wheat flour. .

From whea
"shorts"....

Average of preceding
figures

Carbon.

P.ct.

52.52
52.77
52.67
52.55
52.74
52.39
52.84
53-02

52.33
52.38

52.69

52.72
52.71
52.65

52.58
52.68
52.84

52.90
52.89
53- 16
52.24

52.82
52.68

53-25
52.85
52.74

52.72

Hydrogen.

P.ct.

6.78
6.78
6.70
6.85
6-77
6-93
6.S1
6.79

6.91
7-13

6.84
6.86
6.81
6.83

6.67
6.7S
7.18

6.99
6.87
6.83
6.71

6.S8
6.S1

7.02
6.81
6.8-

6.86

Nitrogen.

Sulphur. Oxygen. Total.

P.ct.
17.18
17.26
17.64

17.77
17.66

17-94
17.62

17-31
17.67
17.32
17.69
17.70
17.82

17-73
17.89

17-75
17.79

17-65
17-65
17-57

17-52
18.06

17-75
17-57

17-55
17-63

I7-38
17.48
17.67

17.66

P.ct.

1.08
1.26
1.22
1. 21
I.23

1.38
I. II
I.05

P.ct.

21.98
21.42

21.75
21-45
21.64
21.99

21.57
21.82

23.06
22.67

1.02

0-95
1. 10
1.08

,08
.09

21.72
21.58
21.63
21.65

22.02
21.80

22.41

1-43
0.92

0.96
1.0S

21.16
21.26
21.30

22.40

22.75
22.8S

1-37

22.86
22.72

20.98

1. 14

21.62

P.ct.

100
100
loo
IOO
IOO

IOO
IOO
IOO

IOO
IOO

IOO
IOO
IOO
IOO

IOO
IOO
IOO

IOO
IOO
IOO
IOO

IOO
IOO

IOO
IOO
IOO

IOO

1 Omitted in average.

THE PROPORTION OP GLUTAMINIC ACID YIELDED BY VARIOUS FRACTIONS

OF THE ALCOHOL-SOLUBLE PROTEIN.

The results of this extensive fractionation of the alcohol-soluble protein
give no evidence, based on the ultimate composition of the many fractions,
of the presence of more than one protein substance. This, however, does
not justify the conclusion that only one such substance exists. In view of
the recent positive statements of Kossel & Kutscher and of Kbnig & Rintelen
it is important to obtain, if possible, further evidence based on the propor-
tion of some of the decomposition products yielded by the various fractions
of this protein. As gliadin yields a relatively large amount of glutaminic

EXPERIMENTAL. 65

acid when decomposed by boiling with strong hydrochloric acid, determina-
tions of the quantity of glutaminic acid obtained from various fractions
have been made.

The first difficulty encountered in examining the evidence that has been
offered respecting the existence of several alcohol-soluble proteins in wheat
flour lay in the impossibility of following Ritthausen's directions for pre-
paring these, since many evidently important details are omitted in the
description of his methods.

Kossel & Kutscher state that their products were made according to Ritt-
hausen's directions, but give no details, nor do they state which method
they employed. Kutscher concludes another paper by the statement that
' ' the wheat gluten consists of gluten-casein wholly insoluble in cold 60 per
cent alcohol; gluten-fibrin, but little soluble, and gliadin, easily soluble in
cold 60 per cent alcohol."

Although the writer has made a very large number of preparations repre-
senting fractions of this protein substance dissolved by alcohol of various
degrees of strength, he has never obtained any that were not either com-
pletely soluble in cold alcohol of 60 per cent by volume, or else contained
such insignificant quantities which did not dissolve that he has found it
impossible to make from them a preparation of ' ' gluten-fibrin ' ' suitable
for further examination. He has, therefore, been unable to repeat the
work of Kossel & Kutscher and is entirely at a loss to understand how their
preparation of " gluten-fibrin " was obtained.

Konig & Rintelen describe their procedure in more detail. These inves-
tigators extracted wheat gluten with absolute alcohol, added ether to the
alcoholic extract, and united the precipitate produced with the extracted
gluten. This latter was then extracted with 65 per cent alcohol, and to the
extract alcohol was added until the mixture contained 88 to 90 per cent of
alcohol.

After decanting from the precipitate that had formed, the solution was
filtered clear and evaporated to dryness on the water-bath, finely pulverized,
and extracted with ether to remove fat. As all of the fat could not be thus
removed, the mass was again dissolved in alcohol, to which some potassium
hydroxide was added, and this solution shaken out several times with ether.
The weakly alkaline solution was then exactly neutralized with hydrochloric
acid and evaporated on the water-bath. The product thus obtained was
their "gluten-fibrin."

The precipitate produced by 88 to 90 per cent alcohol was washed with
alcohol of the same strength and dissolved in a little 65 per cent alcohol.
From this one-half of the alcohol was distilled off and the residual solution
cooled, when a precipitate separated. From this the solution was decanted,
leaving a mass of gliadin. The solutions which remained from several such
5

66 THE PROTEINS OF THE WHEAT KERNEL.

precipitates were united and distilled until one-third of the solvent was
removed. On cooling the residual solution, a deposit formed which they
considered to be a mixture of gliadin and mucedin. The solution decanted
from this deposit was evaporated to dryness, and yielded a considerable
residue of " mucedin."

The analyses of these products showed that the gliadin thus prepared had
the same composition as that obtained by the writer, as well as that made
by Ritthausen, while the "gluten-fibrin" and mucedin contained about i
per cent less nitrogen and much more carbon.

Konig & Rintelen obtained their mucedin from the nearly aqueous solu-
tions remaining after separating the gliadin by evaporating to dryness. The
writer assumes that the residue which remained was subjected to some fur-
ther purification, but concerning this they say nothing. The solution con-
tains many impurities, and when the protein substance in it has been
properly purified it has the properties and composition of gliadin.

Although large quantities of gliadin have been made in this laboratory
and subjected to very careful and extensive fractionation, no evidence what-
ever of the existence of " mucedin " has been obtained.

Furthermore, it would seem improbable that our gliadin could be con-
taminated by " gluten-fibrin " and " mucedin," which the writer certainly
did not succeed in separating from it, and at the same time show so close
an agreement in composition with that of Konig & Rintelen, from which
they suppose that both of these proteins had been carefully removed. It is
also improbable that Kjeldahl should have found the specific rotation uniform
for numerous preparations of the alcohol-soluble protein if the material
which he examined was a mixture of three different substances ; nor, if this
were the case, could his determination of («)D — 92 ° be expected to agree

20°

so closely with that made by Osborne & Harris,1 («) -=: 92. 30, and by

Mathewson,2 («) -° 9i-95°-

The composition of both "gluten-fibrin" and "mucedin" differs from
that of gliadin just as one would expect if these former substances were
slightly altered, and somewhat impure products obtained from gliadin.

Until more convincing evidence of the existence of " gluten-fibrin " and
"mucedin" as distinct protein substances is brought forward, they can not
be considered to be original constituents of the wheat kernel.

Kutscher has stated that there is a wide difference iu the proportion of
glutaminic acid which is yielded by gliadin and gluten-fibrin. The writer

1 Osborne & Harris, Journal American Chemical Society, 1903, xxiv, p. 844.

2 Mathewson, Journal American Chemical Society, 1906, xxxviii, p. 1482.

EXPERIMENTAL. 67

has therefore determined the amount of this amino-acid which was yielded
by several preparations of the alcohol-soluble protein, one of which repre-
sented the fraction of the protein soluble in the strongest alcohol, and should
therefore, according to Ritthausen, consist chiefly of gluten-fibrin. The
amount of glutaminic acid in gliadin was first determined by boiling 100
grams of gliadin, equal to 93 grams moisture free, for 14 hours with 200 cc.
of concentrated hydrochloric acid. After standing on ice for 3 days, the
entire solution solidified to a thick mass of crystals, which was sucked out
with a pump and washed with ice-cold alcoholic hydrochloric acid. When
dried over sodium hydroxide, this crude glutaminic acid hydrochloride
weighed 58.33 grams. The filtrate and washings on concentration gave by
similar treatment 2.58 grams more, making the total crude glutaminic acid
hydrochloride 60.01 grams. This product was then dissolved in water freed
from color with animal charcoal and recrystallized. After removing ammo-
nium chloride by boiling with a slight excess of barium hydroxide and the
barium with an equivalent quantity of sulphuric acid, 43.02 grams of pure
glutaminic acid hydrochloride were obtained, which are equal to 34.42 grams
of the free acid, or 37 per cent of the gliadin.

Nitrogen: 0.5092 gram substance, dried at ioo°, gave NH3 = 3.86 HC1 (1 cc. HC1 =

0.01 gram N) = 7.58 p. ct. N.
Calculated for C5HI0O4NCl, 7.64 p. ct. N.

In confirmation of these figures this determination was repeated with
two fractions of the alcohol-soluble protein of wheat gluten which had been
separated from relatively strong alcoholic solutions, and should therefore
have contained a large proportion of "gluten-fibrin" if the statements
respecting the solubility of this substance are correct.

Two portions of different preparations of the air-dry substance, equivalent
to 18.62 and 14.65 grams dried at no°, were hydrolyzed as before, their
solutions saturated with hydrochloric acid, and kept for some time on ice.
The glutaminic acid hydrochloride which separated was filtered out, washed
with alcoholic hydrochloric acid, and freed from ammonia by evaporating
with an excess of baryta and from barium by an equivalent quantity of
sulphuric acid. The solution was then decolorized with animal charcoal
and evaporated with an excess of hydrochloric acid until crystallization
began. The glutaminic acid hydrochloride which separated, when washed
with ice-cold alcoholic hydrochloric acid and dried, weighed, respectively,
8.69 and 6.27 grams, equivalent to 37.33 and 34.2 percent of free glutaminic
acid in the protein.

Another attempt was made to isolate a fraction of ' ' gluten-fibrin ' ' by
dissolving 200 grams of a preparation representing the total alcohol-soluble
protein of wheat gluten in a mixture of 900 cc. of absolute alcohol and

68 the; proteins oe the wheat kernel.

600 cc. of water — that is, in alcohol of 60 per cent by volume. Although
the solution was somewhat turbid, nothing was deposited, even on long
standing. 300 cc. of water were then added to the solution, making the
alcohol 50 per cent, but still nothing separated. The strength of the alcohol
was therefore raised to 75 per cent by adding 1800 cc. of absolute alcohol,
and a very large precipitate, A, at once separated. By adding 2000 cc. of
absolute alcohol to the clear solution from which A separated another large
precipitate (B) was produced, and in the filtrate from B a third precipitate
(C) resulted, when a further large quantity of absolute alcohol was added.
This last product weighed 20 grams and constituted only 10 per cent of the
total protein. The solution from which C separated contained only traces
of protein, and C, therefore, represented the fraction of the whole protein
soluble in the strongest alcohol, and should consequently contain much
"gluten-fibrin." In 17.5 grams of this substance dried at no° the gluta-
minic acid produced by decomposing with hydrochloric acid was determined.
By proceeding in the same manner as in the experiment last described 7.8914
grams of pure glutaminic acid hydrochloride were obtained, which are equiv-
alent to6.2i3i grams of the free acid, or 35.5 per cent.

Nitrogen: 0.7296 gram substance, dried at no0, gave NH3 = 5.66 cc. HC1 (1 cc. HC1

= 0.0100 gram N) = 7.75 p. ct. N.
Calculated for C5H9N04HC1, 7.64 p. ct. N.

This result seems to furnish conclusive evidence that there is no fraction,
soluble in very strong alcohol, to be obtained from the alcohol-soluble pro-
tein of wheat gluten that is characterized by yielding a relatively small
proportion of glutaminic acid.

Since Kutscher decomposed his proteins with sulphuric acid, while hydro-
chloric acid was used in the preceding experiments, the following experi-
ment was made in order to determine whether the higher yield obtained by
us might not be due to this fact. Accordingly 50 grams of one of the
preparations of gliadin, from which 37 per cent of glutaminic acid had pre-
viously been isolated, were boiled for 14 hours with a mixture of 150 grams
of sulphuric acid and 300 cc. of water.

The resulting solution was treated with an excess of baryta and the ammo-
nia expelled by evaporation. The barium was then removed by an equiv-
alent amount of sulphuric acid and the filtered solution evaporated. Some
tyrosine separated, which was filtered out, and the evaporation continued
until the volume was quite small. On standing, an abundant quantity of
crystals of free glutaminic acid separated, and from the mother-liquor, by
further concentration and standing, a second crop of crystals was obtained.
After recrystallizing several times, 8.48 grams of pure glutaminic acid were
obtained, which contained 9.43 per cent of nitrogen.

EXPERIMENTAL.

Nitrogen; 0.6094 gram substance, dried at no0, gave NH3 = 5.75 cc. HC1 (1 cc. HC1
= 0.0100 gram N) =9.43 p. ct.; calculated for C5H9N04) 9.48 p. ct.

From the mother-liquors which yielded this glutaminic acid there were
further separated, by saturating with hydrochloric acid and proceeding in
the manner already described, 4.055 grams of well-crystallized hydrochloride,
which contained 7.73 per cent of nitrogen.

Nitrogen: 0.4983 gram substance, dried at no°, gave NH3 = 3.85 cc. HC1 (1 cc. HC1
= 0.0100 gram N) = 7.73 p. ct. N ; calculated for C3H9N04HC1, 7.64 p. ct. N.

The free glutaminic acid corresponding to this amount of the hydro-
chloride is 3.244 grams, making a total of 1 1.724 grams. Since the 50 grams
of air-dry gliadin were equivalent to 46.33 grams dried at no°, this amount
of glutaminic acid is equal to 25.3 per cent, or about the same proportion as
Ritthausen found in his preparation of ' ' mucedin ' ' after decomposing with
sulphuric acid, but much more than the 19.81 per cent found by Kutscher
in the same substance. The amount, however, was only about two-thirds
as much as that found after decomposing with hydrochloric acid, and
although the separation was not complete there was no reason to suppose
that more remained in the mother-liquors in one case than in the other. It
is possible, however, that the very large precipitate of barium sulphate that
formed on removing the sulphuric acid retained a considerable part of the
glutaminic acid even after extensive washing with hot water.

In conclusion, the results of these determinations are here brought together
that they may be more readily compared :

Table 16. — Percentage of glutaminic acid yielded by gliadin.

Preparation.

Decomposed by —

HG1.

H2S04.

P.ct.
37.00

37-33
34.20

35-5Q

P.ct.
25-3

All these are minimal figures, since in each case some glutaminic acid
still remained in the mother-liquors, but it does not seem probable that more
than relatively insignificant quantities were thus lost.

From these results it would appear that Kutscher' s determinations of
glutaminic acid fall far short of the actual quantity of this substance yielded
by the alcohol-soluble protein of wheat, and that they therefore afford no

/O the; proteins of the wheat kernel.

evidence which justifies the conclusion that this substance consists of two
distinct protein bodies. Fractional precipitations of this alcohol-soluble
protein yield practically the same large proportion of glutaminic acid, so
that, in view of their very close agreement in composition and properties,
both physical and chemical, there is every reason to believe that only one
such protein is present. Gliadin yields a remarkable proportion of gluta-
minic acid, much in excess of that from any other known protein and
greater than that of any single decomposition product yet obtained in a
pure state from any other true protein substance, the protamines, of course,
excepted.

HYDROLYSIS OF GLIADIN.

There being no sufficient evidence that more than one alcohol-soluble
protein occurs in the wheat kernel, no attempt has been made, in preparing
large quantities of gliadin for the present investigation, to subject the pro-
tein matter extracted by alcohol to any fractional precipitation, but it was
separated as completely as possible from all other substances soluble in water,
alcohol, and ether.

The gliadin for this investigation was prepared entirely from gluten, as
thereby the water-soluble constituents of the seed are more completely re-
moved than by any other method of preparation which can be readily used
on a large scale. The wheat flour was kneaded into dough in a domestic
"bread-mixer," and then under water in a specially constructed kneading-
machine. After frequently decanting and renewing the water, a thoroughly
coherent gluten was obtained. This was washed practically starch free in
a current of water and, while moist, was ground by passing through a
special form of "drug-press," which was a ready means of reducing it to
comparatively small pieces. The ground gluten was then extracted with
alcohol of such strength that, with the combined water of the gluten, a
solvent of 60 to 70 per cent by volume resulted. The extracts were filtered
perfectly clear through thick felts of filter paper pulp, and the water-clear
solution, free from any trace of opalescence or turbidity, was evaporated to
a small volume on a water-bath. The thick sirup that resulted was cooled
and then poured, with constant and rapid stirring, into a large volume of
distilled ice-water containing a very little sodium chloride. The gliadin
was thus precipitated as a filament, which, on stirring, united to a coherent
plastic mass. This gliadin was next dissolved by stirring with strong alcohol
until all had gone into solution, the water combined with the precipitated
gliadin being sufficient to dilute the alcohol to the proper degree. The
resulting solution was evaporated to a thick sirup, absolute alcohol being
added from time to time in order to hold the gliadin in the solution, since
this, during the evaporation, became constantly more aqueous. The thick

EXPERIMENTAL,. 71

sirup was then poured, in a very fine stream, into a large volume of absolute
alcohol under rapid and constant stirring. In this way a porous mass of
protein was obtained which was at once reduced to small pieces and digested
under fresh absolute alcohol. When well dehydrated, the gliadin was
digested with ether, partially dried over sulphuric acid, ground to a coarse
powder, and then dried thoroughly over sulphuric acid. When thus pre-
pared, gliadin forms a snow-white, friable mass which is easily reduced to
a powder.

1 100 grams of gliadin, equal to 998.6 grams dried at noc, were heated
with a mixture of 1000 cc. of concentrated hydrochloric acid and 1000 cc.
of water on the water-bath for several hours, until the gliadin had dissolved
and frothing had ceased. The solution was boiled in an oil-bath having a
temperature of 1 15° for 10 hours, cooled with ice, and saturated with gaseous
hydrochloric acid. After remaining on ice for 2 days, the glutaminic acid
hydrochloride that had separated was filtered out, washed with ice-cold
alcoholic hydrochloric acid dissolved in water, the solution treated with
bone-black and freed from ammonia by boiling with an excess of barium
hydroxide. After removing the barium with an equivalent amount of sul-
phuric acid, the glutaminic acid was separated as hydrochloride, and, when
recrystallized and thoroughly dried, weighed 374.3 grams, equivalent to 300
grams of free glutaminic acid. This was converted into the free acid, which
melted at 2020 to 2030.

Carbon and hydrogen : 0.6218 gram substance gave 0.9276 gm. C02 and 0.3494 gtn. H20.
Nitrogen : 0.4574 gram substance gave NH3 = 4.3 cc. HC1 (1 cc. HCl = o.oi gram N).
Calculated for C5H904N,C 40. 78, H 6. 18, N 9.54 p. ct. ; found, C 40. 69, H 6.24, N 9.40 p. ct.

The mother-liquor from the recrystallized glutaminic acid was added to
the filtrate from the first separation of the glutaminic acid, and the entire
solution concentrated to a sirup under strongly reduced pressure. 3 liters
of alcohol, previously saturated with hydrochloric acid at a low temperature,
were added to the sirup, and dry hydrochloric acid gas was passed into the
solution until it was saturated. The mixture was again concentrated as
before, under reduced pressure, the sirup again taken up in 3 liters of alco-
holic hydrochloric acid, and after standing several hours again concentrated
to a sirup, taken up a third time in alcoholic hydrochloric acid, and after some
hours concentrated to a sirup on a bath of 400 and under a pressure of from
5 to 10 mm. The neutralization, extraction, and drying of the esters were
conducted according to the method described by Emil Fischer.1

The undistilled residue, after distillation A, weighed 180 grams.

The residue which remained after extracting the esters with ether was
made strongly acid with hydrochloric acid, freed from sodium and potassium

Fischer, Emil, Zeitschrift fiir physiologische Chemie, 1901, xxxiil, p. 151.

THE PROTEINS OF THE WHEAT KERNEL.

salts by repeated evaporations with alcoholic hydrochloric acid and thor-
ough extraction of the precipitated chlorides with the latter. The alcoholic
solution of the chlorides of the amino-acids was evaporated to a sirup, and
esterificatiou repeated as in the first instance. After extracting the esters
with ether and drying them, the entire process was repeated, and the ethe-
real solution of the esters resulting from this third treatment was united
with that from the second, thus following the method applied by Abderhalden

to oxyhemoglobin.1

Distillations A and B.

Fraction.

Distillation A.

Distillation B.

Temper-
ature of

bath
up to —

Vapor.

Press-
ure.

Weight.

Temper-
ature of

bath
up to —

Press-
ure.

Weight.

Ill

IOO

1 20
160

0
75-76

vim.

12.0
12.0

0.8
0.8

Grams.
28.18
4703
64.68
40.00

83
100

120

200

mm.

12.0

0.8

G> ams.
20.13

36 s.

62.70
33 °°

179.89

152.64

Fraction I.

Distillation A. — This was saponified at once by evaporation with con-
centrated hydrochloric acid on a water-bath, the residue taken up in
alcohol, the solution saturated with dry hydrochloric acid gas, and a crystal
of glycocoll ester hydrochloride added. After prolonged standing on ice,
no separation occurred. The solution was then evaporated on the water-
bath with concentrated hydrochloric acid, the latter removed with lead oxide
and the lead with hydrogen sulphide. The amino-acids were subjected to
fractional crystallization.

Distillation B was treated in substantially the same way, but although
several attempts were made to isolate the hydrochloride of glycocoll ester,
none was found. By systematic fractionation there were obtained from
fraction I of the two distillations, A and B, 6.68 grams alanine and 0.S6
gram leucine.

The leucine, when recrystallized from dilute alcohol, decomposed at

about 2980.

Carbon and hydrogen: 0.1778 gram substance, dried at 1100, gave 0.3577 gram C02 and

0.160S gram H20.
Calculated for C6H1302N, C 54.89, H 10.01 p. ct.; found, C 54.87, H 10.04 p. ct.

'Abderhalden, Zeitschrift fur physiologische Cheinie, 1903, xxxvn, p. 484.

EXPERIMENTAL. 73

The alanine, when recrystallized by dissolving in a little hot water and
gradually adding alcohol, decomposed at about 2900.

Carbon and hydrogen: 0.2404 gram substance, dried at no°, gave 0.3571 gram CO,, and

o. 1712 gram H20.
Nitrogen: 0.3980 gram substance gave NH3 = 6.2 cc. HC1 (1 cc. HCl = o.oi gram N).
Calculated for C3H702N, C 40.40, H 7.93, N 15.75 P- ct.; found, C 40.51, H 7.91,

N 15.58 p. ct.

( Fraction II. Temperature of bath up to ioo°.
(Pressure, 12 mm. Weight, 83.84 grams.

Distillations A and B. — Each was saponified by boiling for 5^ hours with
5 parts of water.

The solution was evaporated to dryness under reduced pressure, the dried
residue boiled up with absolute alcohol, and 13.37 grams were dissolved.
This solution was united with a similar one obtained from fraction III.
The substance, insoluble in alcohol, after systematic fractional crystalliza-
tion, gave —

(I) 22.1 grams of leucine, which decomposed at about 2980 and had the
following composition :

Carbon and hydrogen : 0.3128 gram substance, dried at no0, gave 0.6310 gram C02 and

0.2794 gram H20.
Calculated for C6H1302N, C 54.89, H 10.01 p. ct.; found, C 55.01, H 9.92 p. ct.

(II) A fraction of 3.45 grams which by fractional crystallization could
not be further separated and gave results on analysis which agreed best for
a mixture of leucine and amino-valerianic acid.

Carbon and hydrogen : 0.2857 gram substance, dried at no0, gave 0.5519 gram C02 and

0.2577 gram H20.
Calculated for equal molecules of leucine and amino-valerianic acid, C 53.05, H 9.74

p. ct. ; found, C 52.68, H 10.02 p. ct.

(III) 2.1 grams substance which had the properties and composition of
amino-valerianic acid.

Carbon and hydrogen: (I) 0.2115 gram substance, dried at 110°, gave 0.3957 gram CO,
and 0.1870 gram H20 ; (II) 0.4597 gram substance, dried at no0, gave 0.8643 gram
CO, and 0.3956 gram H20.

Calculated for C5Hu02N, C 51.22, H 9.48 p. ct; found, (I) C 51.03, H 9.82 p. ct.;
(II) C 51-27, H 9.56 p. ct.

20°

Specific rotation. — Dissolved in 20 per cent hydrochloric acid, (a) — — =

+ 25. 790. E. Fischer and Dorpinghaus1 found + 25. 90 for their preparation
from horn, and Schulze and Winterstein* found +28.20 and + 27.90 for
preparations from lupine seedlings.

1 Fischer & Dorpinghaus, Zeitschrift fur physiologische Chemie, 1902, xxxvi, p. 462.

2 Schulze & Winterstein, ibid., 1902, xxxv, p. 300.

74 T II Ii PROTE] -S OF THE WHEAT KERNEL.

The chlorine was removed from the solution used for determining the
specific rotation and the substance racemized by heating with 20 cc. of
water and 7 grams of crystallized barium hydroxide for 19 hours in an
autoclave at 1750. The barium was quantitatively removed with sulphuric
acid, and the «-naphthyl-hydantoic acid prepared according to the directions
of Neuberg & Manasse.1 This crystallized in long needles and melted
constantly on repeated recrystallization from 40 per cent alcohol at 1800
to 1810.

Carbon and hydrogen: 0.3235 gram substance, dried at 900, gave 0.7936 gram C02 and

0.1909 gram H20.
Calculated for C16H1803N2, C 67.07, H 6.35 p. ct. ; found, C 66.90, H 6.56 p. ct.

By racemizing the remaining mixture of undetermined amino-acids we
were unable to isolate any more amino-valerianic acid.

(IV) 8.6 grams alanine. This was racemized by heating with an excess
of barium hydroxide and coupled with a-naphthyliso-cyanate according to the
directions of Neuberg & Manasse. The hydantoic acid, which crystal-
lized in prisms, melted at 1970.

Carbon and hydrogen: 0.3146 gram substance, dried at no°, gave 0.7480 gram C02 and

0.1560 gram H20.
Calculated for CuH1403N2, C 65.06, H 5.48 p. ct.; found, C 64.84, H 5.51 p. ct.

f Fraction III. Temperature of bath, up to 1200. )
\ Pressure, 0.8 mm. Weight, 127.38 grams. j

This fraction was boiled for 5^2 hours with 8 parts of water. The solution,
evaporated to dryness under reduced pressure, gave 98 grams of amino-acids
or 80 per cent of the esters. Of this 59.94 grams were soluble in alcohol.
From the part insoluble in alcohol, by systematic fractional crystallization,
there were isolated 33.06 grams of leucine and 4.57 grams of alanine. The
leucine decomposed at about 2980.

Carbon and hydrogen: 0.2416 gram substance, dried at no0, gave 0.4872 gram C02

and 0.2166 gram H,0.
Nitrogen : 0.2690 gram substance gave NH3 = 2.85 cc. HC1 ( 1 cc. HC1 = 0.01 gram N).
Calculated for C6H1302N, C 54.89, H 10.01, N 10.70 p. ct ; found, C 54-99, H 9.96,

N 10.59 P- ct-

The alcohol- soluble substance from fraction II was united with that from
fraction III. The solution was evaporated to dryness under reduced press-
ure, and the residue taken up in water and boiled gently for about an hour

1 Neuberg & Manasse, Berichte der deutscben chemiscben Gesellschaft, 1905, xxxvni,
P- 2359-

EXPERIMENTAL. 75

with an excess of copper hydroxide. The filtered solution was evaporated
to dryness under reduced pressure and the residue boiled with absolute
alcohol. The undissolved part was dissolved in water, freed from copper by
hydrogen sulphide, and the solution again evaporated to dryness under
reduced pressure. The residue was boiled with absolute alcohol, in which
all of it dissolved. The alcohol was evaporated off under reduced pressure,
the residue dissolved in 500 cc. of water and again converted into the copper
salt. By concentration 19.91 grams of crystalline racemic proline copper
salt were obtained, which is equal to 13.98 grams of a-proline. This was
recrystallized from water and dried in the air.

Water : 0.7789 grain substance lost 0.0856 gram H20 at no°.

Calculated for C10Hi6O4N2 Cu ■ 2H20, H20 11.00 p. ct. ; found, H20 10.98 p. ct.

Carbon and hydrogen : 0.6S51 gram substance, dried at no°, gave 1.0310 gram C02 and

0.3450 gram H20.
Copper: 0.2926 gram substance gave 0.0799 gram CuO.
Calculated for C10H16O4N2 Cu, C 41. 11, H 5.54, Cu 21.79 P- ct \ found, C 41.04, H 5.59,

Cu 21.81 p. ct.

The r-proline copper salt was freed from copper with hydrogen sulphide,
its solution evaporated to dryness, and the residue recrystallized from alcohol.
After drying in vacuo over sulphuric acid the r-proline melted at 203 ° to
205V

Carbon and hydrogen: 0.3373 gm. substance gave 0.6424 gin. C02 and 0.2453 gm- H20.
Calculated for C5H902 N, C 52.12, H. 7.90 p. ct. ; found, C 51.94, H 8.08 p. ct.

The solution of the alcohol-soluble copper salt was evaporated to dry-
ness and left a residue of /-proline copper salt which, dried at 1200, weighed
71.62 grams, which is equal to 56.51 grams of free /-a-proline.

Copper: 0.2850 gram substance, dried at 110°, gave 0.0760 gram CuO.
Calculated for Ci0Hi6O4N2 Cu, Cu 21.79 P- ct- ; found, Cu 21.30 p. ct.

One-half of this proline copper salt was freed from copper, and the pro-
line racemized by heating with 150 cc. of water containing 80 grams of
crystallized barium hydroxide for 5 hours at 1500. The barium was re-
moved quantitatively with sulphuric acid, the solution concentrated, and the
proline again converted into the copper salt. There was thus obtained 20. 1
grams of very nearly pure racemic a-proline copper.

Water : 0.3356 gram substance, air-dry, lost 0.0369 gram H20 at 1100.
Copper : 0.2640 gram substance gave 0.0640 gram CuO.

Calculated for C10H16O4N2 Cu " 2H20, H20 11.00, Cu 19.40 p. ct. ; found, H20 11.00, Cu
19.35 p. ct.

1 Willstaeter, R., Berichte der deutschen chemischen Gesellschaft, 1900, xxxiii, p. 1160;
Fischer, Emil, ibid., 1901, xxxiv, p. 458.

THE PROTEINS OE THE WHEAT KERNEL.

The total crystalline raceraic copper salt was equal to 41.96 grams of
r-"-proline.

From the other half of /-proline copper salt the free proline was regen-
erated and recrystallized from alcohol. A small part only was obtained in
a crystalline condition, which melted at 2050 to 2060.

Carbon and hydrogen : 0.2729 gram substance, dried over HaS04, gave 0.5198 gram

C02 and 0.1977 gram K.O.
Nitrogen : 0.2453 gram substance gave NH3 = 2.95 cc. HC1 (1 cc. HC1 = 0.01 gram N).
Calculated for C5H902N, C 52.12, H 7.90, N 12.20 p. ct. ; found, C 51.95, H 804,

N. 12.03 P- ct-

From fraction III there were isolated 4.57 grams alanine, 33.06 grams
leucine, and 70.49 grams a-proline, including in this last that from frac-
tion II, which was not weighed separately.

Fraction IV.

Temper-
ature

of bath
to —

Press-
ure.

Weight.

0
160
200

mm.
0.8
0.8

Grams.

39-96
33-oo

72.96

This was treated with water and shaken out with ether according to the
procedure described by Emil Fischer.1

The ether was carefully removed by evaporation and the residual phenyl-
alanine ester saponified by dissolving in concentrated hydrochloric acid
and evaporating on a water-bath. The phenylalanine hydrochloride weighed
29.14 grams, equivalent to 23.87 grams of free phenylalanine. The phenyl-
alanine hydrochloride was recrystallized from strong hydrochloric acid.
It was decomposed by evaporating with an excess of ammonia and the
phenylalanine recrystallized from water. It melted, on slow heating, at
2630 to 265V

Carbon and hydrogen : 0.3051 gram substance, dried at no°, gave 0.7322 gram C02 and

0.1792 gram HaO.
Nitrogen : 0.3020 gram substance gave NH3 = 2.53 cc. HC1 (1 cc. HC1 = 0.01 gram N).
Calculated for C9HnOsN, C 65.40, H 6.73, N 8.50 p. ct. ; found, C 65.44, H 6.53, N 8.38

p. ct.

1 Fischer, E., Zeitschrift fur physiologische Chemie, 1902, xxxvi, p. 274.
'Fischer, Emil, & Abderhalden, Zeitschrift fiir physiologische Chemie, 1902, xxxvi,
p. 268 ; Erlenmeyer & L,ipp, Annalen der Chemie, 1883, ccxix, p. 197.

Experimental,. yy

The aqueous layer was heated with an excess of barium hydroxide on a
water-bath for 5 hours. After standing some time, the barium salt that had
separated was filtered out and decomposed by an equivalent amount of sul-
phuric acid. The solution on concentration yielded 5.76 grams of aspartic
acid.

Carbon and hydrogen : 0.3866 gram substance, dried at no°, gave 0.5109 gram C02 and

0.1852 gram H20.
Nitrogen : 0.3637 gram substance gave NH3 = 3.78 cc. HC1 (1 cc. HC1 = 0.01 gram N).
Calculated for QH704N, C 36.09, H 5.26, N 10.53 P- ct- ; found, C 36.04, H 5.32, N

10.39 P- ct.

The filtrate from the barium aspartate was freed from barium, concen-
trated to small volume, and saturated with hydrochloric acid. On prolonged
standing a trace of phenylalanine hydrochloride separated, but no glutaminic
acid hydrochloride was obtained. After removing the hydrochloric acid
with silver sulphate and the sulphuric acid with barium hydroxide, the
solution was boiled with an excess of copper hydroxide, but no copper salt
could be separated from it, even after concentrating to a very small volume.
The copper was then removed and the solution treated with bone-black and,
when concentrated in a vacuum over sulphuric acid, gave crystals which, on
fractional crystallization from water, gave 0.42 gram of serine, which, in
an open capillary, browned at about 2180 and decomposed to a brownish
mass at about 240. °

Carbon and hydrogen: 0.2577 gram substance, dried at no°, gave 0.3224 gram C02

and o. 1602 gram H20.
Calculated for C3H703N, C 34.24, H 6.73 p. ct. ; found, C 34.12, H 6.91 p. ct.

In the filtrate from the serine there was obtained about 5.32 grams of crys-
talline substance, from which nothing definite could be isolated.

Distillation Residue.

The residues from distillations A and B were dissolved in boiling alcohol
and the solutions united. On cooling, 5.76 grams of long hair-like crystals
separated.

The filtrate from this substance was freed from alcohol and saponified by
heating with 200 grams of crystallized barium hydroxide, the barium re-
moved, the solution concentrated under reduced pressure to small volume,
saturated with hydrochloric acid, and, after standing on ice for some time,
yielded 74.21 grams glutaminic acid hydrochloride, equal to 59.46 grams.1

The glutaminic acid hydrochloride melted at about 1980 with effervescence.

Carbon and hydrogen : 0.4028 gram substance, dried over H2S05, gave 0.4802 gram

C02 and 0.2056 gram H20.
Calculated for C5H10O4 NCI, C 32.67, H 5.50 p. ct. ; found, C 32.51, H 5.67 p. ct.

'Abderhalden & Wells, Zeitscbrift fiir physiologische Chemie, 1905, xi/vi, p. 31.

/S Till- PROTEINS OF THE WHEAT KERNEE.

Specific rotation. — Dissolved in 20 per cent hydrochloric acid, (a) -=- =

+ 31.47°.

Fischer & Dorpinghaus found +31.910 for a preparation from horn,
+ 30. 450 from gelatin, and +28.200 for one from casein.1

The residue which remained after removing the esters with ether from
the original solution of the products of hydrolysis was treated in the
way described by Emil Fischer2 for the isolation of oxy-proline. The
only substance, however, that could be isolated was serine, of which
0.87 gram was obtained, which browned at about 2190 and decomposed at
about 2400.

Carbon and hydrogen: 0.29S7 gram substance, dried at no0, gave 0.3743 gram C02

and 0.1844 gram H20.
Calculated for C3H-03N, C 34.24, H 6.73 p. ct. ; found, C 34. iS, H 6.S5 p. ct.

In the filtrate from this serine ,3-naphthalene-sulphone chloride failed to
give any definite product.

There were thus isolated in the two distillations from fraction I, 6.68
grams ; from fraction II, 8.59 grams, and from fraction III, 4.57 grams of
alanine, or 19.84 grams in all; from fraction II, 2.1 grams of amino-vale-
rianic acid ; from fraction I, 0.86 gram ; from fraction II, 22. 10 grams ; from
fraction III, 33.06 grams of leucine — in all 56.02 grams ; from fractions II
and III, 70.49 grams of proline ; from fraction IV, 23.47 grams of phenyl-
alanine, 5.76 grams of aspartic acid, and 0.42 grams of serine, and from the
residue which remained after extracting the esters 0.87 gram of serine, or
1.29 grams in all.

From the main solution of the total products of hydrolysis there were
obtained 300 grams of glutaminic acid, and from the residues after dis-
tilling the esters 59.5 grams, or in all 3~9-5 grams.

As the amount of proline found in this hydrolysis was so great, the result
was confirmed by a second hydrolysis, and another effort made to obtain
glycocoll, which, if present in very small amount, might have escaped
detection.

For this purpose 500 grams of gliadin, air-dry, equal to 439.6 grams dried
at no°, were hydrolyzed in the same way as in the preceding h}rdrolysis.
After esterifying and shaking out the esters three times, as before, the ether
was removed by distillation at 760 mm. and the esters distilled.

'Cf. Fischer, Emil, & Dorpinghaus, Zeitschrift fiir physiologische Chemie, 1902,
xxxvi, p. 475.
' Fischer, Emil, Berichte der deutschen chemischen Gesellschaft, 1902, xxxv, p. 2660

EXPERIMENTAL.

Tempera-

- ,. ture of
Fraction. bath

up to —

Pressure. Weight.

95
8o

180

200

mm. Grams.
I7.OO 20.20
5.OO 46.08
2.00 44-14
O.88 15.70
O.88 54.7
O.78 29.69

Ill \ A

2IO. 51

Fraction I was immediately evaporated on the water-bath with hydro-
chloric acid, the residue dissolved in alcohol, and the solution saturated with
dry hydrochloric acid gas. The solution was concentrated to a small volume
at a low temperature under a pressure of 10 mm., the residue taken up in
alcohol, its solution cooled to o° and saturated with hydrochloric acid gas.
On prolonged standing 0.22 gram of glycocoll ester hydrochloric separated,
which melted at 1440 to 1450. When mixed with pure glycocoll ester
hydrochloride, the melting-point was unchanged.

Chlorine : 0.1058 gram substance gave 0. 1063 gram AgCl = CI 2X
Calculated for C4HmO,NC1, CI 25.40 p. ct

Neither fraction II nor the ether distilled from the esters gave evidence of
glycocoll.

This preparation of gliadin did, in fact, contain a very small amount of
glycocoll, which is possibly due to a slight contamination with glutenin, in
which there has since been found a notable quantity of this amino-acid.

Fraction II was saponified, and the solution evaporated under highly
reduced pressure from a bath at 400, and the residue extracted with alcohol,
in which about 18 grams dissolved.

Fraction III, by similar treatment, yielded 24 grams of alcohol-soluble
substance. The alcoholic solutions were united and evaporated to dryness
from a bath at 40c. The crystalline residue, when dried to constant weight
in vacuo, weighed 39.59 grams. From this, by extraction with alcohol, 8.7
grams of substance insoluble therein were separated.1 The total proline
thus found was 30. S9 grams, equal to 7.03 per cent.

1 Emil Fischer employs this method for estimating the proportion of pro-ine in pro-
teins, but stated that the result obtained is too high ^Berichte d*er deutschen chemischen
Gesellschaft, 19:6, xxxrx, p. 530).

80 THE PROTEINS OF THE WHEAT KERNEL.

CYSTINE.

300 grams of gliadin were digested for 2 to 3 hours at 85 ° with 900 cc. of
hydrochloric acid, sp. gr. 1.19, and the solution boiled for 3 hours. This
was then concentrated to a sirup under diminished pressure, diluted to 900
cc. with cold water, and neutralized with 50 per cent sodium-hydroxide solu-
tion. After boiling with a large amount of bone-black and concentrating to
800 cc, much substance separated, which was recr y stall ized from about 300
cc. of water. The recrystallized product was dissolved in 5 per cent sul-
phuric acid and precipitated by mercuric sulphate solution.1

The mercury precipitate was decomposed by hydrogen sulphide, the solu-
tion freed from hydrogen sulphide, neutralized with sodium hydroxide, and
acidified with acetic acid. On standing, cystine separated from the solution
in hexagonal plates, and by adding alcohol to the filtrate more was obtained.
When no more cystine could be thus obtained, the precipitation with mercu-
ric sulphate was repeated. By several repetitions of this process 1. 18 grams
of cystine were finally isolated, which, when recrystallized by dissolving in
dilute ammonia and acidifying with acetic acid, gave the following analysis :

Carbon and hydrogen : 0.3063 gram substance, dried at no0, gave 0.3379 gram C0.2 and

0.1444 gram H20.
Calculated for C6Hi204N2S2, C 29.96, H 5.04 p. ct. ; found, C 30.08, H5.23 p. ct.

TYROSINE.

219 grams of gliadin, equal to 200 grams dried at no°, were treated with
600 cc. of concentrated hydrochloric acid, digested for some time on a water-
bath, and the solution boiled for 12 hours on an oil-bath. The solution was
freed from most of the glutaminic acid by saturating with hydrochloric acid,
and the filtrate from the glutaminic acid was diluted, boiled with bone-black,
and then concentrated strongly to remove as much hydrochloric acid as
possible. The rest of the acid was neutralized with 50 per cent sodium-
hydroxide solution. On standing, a considerable precipitate separated,
which was filtered out and dissolved in ammonia. The resulting solution
was boiled until most of the ammonia had been removed and the tyrosine
that separated was filtered out. When dried, this weighed 2.4 grams, equal
to 1.2 per cent of the gliadin. Recrystallized from boiling water, this gave
the following results on analysis :

Carbon and hydrogen: 0.3661 gram substance, dried at no°,gave 0.7981 gram C02 and

0.2160 gram H20.
Calculated for C9Hn03N, C 59.62, H 6.13 p. ct. ; found, C 59-45. H 6.56 p. ct.

Tyrosine separated from our hydrolysis solutions of gliadin with very
great difficulty. Two other attempts to determine its proportion, which
were made by hydrolyzing with sulphuric acid, gave lower results, and the

1 Cf. Hopkins & Cole, Journal of Physiology, 1901, xxvn, p. 418.

EXPERIMENTAL. 8 1

solutions from which the tyrosine separated still continued to give a strong
Millon's reaction. Kutscher1 found 2.09 per cent of tyrosine in gliadin,
and Abderhalden & Samuely2 found 2.37 per cent.

HISTIDINE.

Fifty grams of gliadin, equal to 43.97 grams dried at 1 io°, were hydrolyzed
according to the directions of Kossel & Kutscher3 and the determination
of the bases carried out according to the method of Kossel & Patten.4

The solution of the histidine was made up to 500 cc. and nitrogen deter-
mined in 100 cc. of it.

100 cc. of solution gave ammonia = 1.37 cc. HC1 (1 cc. HC1 = 0.01 gram N) = 0.0137
gram N = 0.0685 gram N in 500 cc. = 0.2524 gram histidine = 0.58 p. ct. of the
gliadin.

The identity of this histidine was not established, as the quantity was too
small to permit the preparation of a satisfactory product.

ARGININE.

The arginine solution was made up to 500 cc. and nitrogen determined in
50 cc. of it.

50 cc. of solution gave ammonia=4.i5 cc. HC1 (1 cc. HCl = o.oi gram N) = 0.0415
gram N or 0.415 gram in 500 cc. = 1.39 gram arginine or 3.16 p. ct. of the gliadin.

The remaining solution was treated as Kossel & Kutscher direct and the
arginine obtained as carbonate. A portion of this carbonate was converted
into the picrolonate according to the directions of Steudel.5 This melted at
2260 to 2270, while Steudel gives 2250.

Nitrogen: 0.0832 gram substance, dried at no0, gave 18.8 cc. moist N2 at 765 mm.

and 250.
Calculated for C6Hu02N4 ■ C10H8O5N4, N 25.62 p. ct.; found, N 25.40 p. ct.

LYSINE.

The filtrate from the silver precipitate of arginine and histidine was freed
from silver, precipitated with phosphotungstic acid, and lysine tested for
with picric acid in the usual way, but none was found.

Kossel & Kutscher6 found, in the three fractions of the alcohol-soluble
protein of the wheat kernel which they examined, 1.2, 0.43, and 1.53 per
cent of histidine. The writer's determination falls between these. Kutscher,7
in discussing the individuality of these three fractions, considers the differ-

1 Kutscher, Zeitschrift fiir physiologische Chemie, 1903, xxxviii, p. in.

2 Abderhalden & Samuely, ibid., 1905, xi.iv, p. 276.
3 Kossel & Kutscher, ibid., 1900, xxxi, p. 165.

4 Kossel & Patten, ibid., 1903, xxxviii, p. 39.

5 Steudel, ibid., 1902, xxxvn, p. 219.

6 Kossel & Kutscher, loc. cit.
' Kutscher, loc. cit.

THE PROTEINS OF THE WHEAT KERNEL.

ences in the amount of histidiue found in them to be within the limits of
accuracy of these determinations.

Kossel & Kutscher also found 3.05 per cent of arginine in the fraction
which they called gluten-fibrin, 2.75 per cent in their gliadin, and 3.13 per
cent in their mucedin. In determining the amount of protein hydrolyzed
they calculated the weight from the nitrogen in solution. If, as seems
probable, only one alcohol-soluble protein exists in this seed, namely,
gliadin, with 17.5 per cent of nitrogen, and that the nitrogen of their solu-
tions belonged to this protein, the proportion of arginine found by them
would be, respectively, 3.13, 2.79, and 3.25 percent, with which 3.16 per
cent agrees very closely. The results of this hydrolysis are given in table 17.

Table 17. — Gliadin.

P.ct.

Glycocoll 0.00

Alanine 2.00

Amino-valerianic acid 0.21

Leucine 5.61

a-proline 7.06

Phenylalanine 2.35

Aspartic acid 0.58

Glutaminic acid 37-33

Serine o. 13

Tyrosine 1.20

P.ct.

Cystine 0.45

Oxy-proline

Lysine 0.00

1 1 istidine 0.58

Arginine 3. 16

Ammonia 5. 11

Tryptophane present

65.78

PROTEIN INSOLUBLE IN WATER, SALINE SOLUTIONS, AND ALCOHOL —

GLUTENIN.

As already stated, extraction with the above-named solvents, applied suc-
cessively, removed but a part of the total protein contained in the wheat
kernel, that remaining being soluble only in dilute acids and alkalis. The
following extractions were next made to determine the nature of this body.

Protein Extracted by Dilute Alkaline Solutions after Extracting the
Flour with 10 per cent Sodium-Chloride Brine and then with Dilute
Alcohol.

After completely extracting from 4000 grams "straight flour" all the
protein soluble in 10 per cent sodium chloride solution, the residue was
freed wholly from protein soluble in cold alcohol of 0.90 sp. gr. The re-
sulting residue was then extracted twice with a large quantity of o. 1 per
cent potassium hydroxide solution. After standing 3 days at a tempera-
ture of 50 with frequent stirring, this extract was filtered off and allowed
to stand in a cold room until the greater part of the fine starch and other
impurities which had escaped filtration had settled. The solution, which
was still turbid, was decanted from the sediment and neutralized as exactly
as possible with 0.2 per cent hydrochloric acid, thereby producing a precipi-

EXPERIMENTAL.

tate which settled rapidly, leaving the filtrate milky in appearance. This
precipitate, after decanting the solution, was dissolved in 0.2 per cent potas-
sium-hydroxide water and set aside to deposit the suspended impurities.
After decantation from the sediment which resulted, the solution was filtered,
but almost nothing was thus removed. It was then neutralized with 0.2
per cent hydrochloric acid and the precipitate produced washed by decan-
tation, first with water and then with dilute alcohol, absolute alcohol, and
ether. No attempt was made in this case to obtain the whole of the protein
soluble in alkali, as the difficulties presented by slow and imperfect filtration
rendered this impossible. The substance thus obtained formed a brownish,
horny mass, which weighed 13 grams. This preparation, 89, when dried
at no°, on analysis gave the results shown in the table at the bottom of this
page.

Subsequent preparations of this body led to the idea that it was far from
pure, this supposition being supported by the fact that the final solution
from which it had been precipitated was turbid. The remainder of the
preparation was then dissolved as completely as possible in 0.2 per cent
potassium-hydroxide solution, and by repeated filtration through very dense
filter-paper obtained perfectly clear. A considerable insoluble residue
remained, which appeared to consist largely of the coagulated form of this
protein. This residue was washed by decantation with water, alcohol, and
ether. It was found to contain but 13.68 per cent of nitrogen, showing
that the preparation, 89, contained much non-nitrogenous matter. The filtra-
tion of the dissolved protein proceeded very slowly, so that it was conducted
at a temperature near o°. A portion of the filtrate first obtained was re-
moved, precipitated by 0.2 per cent hydrochloric acid, washed with water,
alcohol, and ether, and the preparation, 90, analyzed with the following
results. A second portion similarly yielded preparation 91.

Preparatio

w 89, 90,

and 91.

Preparation 89.

Preparation 90.

Preparation 91.

Ash-free.

II.

Average.

Ash-free.

Carbon ....
Hydrogen .
Nitrogen...
Sulphur.. . .

P.ct.

52.47
6.75

15-51
0.86

P.ct.

52.91
6.S1

15.65
0.86

23-77

P. ct.
51.10

6.46
17.01

0.91

P.ct.

O.92

P. Ct.

5i-io
6.46

17.01
0.92

Pel.
52.29
6.61

17.41
0.94

22.75

P.ct.

17.17

17-33

Ash. .

0.88

2.28

0.97

100.00

THE PROTEINS OE THE WHEAT KERNEL.

These results show that it is absolutely necessary to filter the alkaline
solution of this body perfectly clear before the final precipitation, since other-
wise a considerable amount of non-nitrogenous matter will be precipitated
with it. Another extraction was made by treating 200 grams of " patent
flour " from spring wheat with 10 per cent sodium-chloride solution added
in small quantities so as to make a dough. This dough was then washed
with 10 per cent sodium-chloride brine until nearly all the starch was re-
moved and a gluten obtained similar in all respects to that resulting from
treating the flour with water. This gluten was then chopped fine, thoroughly
extracted with alcohol of sp. gr. 0.90, and then dissolved at 200 in o. 1 per
cent potassium-hydroxide water. The resulting solution was filtered, but,
as only a part of the impurities was thus removed, the filtrate was placed in
an ice-box in shallow dishes and allowed to deposit a considerable part of
the suspended matter. The decanted solution, which was only very slightly
turbid, was then precipitated by neutralizing with 0.2 per cent hydro-
chloric acid, and the precipitate washed by decantation with water, extracted
thoroughly with dilute alcohol, digested with absolute alcohol and then with
ether. This preparation, 92, was a snow-white, light, porous mass, easily
reduced to a powder. It had the following composition :

Preparation 92.

II.

Average.

Ash-free.

P.ct.

P. ct
52.18

6.90
16.90

1. 00

P. Ct.

52.50
6.94

17.22
1. 00

22.34

Sulphur . .

17. 11

17.01

Ash .

0.63

100.00

Protein Extracted by Dilute Alkaline Solutions from Gluten after
Extracting the Latter with Dilute Alcohol.

2000 grams of ' ' straight flour ' ' from spring wheat were made into a dough
with distilled water, and this was washed with river- water until the gluten
was freed as completely as possible from starch. This gluten was then ex-
tracted with 75 per cent alcohol as long as anything was removed. The
insoluble residue was dissolved in 0.15 per cent potassium-hydroxide water
and the resulting solution allowed to stand in a cold room for 48 hours.
The solution was thus freed from but a part of the suspended matter. After
decanting from the sediment, the solution was neutralized with dilute hydro-
chloric acid, the precipitate produced, washed several times by decantation

EXPERIMENTAL.

with water, thoroughly extracted with alcohol of 0.90 sp. gr., then with
stronger alcohol, and finally with absolute alcohol and with ether.

The precipitate was again dissolved in o. 1 per cent potassium-hydroxide
solution and allowed to stand over night. It was then filtered, and a part
of the clear filtrate first obtained was precipitated by neutralization with 0.2
per cent hydrochloric acid. This precipitate was washed with water, alco-
hol, absolute alcohol, and ether, yielding preparation 93. A part of this
preparation, 93, was redissolved in 0.2 per cent potassium-hydroxide water
and found to contain a considerable amount of substance which had become
insoluble in consequence of drying. This insoluble portion was filtered off,
washed with water, alcohol, and ether, and gave preparation 94. The filtrate
from this substance was precipitated with 0.2 per cent hydrochloric acid,
and the precipitate filtered off and washed with water, alcohol, and ether.
Through an accident this preparation dried on the filter and could not be
removed from the paper. It was then again dissolved in dilute potassium-
hydroxide water and treated exactly as before, yielding preparation 95.
The following analyses show the composition of these three preparations :

Preparations 93, 94, and 95.

II.

Average.

Ash-free.

Preparation 93 :

Carbon

P.ct.

51-59
6.72

17-34
\

P. ct.

P.ct.

51-59
6.72

17.37

P.ct.
52.32

6.82
17.61

23-25

Hydrogen

Nitrogen

17.39

Sulphur

Oxygen

I.40

Ash

Preparation 94 :

Carbon

100.00

50.79
6.62

16.38
}

50.79
6.62

16.38

52.87

6.88

17.05

23.20

Hydrogen

Nitrogen

16.38

Sulphur

3-94

Ash

Preparation 95 :

Carbon

100.00

51-50

6.70

16.70

}

51-37
6.57

51.44

6.64

16.70

52.62

6.80

17.08

23-5o

Hydrogen

Nitrogen

Sulphur

Oxygen

2.25

Ash

100.00

THE PROTEINS OF THE WHEAT KERNEL.

Another lot of gluten made from 1000 grams of "straight flour" was
treated in exactly the same way as that last described. This gluten was
thoroughly extracted with alcohol of 0.90 sp. gr. and the residue dissolved
in about 500 cc. of 0.2 per cent potassium-hydroxide water. After standing
over night, a very turbid liquid was decanted from the sediment which had
formed and treated with very dilute acetic acid added to slightly acid reac-
tion. The precipitate produced was washed with water, alcohol, and ether
and dissolved again in 0.2 per cent potassium-hydroxide water. The result-
ing solution, filtered perfectly clear, was precipitated with 0.2 per cent
hydrochloric acid, washed by decantation with water, then with dilute
alcohol increased gradually in strength up to absolute alcohol, and finally
with ether. When dried over sulphuric acid, a pure white, light mass was
obtained. This preparation, 96, was analyzed with the results shown in the
table below.

Again, gluten was prepared in the usual manner and extracted with alcohol
until everything soluble in that reagent was removed. The residue was then
dissolved in 0.2 per cent potassium-hydroxide water and, in order to carry
the operation to an end as rapidly as possible, the solution was at once
thrown into a filter. As soon as the filtrate ceased to pass through turbid
it was returned to the funnel and the filtration continued in an ice-chest.
A considerable portion of the solution was obtained perfectly clear after 20
hours. This was then precipitated with o. 2 per cent hydrochloric acid and
treated in the usual manner, giving preparation 97.

Preparations 96 and gj.

Preparation 96.

Preparation 97.

II.

Average.

Ash-free.

Carbon

Hydrogen . .
Nitrogen. . . .
Sulphur ....

P. Ct.

52.07
6.71

17.23
1.07

P.ct.
52.23
6.88
17.42

P.ct.

52.15
6.80

17.33
I.07

P.ct.

52.54
6.85

17.46

1.07

22.08

P.ct.
52.07
6.77

17.49
1.24

P.ct.

52.38

6.81

17.59
1.24

21.98
100.00

Ash

0.74

0.76

o.75

0.61

100.00

In order to determine whether a loss of nitrogen occurred through pro-
longed contact with the alkaline solution, the following experiment was tried,
in which the conditions under which preparation 97 was made were repeated
and preparation 98 obtained. The rest of the alkaline solution was kept in
the ice-box for 3 days longer, during which time the ice melted and the

EXPERIMENTAL.

temperature rose to 200. The preparation, 99, obtained by neutralizing the
solution with hydrochloric acid contained the same amount of nitrogen as
preparation 98, from which it is evident that prolonged solution in dilute
potassium hydroxide solution caused no loss of nitrogen.

Preparations 98, 99, and 100.

Preparation 98.

Preparation 99.

Preparation 100.

Ash-free.

II.

Average.

Ash-free.

Nitrogen . . .
Ash

P.ct.

17.32

1.23

P.ct.
17-53

P.ct.

i7-5o

0.16

P.ct.
17.53

P.ct.
17.04
1.22

P.ct.
16.93

P. ct.
16.99

P.ct.
17.20

Another preparation of this substance (preparation 100) was made by
completely extracting 200 grams of "patent flour" (from spring wheat)
with large quantities of alcohol of 0.90 sp. gr. The flour was then washed
with absolute alcohol and air-dried. The dry material was then powdered
and made into a dough with distilled water which had considerable coher-
ence, showing that the protein insoluble in alcohol played an important part
in its formation. This dough was then washed on a fine hair-sieve under a
stream of running water, but no coherent gluten resulted. The washings
were then allowed to settle, and the sediment, after decanting the solution,
was treated with 0.2 per cent potassium-hydroxide water. The solution so
obtained, after standing over night, was decanted from the sediment and
precipitated with o. 2 per cent hydrochloric acid and the separated protein
allowed to settle. The solution was then decanted, the precipitate dissolved
in 0.2 per cent potassium-hydroxide water, and filtered clear in the ice-box.
The filtered solution was then precipitated and the separated substance
treated in the usual manner. This preparation, 100, contained nitrogen, as
shown in the above table.

In order to learn whether any change in the protein occurred in conse-
quence of contact with aqueous solutions before extracting with potassium-
hydroxide water, the following experiment was made :

1000 grams of "straight flour" from spring wheat were repeatedly ex-
tracted with alcohol of 0.90 sp. gr., and after removing everything soluble
in that liquid the alcohol was squeezed out as completely as possible in a
screw-press and the residue extracted with 0.2 per cent potassium-hydroxide
water. It was found impossible to separate the solution from the undis-
solved portion of the meal, either by filtration or subsidence, on account of
the presence of gummy matter. An equal volume of alcohol of sp. gr. 0.820

THE PROTEINS OE THE WHEAT KERNEL.

was therefore added, and on long standing the insoluble substance gradually
settled, leaving a comparatively clear, yellow solution. This was then
siphoned off and filtered. On account of its gummy character it filtered
very slowly. The clear solution finally obtained was then precipitated with
0.2 per cent hydrochloric acid, the precipitate filtered off and dissolved in
0.2 per cent potassium-hydroxide water. The solution so obtained was
filtered clear and precipitated with 0.2 per cent hydrochloric acid, the result-
ing precipitate washed by decantation with water, alcohol — at first dilute,
then gradually increased to absolute — and finally with ether. Preparation
101 was thus obtained, having the composition shown in the following table :

Preparations 101 and 102.

Preparation 101.

Preparation 102.

Ash-free.

II.

Average.

Ash-free.

Carbon

Hydrogen . .
Nitrogen . . .
Sulphur ....

Oxygen

Ash

P. Ct.

52.14
6.91

17-54

}

0. 10

P. ct.

52.19
6.92

17-56
23-33

P.ct.

51-59

6.S5

17.21

i 0.89

P.ct.

5I-67

6.S7

17.32

P.ct.

5I-63
6.86

17.27
0.89

P.ct.
52.19

6-93
17-45

0.90

23-43

1.07

100.00

This analysis shows that the protein extracted by potassium hydroxide
water from the flour which has not been in contact with water has the
same composition as that obtained in the other experiments.

Protein Extracted by Dilute Alkaline Solutions after Complete Extraction
with Dilute Alcohol of Gluten from Whole-wheat Flour.

1000 grams of flour made by grinding the entire kernel of spring wheat
were made into a dough, washed with water till free from starch, and the
gluten obtained extracted thoroughly with dilute alcohol. The residue was
then dissolved in 0.2 per cent potassium-hydroxide water, and after the
resulting solution had stood for some time it was decanted from the sedi-
ment and precipitated by 0.2 per cent hydrochloric acid. The precipitate
was washed by decantation with water, thoroughly extracted with dilute
alcohol and then with absolute alcohol, and finally with ether, and then
redissolved in 0.2 per cent potassium-hydroxide water.

The solution, after filtering perfectly clear, was precipitated and the pre-
cipitate treated in the same manner as all the preceding preparations. When

EXPERIMENTAL.

analyzed, preparation 102 was found to have the composition shown in the
table on page 88.

The filtrate from this preparation, as well as all the others previously-
described, contained a small amount of protein matter. This was then
treated with a solution of copper sulphate and the small precipitate thereby-
produced filtered off, washed with water, alcohol, and ether (preparation
103), and found to contain the following amount of nitrogen :

Preparation 103.

Ash-free.

Nitrogen

Ash

P. ct.
13.28
23.88

P.ct.
17-45

In the same way as 102 a preparation of this protein was made from
flour from the entire kernel of winter wheat. The composition of this
preparation is shown by the figures in the following table for preparation
104.

Preparations 104 and 105.

Preparation 104.

Preparation 105.

II.

Average.

Ash-free.

Hydrogen . .
Nitrogen. . . .
Sulphur ....

Ash

P. ct.

5I.7I
6.79

17.32
}

P.ct.

P.ct.
5I-7I
6.79

17.38

P.ct.
52.03
6.83

17.48

23.66

P.ct.

51-17
6.70

17-35

P.ct.
52.44
6.86
17.81

22.89
100.00

17.44

0.62

2.62

100.00

A second portion of the same solution from which preparation 104 had
been precipitated was obtained 2 days later. This was then treated in
the same way and yielded preparation 105, which was found to have the
composition shown in the table above.

If preparations 89 and 92 are omitted on account of being obtained from
unfiltered solutions, 94 as an altered and insoluble product, and 95 as having
been subjected to an exceedingly prolonged action of an alkaline solution,
then will be had, in table 18, the analyses which, in the opinion of the
writer, most nearly represent the true composition of this protein.

THE PROTEINS OF THE WHEAT KERNEL.
Table 18. — Protein of the wheat kernel soluble only in dilute acids and alkalis.

90.

91.

93-

96.

97.

99-

Carbon

Hydrogen

Sulphur

P.ct.

52.29

6.61

I7-4I

•94

22.75

100.00

P.ct.

P. ct.

52.32

6.82

17.61

} 23.25
100.00

P. ct.

52.54
6.85

17.46
f 1.07
\ 22.08

100.00

P.ct.

52.38
6.81

17.59
1.24

21.98

100.00

P.ct.

P. ct.

x7-33

17-53

100.

101.

102.

103.

104.

105.

Average.

P.ct.

52.19
6.92

17.56
} 23.33

P.ct.
52.19
6-93
17-45

23-43
IOO.OO

P.ct.

52.03

6.83

17.48

23.66

P. ct.

52.44

6.86

17.81

22.89
100.00

P.ct.

52.34
6.83

17-49

r 1.08

\ 22.26

Sulphur

17.20

17.45

Oxveen

IOO.OO

100.00

There can be no doubt that the earlier analyses of this protein, with the
exception of those made by Ritthausen and Chittenden & Smith, are incor-
rect, for the substance analyzed contained a large part of the impurities of
the gluten, since after extraction with alcohol the residue was not dissolved
in any solvent, and the starch, bran, etc., contained in it filtered off. Ritt-
hausen' s figures are the average of two analyses of preparations obtained
from solutions in 0.2 per cent potassium-hydroxide water which had been
filtered, one perfectly clear, giving a product containing 17.21 per cent N,
the other very nearly clear and containing 17.08 per cent N.

It is evident that the alkaline solution of the gluten that has previously
been extracted with cold dilute alcohol contains something which requires
filtration through very dense paper to effect its separation. Wheat gluten
contains a considerable amount of phytocholesterin and lecithin, together with
fat, and it is probable that all these bodies are held suspended in the solution
of the dissolved protein as an emulsion, and can only be removed by pre-
cipitating the first solution in potassium-hydroxide water, extracting the
precipitate with alcohol and ether, redissolving, and filtering the resulting
solution perfectly clear. The first alkaline solution before precipitating and
extracting with alcohol and ether can not be filtered, as it either passes
through turbid or does not run through at all.

On the ground of priority and the fact that the relations to animal proteins
which gave rise to the various names subsequently applied to this body have

EXPERIMENTAL. 91

been proved to have no foundation, it would be desirable to return to Taddei's
original name and in future call this protein zymon. Unfortunately this
name is derived from the Greek word fo^, a ferment, and, as the results of
this investigation show that the supposed ferment-changes do not occur in
the formation of gluten, this name is undesirable. As this protein is espe-
cially characteristic of gluten, it seems appropriate to call it glutenin, a name
suggested by Prof. S. W. Johnson.

Hydrolysis of Glutenin.

The large quantity of glutenin which was required for the quantitative
determination of its decomposition products was prepared from the residue of
the wheat gluten after extracting the gliadin with alcohol. This residue was
dried at room temperature and then ground to a powder, which was extracted
first with absolute alcohol and then with ether as long as either solvent re-
moved anything from it. The alcohol was then removed at room temperature
and the residual powder treated with just enough 0.2 per cent solution of
potassium hydroxide to dissolve it. The resulting turbid solution was then
filtered perfectly clear and neutralized with very dilute hydrochloric acid.
The precipitate produced was extracted with 70 per cent alcohol as long as
any gliadin was removed, then thoroughly dehydrated with absolute alcohol,
and dried over sulphuric acid.

940 grams, equal to 839.32 grams of glutenin, ash and water free, were
hydrolyzed by heating for several hours on a water-bath with a mixture of
950 cc. of concentrated hydrochloric acid and 950 cc. of water. After standing
over night, the solution was boiled on an oil-bath for 9 hours, and then
saturated with hydrochloric acid gas. By the same treatment as that applied
to gliadin (p. 71) 202.73 grams glutaminic acid hydrochloride, equal to
162.40 grams of the free acid, were obtained. Recrystallized once from
concentrated hydrochloric acid, this melted at 1980.

Chlorine: 0.5386 gram substance gave 0.421 1 gram AgCl.

Nitrogen: 0.5911 gram substance gave NH3 = 4.53cc. HC1 (1 cc. HCl = o.oi gram N)„

Calculated for C5H10O4 NCI, CI 19.35, N 7.65 p. ct. ; found, CI 19.33, N 7.66 p. ct.

The united filtrates and washings were concentrated to a sirup on a water-
bath under reduced pressure and the hydrochlorides of the amino-acids
esterified three times, as in the case of gliadin. The hydrochlorides of the
esters were neutralized and the free esters shaken out with ether, as for
gliadin. After drying the ether solution of the esters with potassium car-
bonate, it was kept 2 days over anhydrous sodium sulphate. The ether
was then removed by distillation from a water-bath at atmospheric pressure
and the esters distilled, with the results shown in the table on page 92 for
distillation A. The undistilled residue weighed 21 1.5 grams.

THE PROTEINS OF THE WHEAT KERNEL.
Distillations A and B.

Fraction.

Distillation A.

Distillation B.

Temper-
ature of

bath
up to —

Pressure.

Weight.

Fraction.

Temper-
ature of

bath
up to —

Pressure.

Weight.

65
IOO
IOO
IIO

155
200

m m .

12.0
12.0

4.0
1.5

1.5
0.8

Gravis.
61.OO

43-74
45-94

33-Qi
43-32
32-56

I..

IOO

120

180

mm.

12.0

10.0

0.8

Grams.
28.74
24.27
19.18

23-23
23.92

II.. .

™{»

IV..

259-57

H9-34

The residue, from which the esters had been removed by ether, was sub-
jected to two or more esterificatious, as in the case of gliadin, and the esters
distilled. (See distillation B in the table above.) The undistilled residue
weighed 93 grams.

The different fractions from the two distillations were worked up as follows:

Fraction.

Temper-
ature of

bath
up to —

Pressure.

Weight.

, r A

65
65

mm.
12
12

Grams.
61.OO

28.74

This was saponified at once by evaporating to a sirup with concentrated
hydrochloric acid and the residue dissolved in alcohol and esterified with
dry hydrochloric acid gas. The glycocoll ester hydrochloride which sepa-
rated weighed 6.68 grams. Recrystallized from alcohol, this melted at 1440.

Carbon and hydrogen: 0.3179 gram substance gave 0.3984 gram C02 and 0.2151 gram

H20.
Calculated for C4H10O2NCl, C 34.39, H 7.23 p. ct.; found, C 34.18, H 7.52 p. ct.

The filtrate from the glycocoll ester hydrochloride was saponified by
evaporating on the water-bath with hydrochloric acid, the latter removed
with silver sulphate, and the solution freed from sulphuric acid with an

EXPERIMENTAL.

equivalent quantity of barium hydroxide. By fractional crystallization this
solution yielded 11.83 grams of alanine, which melted at about 2900.

Carbon and hydrogen: 0.2701 gram substance, dried at no0, gave 0.4001 gram C02

and 0.1946 gram H20.
Nitrogen: 0.3460 gram substance, dried at no°, gave NH3 = 5.44 cc. HC1 (1 cc.

HCl = o.oi gram N).
Calculated for C3H702N, C 40.40, H 7.93, N 15.75 P- ct.; found, C 40.40, H 8.or, N

15.73 P- ct.

Fraction.

Temper-
ature of

bath
up to —

Pressure.

Weight.

«{tv::::::

100
88

mm.
12
IO

Grams.

43-74
24.27

The united esters were saponified by boiling with 10 parts of water for 5
hours, when their solution reacted neutral to litmus. This solution was
evaporated to dryness under reduced pressure and boiled up with absolute
alcohol, whereby 0.5 gram substance was dissolved. By repeated fractional
crystallization of the substance insoluble in alcohol, 3.5 grams leucine were
obtained. The isolated leucine decomposed at about 2980.

Carbon and hydrogen: 0.240S gram substance, dried at no°, gave 0.4852 gram C02 and

0.2169 gram H2C
Calculated for C6H1302N, C 54-89, H 10.01 p. ct. ; found, C 54.95, H 10.01 p. ct.

After adding to the filtrate from this leucine the most soluble portion of
fraction III, and further fractiouing, 25.76 grams of alanine and 3.84 grams
of glycocoll were obtained. The latter was isolated as the hydrochloride
of the ester, which melted at 1440. The alanine was racemized and the
a-naphthyl-hydantoic acid prepared according to the directions of Neuberg &
Manasse.1 When recrystallized from dilute alcohol, this melted at 1970.

Carbon and hydrogen: 0.2527 gram substance, dried at 900, gave 0.5998 gram C02

and 0.1280 gram H20.
Nitrogen: 0.3515 gram substance, dried at 900, gave NH3 = 3.74 cc. HC1 (1 cc.

HC1 = 0.01 gram N).
Calculated for CuH1403N,, C 65.06, H 5.48, N 10.87 p. ct.; found, C 64.74, H 5.61,

N 10.61 p. ct.

1 Neuberg & Manasse, Berichte der deutschen chemischen Gesellschaft, 1905, xxxviii,
P- 2359.

THE PROTEINS OF THE WHEAT KERNI'J..

Fraction.

Temper-
ature of

bath
up to —

Pressure.

Weight.

™{i

1 20

in m .

1-5
0.8

Grains.

78.95
42.41

These esters were saponified by boiling with 6 parts of water for 5 hours,
when their solution reacted neutral to litmus. After evaporating to dryness
under reduced pressure, the dried residue was extracted with boiling absolute
alcohol. The part insoluble in alcohol, by a systematic fractional crystal-
lization, gave 32.42 grams of leucine, which decomposed at about 2980.

Carbon and hydrogen: 0.4255 gram substance, dried at no0, gave 0.8535 gram C02

and 0.3926 gram H20.
Nitrogen: 0.1488 gram substance gave NH,= 1.60 cc. HC1 (1 cc HCl = o.oi gram N).
Calculated for CGH130,N, C 54.89, H 10.01, N 10.70 p. ct.; found, C 54.71, H 10.251

N 10.77 P- ct-

The filtrate from this leucine yielded 2 grams of substance, which had the
composition and properties of amino-valerianic acid.

Carbon and hydrogen: 0.2216 gram substance gave 0.4144 gram C02 and 0.1967 gram

H20.
Calculated for C5Hu02N, C 51.22, H 9.48 p. ct.; found, C 51.00, H 9.86 p. ct.

Specific rotation. — Dissolved in 20 per cent hydrochloric acid,

0) -^ = + 25.63°

A similar preparation from gliadin gave +25.790. E. Fischer & Dor-
pinghaus1 found +25.90 for a preparation from horn, and E. Schulze &
Winterstein2 found +28.20 and + 27.90 for a preparation from lupine
seedlings.

The solution used for determining the specific rotation was freed from
hydrochloric acid with silver sulphate, and the amino-acids racemized by
heating with an excess of barium hydroxide in an autoclave at 1750 for 19
hours. After removing the barium quantitatively with sulphuric acid, the
substance was coupled with a-naphthylisocyanate. The hydantoic acid
melted constantly, on repeated recrystallization from dilute alcohol, at 1830 to

'Fischer, E., & Dorpinghaus, Zeitschrift fur physiologische Chemie, 1902, xxxvi,
p. 462.
2 Schulze, E., & Winterstein, ibid., 1902, xxxv, p. 300.

EXPERIMENTAL. 95

1 84°. Heated side by side with the corresponding substance obtained from
gliadin, the hydantoic acid from glutenin melted at 20 higher.

Carbon and hydrogen: 0.3277 gram substance, dried at 8o°, gave 0.S067 gram C02 and

o. 1891 gram H20.
Calculated for C16H18OjN2, C 67.07, H 6.35 p. ct.; found, C 67.14, H 6.42 p. ct.

From the filtrate from the amino-valerianic acid there was further obtained
1. 4 1 grams of alanine. The alcoholic solution which contained the proline
was evaporated to dryness under reduced pressure and the dried residue
again treated with boiling absolute alcohol. Even after several repetitions
of this process no substance insoluble in alcohol could be obtained. The
alcohol- soluble substance, when dried, weighed 35.54 grams. A copper salt
was prepared from this in the usual manner, and its solution evaporated to
dryness under reduced pressure. The dried residue was extracted with boil-
ing absolute alcohol in order to remove the /-proline copper. The residue
insoluble in alcohol, when recrystallized from water, gave 15.53 grams of
racemic proline copper, equivalent to 10.9 grams of proline.

Water : 0.361S gram substance lost at no° 0.0399 gram of H20.
Copper : 0.3176 gram substance, dried at no°, gave 0.0856 gram CuO.
Calculated for C10H16O4N2 Cu ■ 2H.O, H20 11.00 p. ct. ; found, H20 11.03 P- ct-
Calculated for C10H16O4N2 Cu, Cu 21.79 P- ct- I found, Cu 21.54 p. ct.

The alcoholic solution of the /-proline copper salt was evaporated to dry-
ness, the copper removed, and the proline identified as the phenylhydantoin
which was prepared according to the directions of Fischer.1 The substance
thus prepared was at once pure and melted at 143°.

Carbon and hydrogen: 0.2822 gram substance, dried in vacuo over H,S04, gave 0.6866

gram CO, and o. 1494 gram H20.
Nitrogen: 0.1025 gram substance gave NH3 = 1.33 cc. HC1 ( 1 cc. HC1 = 0.01 gram N).
Calculated for C12H1202N2, C 66.60, H 5.61, N 12.99 P- ct- \ found, C 66.36, H 5.88,

N 12.98 p. ct.

f Fraction IV, A. Temperature of bath, up to 1550.
(Pressure, 1.5 mm. Weight, 43.32 grams.

This fraction was shaken out with ether in the usual way and the ether
allowed to evaporate spontaneously. No evidence of the presence of phenyl-
alanine was obtained. The residue of ester was saponified with concentrated
hydrochloric acid and the hydrochloride decomposed with ammonia. The
free acid crystallized in the characteristic form of leucine and decomposed at
298 °. There were obtained 13.99 grams of leucine.

Carbon and hydrogen: 0.2906 gram substance, dried at no°, gave 0.5825 gram C02

and 0.2690 gram H20.
Calculated for C6H130,N, C 54.89, H 10.01 p. ct. ; found, C 54.67, H 10.2S p. ct.

Fischer, E., Zeitschrift fur physiologische Chemie, 1901, xxxiil, p. 151.

the; proteins of the wheat kernel.

The aqueous layer was'saponified by heating on a water-bath with an excess
of barium hydroxide for 5 hours. The barium aspartate, which separated in
considerable quantity on standing, was united with that obtained from frac-
tion V and treated as will be described later.

From the filtrate from the barium aspartate no definite substance could be
obtained. It appeared to contain serine, but none could be isolated, even by
the use of ,3-naphthalene-sulphone-chloride.

Fraction.

Temper-
ature of

bath
up to —

Pressure.

Weight.

V, A

200
180

mm.
0.8
0.8

Grams.
32-56
23.92

IV, B

These esters were shaken 'out with ether, and the substance extracted
was saponified with hydrochloric acid. The hydrochloride thus obtained,
which weighed 20.21 grams, equal to 16.55 grams free phenylalanine, was
converted into the free acid with ammonia and then into the copper salt by
boiling its solution with copper hydroxide.1

Copper : 0.2099 gram substance, dried at 1100, gave 0.0425 gram CuO.

Nitrogen: 0.22S3 gram substance gave NH3 = 1.63 cc. HC1 (1 cc. HCl = o.oi gram N)-

Calculated for C18H20O4N2Cu, Cu 16.23, N 7.17 p. ct. ; found, Cu 16.18, N 7.14 p. ct.

The free phenylalanine isolated from this copper salt melted at 2630 to 2650.

Nitrogen: 0.1085 gram substance gave NH3 = 0.92 cc. HC1 (1 cc. HC1 = 0.01 gram N).
T Calculated for C9Hu02N, N 8.50 p. ct.; found, N 8.48 p. ct.

The aqueous layer was saponified by heating for 5 hours with an excess
of barium hydroxide on the water-bath. The barium aspartate, which sepa-
rated on standing, was united with that previously obtained from fraction
IV, A, decomposed with an equivalent amount of sulphuric acid, and 7.12
grams of free aspartic acid were obtained from the solution.

Carbon and hydrogen: 0.3293 gram substance, dried at no°, gave 0.4335 gram C02and

0.1625 gram H20.
Nitrogen: 0.2997 gram substance gave NH3 = 3.i7 cc. HC1 (1 cc. HCl = o.oi gram N).
; Calculated for C4H704N, C 36.05, H 5.31, N 10.55 P- ct. ; found, C 35.90, H 5.48,
N 10.58 p. ct.

The filtrate from barium aspartate was freed from barium, concentrated
under reduced pressure, and saturated with hydrochloric acid. On long
standing on ice a trace of phenylalanine hydrochloride separated, but no
glutaminic acid hydrochloride could be obtained. After removing the

*Cf. Schulze & Winterstein, Zeitschrift fur physiologische Chemie, 1902, xxxv, p. 210.

EXPERIMENTAL. 97

hydrochloric acid with silver sulphate, the solution was boiled with a solu-
tion of copper hydroxide and 1.1 grams of copper aspartate were isolated.

Copper : 0.3991 gram substance, air-dried, gave o. 1163 grain CuO.

Nitrogen : 0.2066 gram substance gave NH.= 1.06 cc. HC1 (1 cc. HC1 =0.01 gram N).
Calculated for C4H504N, Cu • 4^ H.,,0,1 Cu 23.06, N 5.09 p. ct. ; found, Cu 23.27, N 5.13
p. ct.

The filtrate from this copper aspartate, when freed from copper by hydro-
gen sulphide and concentrated to small volume, yielded, by fractional crys-
tallization, 4.35 grams of nearly pure serine.

Carbon and hydrogen: 0.2847 gram substance gave 0.3559 gram C02 ando.1751 gram

H20.
Calculated for C3H703N, C 34.29, H 6.67 p. ct. ; found, C 34.09, H 6.83 p. ct.

This substance browned at about 213° and decomposed to a brownish mass
at about 2430.

Residue from Distillation.

The residues which remained after distilling off the esters weighed 304.5
grams. These were dissolved in hot alcohol, and from their united solutions
3.16 grams of substance separated on cooling. The filtrate from this sub-
stance was evaporated under reduced pressure, the residue dissolved in water
and saponified by heating with an excess of barium hydroxide for 9 hours.
After removing the barium quantitatively, the solution was concentrated and
saturated with hydrochloric acid. On standing for some time on ice, 38.25
grams of glutaminic acid hydrochloride, which melted at 1980, were ob-
tained. This was equivalent to 30.64 grams of free glutaminic acid, and,
with that previously isolated, made a total of 193.04 grams, or 23 per cent
of the glutenin. The glutaminic acid hydrochloride was decomposed with
an equivalent quantity of potassium hydroxide, and the free glutaminic acid
was recrystallized from water. It melted at 202 ° to 203 ° with effervescence.

Carbon and hydrogen: 0.3504 gram substance, dried at 1100, gave 0.5275 gram C02

and 0.2003 gram H20.
Calculated for C5H904N, C 40.82, H 6.12 p. ct. ; found, C 41.06, H 6.35 p. ct.

Residue after Esterification.

The residue which remained after the third esterification and extraction
of the esters was treated in the same way as the corresponding residue from
gliadin (p. 77), and the solution, freed from all mineral salts and bases
precipitable by phosphotungstic acid, was concentrated under reduced press-
ure to a small volume and then left for some time over sulphuric acid.

1Ritthausen, Die Eiweisskorper, etc., Bonn, 1872, p. 219.

98 THE PROTEINS OF THE WHEAT KERNEL.

After removing a little tyrosine that first separated, 1.87 grams of serine
crystallized out, which, when recrystallized from water, browned at about
2100 and decomposed at 2400.

Carbon and hydrogen: 0.2308 gram substance, dried at no°, gave 0.2894 gram C02 and

o. 1407 gram H20.
Calculated for C,H703N, C 34.29, H 6.67 p. ct. ; foun.l, C 34.19, H 6.77 p. ct.

This, with the serine obtained from fraction V, gives a total of 6.22 grams
of serine isolated. The mother-liquor from the serine contained consider-
able substance, but no oxy-proline or other definite substance could be

obtained from it.

cystine.

300 grams of glutenin were hydrolyzed in the way described for gliadin
(p. 80). After evaporating at low pressure to a sirup, neutralizing the
remaining excess of acid with sodium hydroxide, and decolorizing the solu-
tion with bone-black, a considerable quantity of tyrosine separated out,
which, on examination, was found to contain nearly all the cystine that
could be detected in the solution. It was therefore dissolved in 5 per cent
sulphuric acid and the cystine precipitated with mercuric sulphate. The
mercury precipitate was decomposed with hydrogen sulphide, the solution
concentrated somewhat, made alkaline with ammonia and then acid with
acetic acid, and an equal volume of alcohol added. The cystine, which sep-
arated on standing in characteristic hexagonal plates, weighed only 0.17
gram. No more could be obtained. This was dissolved in ammonia and
reprecipitated by acetic acid.

Sulphur : 0.0897 gram substance, dried at no0, gave 0.1730 gram BaS04.
Calculated for C6H1,04N2S2, S 26.68 p. ct. ; found, S 26.53 P- ct.

Although glutenin contains about the same amount of sulphur as gliadin,
the amount of cystine obtained from the latter under similar conditions was
very much greater. It would seem as if glutenin in fact yields less cystine,
though the uncertainties attending the isolation of this substance will not
permit of a positive conclusion.

TYROSINE.

250 grams of glutenin were boiled with a mixture of 750 grams sulphuric
acid and 1500 grams of water for 12 hours. The solution was freed from
sulphuric acid by an equivalent amount of barium hydroxide, and after
concentrating to 800 cc. allowed to stand for some time. A considerable
quantity of tyrosine separated, which was filtered out, the filtrate boiled
with barium carbonate in order to expel ammonia, and then concentrated to
one-half its original volume. After cooling, the residue of barium carbonate

EXPERIMENTAL. 99

and other substances which had separated were extracted with hot dilute
ammonia and the filtered extract concentrated and cooled. On standing, a
little more tyrosine separated, which was added to that first obtained. No
more tyrosine could be isolated from the solution of the hydrolytic decom-
position products. All of the tyrosine which had separated was dissolved
in 5 per cent sulphuric acid, and phosphotungstic acid was added to the
solution. Only a small precipitate resulted. After removing the phospho-
tungstic acid with barium hydroxide, the solution was concentrated strongly
and allowed to cool. After standing for some time, 9.62 grams of tyrosine
were obtained, which is equal to 4.25 per cent of the glutenin.

Carbon and hydrogen: 0.2922 gram substance, dried at uo°, gave 0.6370 gram CO, and

0.1634 gram H20.
Calculated for C9HU03N, C 59.62, H 6.13 p. ct.; found, C 59.45. H 6.21 p. ct.

Kutscher1 found 2.75 per cent of tyrosine in " gluten- casein."

HISTIDINE.

Fifty grams of glutenin, equal to 43.39 grams dried at no°, were hydro-
lyzed, and the arginine and histidine separated in the same way as that
described for gliadin (p. 81). The solution containing the histidine was
made up to 500 cc.

Nitrogen: 100 cc. solution gave NH3 =4.15 cc. HC1 (1 cc. HC1 =0.01 gram N) =
0.0415 gram N = 0.2075 gram in 500 cc. = 0.7645 gram histidine = 1.76 p. ct. of
the glutenin.

The amount of histidine in the remaining solution was too small for

identification.

arginine.

The filtrate from the histidine precipitate yielded 500 cc. of solution con-
taining the arginine, in which was found the following amount of nitrogen :

Nitrogen: 50 cc. solution gave NH3 = 6.79 cc. HC1 (1 cc. HC1 = 0.01 gram N) =
0.0679 gram N, or 0.679 gram in 500 cc, or 2.107 gram arginine = 4.72 p. ct. of the
glutenin.

The remaining solution, treated as Kossel directs, yielded the arginine as
carbonate. This was converted into the copper salt, which gave the fol-
lowing results on analysis :

Carbon and hydrogen: 0.2118 gram substance, air-dried, lost 0.0210 gram H20 at ioo°.
Calculated for C12H28O10N10 Cu ■ 3 H20, H20 9.15 p. ct. ; found, H20 9.92 p. ct.
Copper : (I) 0.1858 gram substance, dried at ioo°, gave 0.0275 gram CuO ; (II) 0.1808

gram substance, dried at ioo°, gave 0.0267 gram CuO.
Calculated for C12H28O10N10 Cu, Cu 11.85 p. ct. ; found, Cu (I) 11.84, (II) 11.78 p. ct.

1 Kutscher, Zeitschrift fur physiologische Chemie, 1903, xxxvi, p. 114.

IOO THE PROTEINS OF THE WHEAT KERNEL.

Kossel & Kutsclier1 found in three separate determinations of arginine
in glutenin 4.50, 4.02, and 4.54 per cent. They base their determinations
on the supposition that glutenin contains 16.2 per cent nitrogen. If their
results are recalculated to a basis of 17.5 per cent of nitrogen which has been
found in this protein,2 they become 4.82, 4.52, and 4.84 per cent, with
which 4.72 per cent agrees very closely.

lysine.

The filtrate from the arginine silver was treated as Kossel directs, and
after precipitating the lysine with phosphotungstic acid it was converted
into the picrate, and 2.33 grams, equivalent to 0.907 gram of free lysine,
were obtained. This is equal to 1.92 per cent of the glutenin. Kossel &
Kutscher found in the three determinations of this substance which they
made in this protein 1.9, 2.29, and 2 per cent, or, recalculating to a basis of
17.5 per cent of nitrogen in this protein, 2.05, 2.15, and 2.40 per cent.

Nitrogen: 0.1225 gram substance, dried at 1200, gave 23.1 cc. moist N2 at 760 mm.

and 290.
Calculated for C6Hu02N2 • C6H307N3) N 18.70 p. ct. ; found, N 18.76 p. ct.

The results of this hydrolysis are given in table 19.

Table 19. — Glutenin.

P.ct.

Glycocoll 0.89

Alanine 4.65

Amiuo-valerianic acid 0.24

Leucine 5.95

a-proline 4.23

Phenylalanine 1.97

Aspartic acid 0.91

Glutaminic acid 23.42

Serine 0.74

P.ct.

Tyrosine 4. 25

Cystine 0.02

Lysine 1.92

Histidine 1 .76

Arginine 4.72

Ammonia 4.01

Tryptophane present

Total 59.66

THE AMOUNT OF THE VARIOUS PROTEINS CONTAINED IN THE

KERNEL OF WHEAT.

1000 grams of fine meal obtained by freshly grinding the entire kernel of
spring wheat and a like quantity of similar flour from winter wheat were
each extracted with 4000 cc. of 10 per cent sodium-chloride solution, and
2500 cc. of the clear extract were obtained from the spring-wheat flour and
2600 cc. from the winter- wheat flour. As 100 cc. of solution were used for
every 25 grams of flour taken, the amount of extract obtained was approx-
imately equal to that from 625 grams of spring- wheat flour and 650 grams of
winter-wheat flour. These extracts were then dialyzed until all the chlo-

1 Kossel & Kutscher, Zeitschrift fur physiologische Chemie, 1900, xxxi, p. 165.

2 Osborne & Voorhees, American Chemical Journal, 1893, xv, 392.

EXPERIMENTAL. 101

rides were removed, which required 5 days. The precipitated globulin was
then filtered from each, washed with distilled water, alcohol, absolute alcohol,
and ether, removed from the filter, and dried at no°. From the spring-
wheat extract 3.8398 grams were obtained, equal to 0.624 per cent of the
flour, and from the winter wheat 3.9265 grams, equal to 0.625 per cent.

The filtrates from the globulin were then heated to 65 ° in a water-bath,
and after being held at this temperature for some time the coagulum was
filtered off and washed with hot water, alcohol, and ether, removed from
the paper, and dried at no0. From the spring wheat 1.97 14 grams were
obtained, being 0.315 per cent of the flour, and from the winter wheat
1.9614 grams, equal to 0.302 per cent.

The solutions filtered from each of these coagula were next heated just
to boiling and the resulting coagulum filtered off, washed thoroughly, and
treated as the preceding preparations had been. The spring- wheat extract
thus yielded 0.4743 gram, equal to 0.076 percent ; the winter-wheat extract
0.3680 gram, equal to 0.057 Per cent.

The two extracts were next concentrated by boiling down over a lamp.
They remained clear at first, but when somewhat concentrated the protein
began to separate as a skin on the surface of the solution. When reduced
to about one-fourth its original volume, the coagulum was filtered off, washed
with boiling water, alcohol, and ether, and dried at 1 io°. The spring wheat
thus yielded 0.8737 gram> equal to 0.139 per cent ; the winter wheat 0.8721
gram, equal to 0.134 per cent. The filtrates from these two preparations
were evaporated very nearly to dryness on water-baths. On cooling, much
substance separated, which, when treated with hot water, dissolved again.
The insoluble coagulum was filtered from each, washed, and dried in the
usual manner. The spring wheat gave 0.8149 gram of substance, being
0.130 per cent of the flour ; the winter wheat 0.5795 gram, being 0.089 Per
cent. The total amount of protein coagulating on concentration was there-
fore 0.269 Per ceQt for the spring wheat and 0.223 per cent for the winter
wheat.

The filtrates from these second coagula were then again evaporated to a
sirup, and as no more insoluble matter separated they were each precipitated
by pouring into strong alcohol. Large precipitates resulted in each case,
which, after settling and decantation of the alcohol, were dissolved again in
a little water and precipitated by pouring into strong alcohol. Much color-
ing matter and sugar was held in solution, as proved by evaporation of the
alcoholic mother-liquors. The precipitates were then thoroughly dehydrated
with absolute alcohol, washed with ether, and dried at no°. The spring-
wheat extract thus yielded 6.9289 grams of substance, the winter wheat
8.7517 grams. As these preparations were unquestionably very impure

102

THE PROTEINS OE THE WHEAT KERNEL.

and no practicable method of purification existed which could be carried out
without great loss of substance, the nitrogen in each was determined and
the protein calculated by multiplying the result obtained by 6.25. The
spring-wheat preparation was thus found to contain 3.07 per cent of nitro-
gen, equal to 19.19 per cent of protein, and the winter-wheat preparation
5.15 per cent nitrogen, equivalent to 32.18 per cent. The amount of pro-
teose and peptone thus found in the extract from the spring wheat was
1.3297 grams, which equals 0.213 per cent of the flour, and in the winter-
wheat extract 2.8063 grams, equal to 0.432 per cent of the flour.

The sodium chloride extract contained, therefore, the following amounts
of protein :

Spring
wheat.

Winter
wheat.

Globulin

P.ct.
0.624
0.391
0.269
0.213

P. ct.
0.625

o-359
0.223
0.432

Albumin

Coaguluru

Proteose

1-497

1-639

The remainder of the protein matter of the seed forms the gluten. The
proportion of gliadin and glutenin in this gluten was determined in the fol-
lowing manner :

200 grams of spring-wheat flour made from the entire seed and a like
quantity of a similar winter-wheat flour were each made into a dough and
thoroughly washed with water as long as starch was removed. The gluten
thus obtained, after freeing from loosely adhering moisture, was weighed
and exactly one-half dried at 1100 to constant weight. The spring wheat
was thus found to yield 12.685 Per cent dry gluten, the winter-wheat flour
11.858 per cent.

The other half of the gluten was cut up very fine and extracted as thor-
oughly as possible with alcohol of 0.90 sp. gr. The alcoholic extract was
then evaporated to small volume, cooled, and the solution decanted from the
precipitated protein. This precipitate was then extracted with ether and
dried at no°. From the spring-wheat gluten 4.3379 per cent and from the
winter wheat 4.2454 per cent of alcohol-soluble protein were obtained.

The residue extracted with alcohol was then dried at no° and weighed.
The spring- wheat gluten contained 7.80 per cent insoluble in alcohol, the
winter wheat 7.504 per cent, reckoned on the wheat. Nitrogen was then
determined in these residues dried at 1100, as well as in the dried gluten

EXPERIMENTAL.

103

and also in the original flours. The washings of the glutens were collected
in jars and allowed to settle, the sediments washed with water and with
very strong alcohol and dried and weighed. The nitrogen in each was then
determined. The results of these determinations and deductions drawn
from them are given in table 20.

Table 20. — Amount of the various proteins of the wheat kernel.

Total nitrogen in the flour

Total gluten in the flour

Part of gluten insoluble in alcohol

Per cent of nitrogen in gluten

Total nitrogen in gluten in per cent of flour

Total nitrogen in residue of gluten insoluble in alcohol

Total nitrogen extracted by alcohol

Gliadin (N X 5-68), assuming 17.60 per cent nitrogen in gliadin

Gliadin by direct weighing

Nitrogen in sediment from washing gluten

Spring
wheat.

Winter
wheat.

P.ct.
1 -950

12.685
7.800

12.010
1.5222
0.8245
0.6977
3-9630

4-3379
0.2239

P.ct.

1.940

n.858

7-504
12.000
1.4230
0.7346
0.6884
3.9100
4-2454
O.I552

Glutenin

Gliadin

Globulin

Albumin

Coagulum

Proteose

From H20 washings of gluten

Spring wheat.

Winter wheat.

Nitrogen.

P.ct.
0.S245 X 5-68
0.6077 X 5.68
o. 124S
0.0657
0.0453
0.0341
0.2239 X 5

2.0050

Protein.

P. a.
4.6S3

3-963
0.624

0.391
0.269
0.213
1.272

n.4'5

Nitrogen.

P.ct.
o.7346 X 5-68
0.6884 X 5 68
o.l 148
0.0603
0.0379
0.0791
0.1552 X 5-68

1.8703

Protein.

P.ct.

4-173
3.910
0.625

o-359
0.223

0.432
c.881

10 603

THK FORMATION OF GLUTEN.

Wheat, so far as known, is the only plant whose seeds contain protein
matter separable in a coherent form from the other constituents by washing
with water. When ground fine and mixed with a suitable quantity of water,
it yields a dough from which a light, porous bread can be made. The im-
portance of this fact in bread-making is so great that considerable attention
has been paid to gluten by the chemists who have studied wheat proteins.

Reference has already been made (p. 45) to the statements of Weyl &
Bischoff that the protein matter of the wheat kernel is chiefly a globulin sub-
stance, which in contact with water undergoes a change, presumably through

104 THE PROTEINS OF THE WHEAT KERNEL.

the influence of a ferment by which gluten results. If the statements of
these investigators are examined no evidence will be found to support their
view. What their reasons were for concluding that ' ' myosin ' ' formed
nearly all the protein of the wheat kernel does not appear. In view of
the results obtained by the writer, this statement is certainly erroneous.
Direct treatment of the meal with alcohol yielded extracts containing gliadin
in exactly the same amount as obtained from the gluten made from an equal
quantity of flour, and extraction of either flour or gluten with alcohol, after
complete exhaustion with sodium-chloride solution, also gave the same pro-
portion of gliadin. This substance must therefore have existed in the seed,
and, as it forms one-half of the gluten, it leaves the other half only as
possibly derived from a globulin body through the influence of a ferment.
If Weyl & Bischoff's view were correct, treatment of the flour with 10 per
cent salt solution ought to alter the character and quantity of the gluten
obtained, if not altogether to prevent its formation. This is not so, for the
usual amount of gluten can readily be obtained from flour made into dough
with ioper cent sodium-chloride solution and then washed with the same
until starch is removed.

Weyl & Bischoff next state that " with the aid of a 15 per cent rock-salt
solution the flour was extracted until no protein could be detected in the
extract; the residue of the meal kneaded with water then gave no gluten.
If the globulin substance is extracted, no formation of gluten takes place." It
has been found that this is true if the flour is stirred up with a large quantity
of salt solution, extracted repeatedly with fresh quantities of the same solu-
tion until no more protein is dissolved, and the excess of solution removed
by allowing the residue to drain on a filter as completely as possible. If,
however, wheat flour is mixed at first with just sufficient salt solution to
make a firm dough, this dough may be washed indefinitely with salt solution,
and will yield gluten as well and as much as if washed with water alone.
This difference is due to the fact that when large quantities of salt solution
are applied at once the flour fails to unite to a coherent mass and can not after-
ward be brought together, as is possible when treated with smaller quantities
of solution.

Weyl & Bi?choff then compare the formation of gluten to that of blood-
fibrin from fibrinogen under the influence of a ferment.

Sidney Martin next advanced a somewhat similar theory of the formation
of gluten from the proteins contained in the seed. He states that alcohol
extracts from gluten but one protein substance ; that this is soluble in hot
water, but not in cold, and he therefore calls it an insoluble phytalbumose.

The residue of the gluten not dissolved by alcohol is uncoagulated pro-
tein, if the alcohol has not been allowed to act too long. This substance he

EXPERIMENTAL. I05

names gluten-fibrin. Martin further says that gluten dissolves almost com-
pletely in 0.2 per cent hydrochloric acid or 0.2 per cent potassium-hydroxide
solution, leaving a small residue of fat. The solution gives a copious pre-
cipitate when neutralized, but the supernatant liquid still contains a quantity
of protein which is the dissolved insoluble albumose. The whole of the
gluten-fibrin is reprecipitated by neutralization — that is, it is wholly con-
verted into an albuminate.

Martin states that by extracting flour with 76 to 80 per cent alcohol only
fat is removed. This statement is certainly erroneous, for the writer has
never failed in many experiments thus to extract this substance (gliadin)
from the flour, and that, too, in the same amount and of the same properties
and composition as from the gluten.

Martin concludes that insoluble albumose is not present as such in the
flour. He then says :

Before proceeding to mention its precursor, it will be well to state that 10 per cent
sodium-chloride solution extracts from flour a large quantity of globulin and of albumose.
This globulin is of the myosin type, coagulating between 550 and 6o° C. , and precipitated
by saturation with sodium chloride and ammonium sulphate. Both the globulin and
albumose are present in a much smaller quantity in the watery extract of the flour.

From this it is evident that Martin has fallen into the same error as Weyl
& Bischoff, mistaking the albumin for a myosin-like globulin, and being
greatly misled as to its amount. Continuing, Martin says :

The direction of the evidence is to show that the insoluble albumose is formed from
the soluble. Moreover, I think that the globulin is transformed into the gluten-fibrin,
for I have been able to obtain from the globulin in solution a body having the same
reactions as the gluten-fibrin.

What this evidence is which by its direction shows that the insoluble
albumose is derived from the soluble is not clear, and Martin makes no
further statements on this point. That a body should be obtained from the
solution containing the globulin which had the same reactions as the
"gluten-fibrin" is not surprising, for the insoluble products derived from
nearly all globulins have no characteristic reactions, being merely soluble
in dilute acids and alkalis and precipitated by neutralization in the same
way as " gluten-fibrin." Martin then states his theory of the formation of
gluten in the following scheme :

Gluten = I Gluten-fibrin — precursor, globulin.

" \ Insoluble albumose — precursor, soluble albumose.

This can not be a correct representation of the formation of gluten, for it
has been shown to be founded on two erroneous observations — first, that
alcohol does not extract protein matter from the flour when applied directly,
and, second, that at least one-half the protein matter of the seed is a myosin-
like globulin.

106 THE PROTEINS OF THiC WHEAT KERNEL.

The results obtained by the author and described in this paper have led
to the conclusion that no ferment-action is involved in the formation of
gluten ; that but two protein substances are contained in the gluten, glu-
tenin and gliadin, and that these exist in the wheat kernel in the same
form as in the gluten, except that in the latter they are combined with
water in an amount equal to about twice the weight of the dried protein.
The reasons for this opinion are, first, that alcohol extracts the same protein
and in the same amount, whether applied directly to the flour, to the gluten,
or to flour previously extracted with 10 per cent sodium-chloride solution ;
second, that 0.2 per cent potassium-hydroxide solution extracts glutenin of
uniform composition and properties from flour which has been extracted with
alcohol or with 10 per cent sodium-chloride brine and then with alcohol as
it extracts from gluten which has been exhausted with alcohol.

Both glutenin and gliadin are necessary for the formation of gluten,
as may be seen from the following experiments : A portion of flour was
washed completely free from gliadin by means of alcohol of 0.90 sp. gr.,
next with stronger alcohol, finally with absolute alcohol, and air-dried.
The residue was then rubbed up fine until all lumps were removed, and
water caref ully added and a dough made of the mass. A tolerably coherent
dough was thus obtained, but much less elastic and tough than that pro-
duced from the untreated flour. This dough was then washed with water
on a sieve, using every precaution to obtain a gluten, but none was formed.

In another experiment 7.5 grams of very finely ground air-dried gliadin
were mixed intimately with 70 grams of fine corn-starch and distilled water
added. A plastic dough was thus produced, but it had no toughness. On
adding a little 10 per cent sodium-chloride solution, the dough became at
once tough and elastic. This was then washed with great care on a sieve
with cold water, a little 10 per cent sodium-chloride solution being added
from time to time, but in spite of every precaution no gluten was obtained.

The following experiment shows that the gliadin used was capable of
forming gluten when glutenin was present, and also that salts have a marked
influence on the toughness of the resulting dough. Two portions of flour
weighing 100 grams each were taken, and after adding 5 grams of gliadin to
one both were made into dough with the same quantity of water. The
two doughs presented marked differences. That to which gliadin had been
added was much tougher and more yellow than the other. They were then
washed with water as long as starch separated. The gluten was dried
superficially by wiping with a cloth and weighed in the moist state. That
from 100 grams of flour to which 5 grams of gliadin had been added weighed
44.55 grams ; that from 100 grams of flour alone weighed 27.65 grams.
The moist glutens were dried at 1 ro° to constant weight, and both yielded

EXPERIMENTAL. 107

the same proportion of dry gluten, viz, 34.6 per cent. The yield of dry-
gluten was accordingly in the first case 15.41 grams and in the second 9.56
grams. The difference, 5.85 grams, shows that the added gliadin was fully
recovered in the gluten.

The figures show that these proteins combine with about twice their
weight of water in forming gluten. The fact that the added gliadin entered
so readily and completely into the formation of gluten indicates that it
exists in the seed as such and undergoes no chemical change during extrac-
tion and drying.

The properties observed in testing the separated gliadin show how it acts
in forming gluten and explain many of the points observed by others and
attributed to a ferment-action.

When treated with distilled water in small amount, the fine-ground air-dry
gliadin at once forms a sticky mass, which, on adding more distilled water,
dissolves to a turbid solution. If, however, a very little sodium chloride is
added to distilled water and this applied to gliadin that has been first
moistened with pure water, a very coherent, viscid mass results, which
adheres to everything it touches and can be drawn out into long threads.
If the gliadin is moistened with 10 per cent sodium-chloride solution and then
treated with a larger quantity of this solution, the substance unites to a
plastic mass, which can be drawn out into sheets and strings, but is not
adhesive. From this it is evident why Ritthausen, in washing flours which
gave a fluid gluten, obtainable only in small quantity and with great diffi-
culty, found that the addition of calcium sulphate to the wash-water ren-
dered the gluten much more coherent and easily obtainable. The gliadin is
thus proved to be the binding material which causes the particles of flour to
adhere to one another, thus forming a dough. But the gliadin alone is not
sufficient to form gluten, for it yields a soft and fluid mass, which breaks up
entirely on washing with water. The insoluble glutenin is probably essen-
tial by affording a nucleus to which the gliadin adheres and from which it
is not mechanically carried away by the wash-water.

The behavior of the gliadin toward 10 per cent sodium-chloride solution
shows why no gluten was obtained by Weyl & Bischoff from flour extracted
with this solvent. The gliadin had under these conditions no adhesive quali-
ties, and therefore was unable to bind the flour into a coherent mass. If,
however, the salt solution is added in small quantities and the flour kneaded
and pressed, the particles are brought together and then adhere tenaciously.

IOS THE PROTEINS OF THE WHEAT KERNEL.

SUMMARY.

The proteins of the wheat kernel are gliadin, insoluble in neutral aqueous
solutions, but distinguished from all the others by its ready solubility in
neutral 70 per cent alcohol ; glutenin? a protein having a similar elementary
percentage composition to gliadin, soluble in very dilute acid and alkaline
solutions, but insoluble in dilute alcohol or neutral aqueous solutions and
yielding a wholly different proportion of decomposition products when boiled
with strong acids ; leucosin, an albumin-like protein, freely soluble in pure
wrater and coagulated by heating its solution to 500 to 6o° ; a globulin similar
in composition and properties to many globulins found in other seeds, and
one or more proteoses which are present in very small quantity. It has
also been shown that the proteins obtained from the embryo of the wheat
are the globulin, albumin, and proteose above mentioned, and that these
form nearly all of the protein substance of this part of the seed. It thus
appears that these three proteins are contained chiefly in the embryo, and
that gliadin and glutenin form nearly the whole of the proteins of the endo-
sperm, or over 80 per cent of the total protein matter of the seed.

It is possible that a part of the albumin, globulin, and perhaps minute
quantities of the proteose are contained also in the endosperm, for these
proteins are always found in flour from which, in the milling process, the
embryo is very nearly completely separated. The uncertainty, however,
as to the completeness of this separation makes it questionable wmether or
not the small amount of these proteins found in the best flour is not due to
the presence of more or less of the embryo that escaped separation in the
milling process.

The flour of wheat differs from that of other seeds in forming a dough
when moistened with sufficient water, which, when washed with more water,
loses its starch, and finally yields a tough elastic mass, long known as wheat
gluten. This gluten contains the greater part of the protein matter of the
seed, together with a little starch, fat, lecithin, and phytocholesterin, and
possibly some carbohydrate substance or substances of as yet unknown char-
acter. These non-protein substances are probably not united with one another
in the gluten, but are mechanically mixed. The quantity of starch that
remains in the gluten depends on the thoroughness of the washing, wmile
the other substances owe their presence largely to their insolubility in water.
The chief constituents of the gluten are the two proteins, gliadin and glu-
tenin, the relative proportions of which vary with the variety of wheat from

1 This is the protein which Ritthausen called " gluten-casein."

SUMMARY. IO9

which the flour is made. The character of the gluten and the commercial
value of the flour depend to a large extent on the proportion of gliadin to
glutenin.

In the moist gluten these proteins are present combined with about twice
their weight of water, which is gradually lost on exposure to dry air or at an
elevated temperature.

The gliadin and glutenin are present as such in the seed and are not, as
was formerly supposed, derived from other protein substances through the
action of an enzyme. This is shown by the fact that they may be obtained
directly from the flour by the same treatment as that which yields them from
the gluten and under conditions which preclude the action of an enzyme.

GLIADIN.

Gliadin is the most important of the five proteins above mentioned, not
only on account of its influence on the character of the gluten, and therefore
on the quality of the flour for domestic purposes, but also on account of its
unusual physical properties and chemical constitution.

Gliadin, unlike most other protein substances, is freely soluble in relatively
strong ethyl alcohol. Although gliadin is wholly insoluble in absolute alcohol
and but slightly soluble in water, it dissolves in dilute alcohol, the solubility
increasing with increasing concentration of alcohol until a certain strength
is reached, when the solubility diminishes until, by absolute alcohol, it is no
longer dissolved. Exactly what strength of alcohol dissolves the largest
proportion of gliadin has never been determined, but the maximum solubility
is attained with about 70 per cent of alcohol by volume.

Gliadin is also soluble in other alcohols, as methyl, propyl, and benzyl
alcohols, in phenol and paracresol, and also in glacial acetic acid.

Gliadin is somewhat soluble in pure water, but less so in water containing
salts, though not wholly insoluble in solutions containing 10 per cent of
sodium chloride. Very dilute acid or alkaline solutions dissolve gliadin to
solutions which, by neutralizing, yield the gliadin apparently unchanged. By
stronger solutions of acids or alkalis the gliadin is altered, as is the case
with all other native proteins.

All the usual color tests given by other protein substances are obtained
with gliadin, which therefore contains the groups that give rise to these re-
actions. Gliadin is much less easily converted into insoluble products than
are most other proteins. Its solution in 70 per cent alcohol can be boiled
for an indefinite time, and even concentrated until much of the alcohol has
been removed, without forming insoluble products. On heating with very
weak alcohol or with water this protein is gradually altered and becomes
insoluble in stronger alcohol, but the coagulated gliadin thus formed is in

THE PROTEINS OF THE WHEAT KERNEL.

appearance unlike the heat coagulum formed by most other proteins. Pro-
teins of similar behavior to gliadin are found in the seeds of other cereals,
such as rye, barley, maize, oats, and sorghum. That found in rye is prob-
ably identical with the gliadin of wheat, for a rigid comparison has not yet
revealed any difference. Both have the same ultimate composition, the
same solubility, the same physical properties, and yield the same amount of
ammonia and glutaminic acid on hydrolysis. The identity of the two, how-
ever, is not certain, and with our present knowledge can not be established.
The proteins of the other cereals above mentioned are distinctly different,
though similar, proteins.

Proteins characterized by such ready solubility in strong alcoholic solu-
tions as are those found in these cereals have not been obtained from the
seeds of any other plants.

The ultimate composition of gliadin has been fixed within narrow limits
by the accordant analyses of several investigators as follows :

Giiusberg.

Ritthausen.

Osborne &
Voorhees.

Nasmith.

Konig &
Rintelen.

Carbon

Hydrogen . .

Nitrogen

Sulphur

P.ct.
52.67
6. S3
17.62

P. Ct.

52.76
7. 10

18.01
0.85

21.28

P. Ct.

52.72
6.86

17.66
1.03

21.73

P. Ct.

52.39
6.84

17-47
1. 12

22.18

P.ct.
52.70
7.62

17.77

095
20.96

Oxygen. ....

22.88

100.00

100 00

Gliadin yields on hydrolysis a larger amount of ammonia, glutaminic acid,
and proline than any other protein yet examined, no glycocoll or lysine, and
a relatively small amount of histidine and arginine, as the following analysis
shows :

Products of hydrolysis of gliadin.

P.ct.

Glycocoll 0.00

Alanine 2.00

Amino-valerianic acid 0.21

Leucine 5.61

Proline 7.06

Phenylalanine 2.35

Aspartic acid 0.58

Glutaminic acid 37-33

Serine o. 13

P.ct.

Tyrosine 1 . 20

Cystine 0.45

Lysine 0.00

Histidine 0.61

Arginine 3. 16

Ammonia 5.11

Tryptophane present

Total 65.81

Although gliadin gives a strong Molisch reaction, which is commonly con-
sidered to indicate the presence of a carbohydrate group, it gives no furfurol
when distilled with hydrochloric acid, as does ovalbumin, which has been

SUMMARY.

Ill

proved to contain such a group. Other evidence than that given by the
Molisch reaction is required before the presence of a carbohydrate group in
this protein can be assumed.

Nearly two-thirds of the total sulphur in gliadin is split off as sulphide
by boiling with alkalis, which would indicate the possibility that all the
sulphur is contained in a cystine complex. The amount of cystine isolated
is far below that required by such an assumption. The determination,
however, of this substance is not quantitative, and the figure given does not
necessarily prove that all of the sulphur may not be contained in a cystine-
yieldiug complex.

The specific rotation of gliadin in 80 per cent by volume ethyl alcohol, ac-
cording to determinations by the writer, which agree closely with those of

20°

others, is («) -^- = — 92.30.

The specific rotation in solvents of alcoholic nature has been recently
determined by Mathewson, who gives the following data :

(«)

40°
D

Methyl alcohol, 70 per cent. — 95. 650
Ethyl alcohol, 70 per cent. — 91-95°
Ethyl alcohol, 60 per cent. — 96. 66°
Ethyl alcohol, 50 per cent. — 98.450
Propyl alcohol, 60 per cent. — 101.100

»£

Phenol, 70 per cent — 123. 15

Phenol, anhydrous — 131.77

Paracresol — 121.00

Benzyl alcohol
Glacial acetic acid.

— 53-ioc

— 78.6oc

The amount of gliadin in different varieties of wheat differs to a large
extent, as shown by a considerable number of determinations that have been
recorded. Thus Teller found in a sample of Canadian white winter wheat
2.74 per cent, in one of Oregon white winter wdieat 2.85 per cent, and
in a red spring wheat from South Dakota 8.15 per cent. In other varieties
of wheat he and others have found quantities falling between these figures.

Although the total quantity of gliadin in the sample of red wheat above
mentioned was nearly three times as great as in that of the white wheat, its
percentage of the total proteins was much more uniform, being 42.5 and 34
per cent respectively. This appears to be generally true for most of the
wheats that have been examined.

Thus Shepard has found in 13 samples of durum wheat that the gliadin
formed from 40.2 to 48.6 per cent of the total protein (N X 5.7), in 9 sam-
ples of Northwestern spring wheat from 44.2 to 49 per cent, in 4 samples
of Kansas hard winter wheat from 40 to 50.7 per cent, and in 3 samples of
soft winter wheat from 40 to 47.4 per cent. The figures given by others are
similar, though a few fall between somewhat wider limits. It would seem
safe to say that in the majority of wheats the gliadin forms from 40 to 50
per cent of the total proteins, and that its absolute amount depends chiefly
on the total protein content of the seed.

112 THE TROTEINS OF THE WHEAT KERNEL.

GLUTENIN.

This protein was first described by Taddei under the name of zytnom.
Iyiebig, as well as Dumas & Cahours, named it " plant-fibrin ; " Ritthausen
called it ' ' gluten-casein ; ' ' Weyl & Bischoff considered it to be an albumi-
nate form of a myosin-like globulin. On the ground of priority and the fact
that the relations to animal proteins, which gave rise to the various names
subsequently applied to this body, have been proved to have no foundation,
it would be desirable to return to Taddei 's original name and in future call
this protein zymom. Unfortunately this name is derived from the Greek
word tofty, a ferment, and as the results of this and all other subsequent
investigations of this subject show that the supposed ferment-changes do not
occur in the formation of gluten, it seems appropriate to call it glutenin, a
name suggested by S. W. Johnson. Glutenin is next in importance to
gliadin, for in most varieties of flour it is present in nearly equal amount. It
is so nearly insoluble in water and alcohol that it is a question whether the
slight traces that are dissolved by these solvents from carefully purified prep-
arations are not due to traces of gliadin, which it is very difficult to separate
from it. In hot dilute alcohol glutenin is slightly soluble and separates
from such solutions on cooling. When freshly precipitated and in the
hydrated condition, it is very readily dissolved by extremely dilute acids or
alkalis and is precipitated from such solutions on neutralization. Glutenin
can be separated from the other constituents of the seed only by solution in
dilute alkalis or acids, and it is necessary to filter the solutions, from which
it is finally precipitated, perfectly clear in order to separate the associated
non-protein substances. This filtration is accomplished with great difficulty
unless it is preceded by thorough extraction of the crude glutenin with
alcohol and ether, whereby fats and lecithins are removed.

Glutenin contains all the groups which give rise to the usual color reac-
tions of the proteins. No protein similar to glutenin in physical and chem-
ical properties has yet been found in any other seed. Although its ultimate
composition is nearly the same as gliadin, the proportion of the various
products of hydrolysis which it yields is very different, as may be seen from
the following analyses :

The ultimate composition of glutenin,

P. ct.

Carbon 52.34

Hydrogen 6.83

Nitrogen 17-49

Sulphur 1.08

Oxygen 22.26

100.00

SUMMARY.

113

The following products of hydrolysis have been obtained by boiling
glutenin with strong hydrochloric acid :

Products of hydrolysis of glutenin.

P.ct.

Glycocoll 0.89

Alanine 4-65

Amino-valerianic acid 0.24

Leucine 5.95

^-proline 4.23

Phenylalanine 1.97

Aspartic acid 0.91

Glutaminic acid 23.42

Serine 0.74

P.ct.

Tyrosine ... 4.25

Cystine 0.02

Lysine 1.92

Histidine 1.76

Arginine 4.72

Ammonia 4-nI

Tryptophane present

59.66

Glutenin is qualitatively distinguished from gliadin by yielding both
glycocoll and lysine, and quantitatively by yielding less proline, glutaminic
acid, and ammonia and more alanine, tyrosine, and arginine. In other
respects the differences are not great.

The amount of glutenin varies greatly in different samples of wheats, but
usually forms about 40 per cent of the total protein of the seed. Teller has
found in a sample of Canadian white winter wheat 3.64 per cent of glutenin and
in an Oregon white winter wheat 3.82 per cent, while in a red spring wheat
from South Dakota he found 8.04 per cent. Although the total amount of
glutenin in these wheats was very different, the relative amounts were nearly
the same, being 45.2 and 46.5 percent of the total proteins in the white
winter wheats and 42 per cent in the red spring wheat. Other winter
wheats differed but little from other spring wheats both in the total and
relative amount of glutenin which they contained. In most of the wheats
that he examined the glutein formed from 40 to 45 per cent of the total
proteins. Shepard, who analyzed a much larger number of samples, found
a wider variation in the relative proportion of glutenin, from 34.2 to 46.8
per cent of the total proteins, and a narrower range in its total amount, the
latter falling between 3.41 and 7. 10 per cent.

LEUCOSIN.

Leucosin forms about 0.3 to 0.4 per cent of the wheat kernel. Although
the entire seed contains but little leucosin, the embryo contains a relatively
large proportion, since about 10 per cent of the commercial " germ meal "
consists of this protein. As this ' ' germ meal ' ' contains more or less of the
endosperm and outer coats of the seed, the embryo contains somewhat more
than 10 per cent. Leucosin is an albumin, for it is soluble in pure water
and coagulated by heating its solution. It has been mistaken by several
observers for a myosin-like globulin, owing to the fact that its temperature
of coagulation falls near to that of such globulins found in animal tissues,
8

114 THE PROTEINS OE THE WHEAT KERNEL.

and also to the fact that its solutions are precipitated by saturating them with
sodium chloride or with magnesium sulphate. That it is soluble in pure
water and not held in solution by small quantities of salts is shown by the
fact that a solution containing a considerable quantity of leucosin when sub-
jected to prolonged dialysis and then evaporated to dryness, and the leucosin
burned off at a low temperature left a residue of mineral matters weighing
less than a milligram. Leucosin is much more readily precipitated by am-
monium sulphate than are the albumins of animal origin, for by adding an
equal volume of a saturated solution of this salt to solutions containing leu-
cosin the latter is almost completely precipitated.

Leucosin resembles the animal proteins in ultimate composition, in the
proportion of its products of hydrolytic decomposition, and in its physical
properties more closely than it resembles most of the seed proteins yet studied.
It seems not improbable that this is because it is a constituent of the tissues
of the embryo, for its physiological functions are unquestionably different
from those of the stored-up food proteins of the endosperm.

Proteins having the same ultimate composition and, so far as known, the
same properties as leucosin are found in the seeds of other cereals. Whether
these are identical or not can not be determined by any means now available.

The ultimate composition of leucosin is shown by the following figures,
which are the average of accordant analyses of several preparations :

Composition of leucosin.

P.ct.

Carbon 53-02

Hydrogen 6.84

Nitrogen . 16.80

Sulphur 1.28

Oxygen 22.06

100.00

Leucosin yields the following amounts of the several products of hydrolysis
when boiled with strong hydrochloric acid :

Products of hydrolysis of leucosin.

P. ct.

Glycocoll 0.94

Alanine 4 . 45

Amino-valerianic acid o. 18

Leucine 1 1 . 34

a-proline 3.18

Phenylalanine 3.83

Aspartic acid 3-35

Glutaminic acid 6 . 73

P. ct.

Tyrosine 3-34

Lysine 2 . 75

Histidine 2 . 83

Arginine : 5-94

Ammonia 1 .41

Tryptophane present

50.32

Leucosin probably contains a relatively large proportion of tryptophane,
for in a series of comparative tests made with a large number of different

SUMMARY. 115

proteins the intensity of its glyoxylic acid reaction was greater than that of

any of the other proteins.

L,eucosin also gave the strongest reaction with the Molisch test of any of a

large number of different proteins examined under like conditions, but

whether or not it contains a carbohydrate group must be shown by other

evidence.

THE GLOBULIN.

The seeds of wheat contain about 0.6 per cent of a protein which is insol-
uble in water, but soluble in neutral saline solutions. This globulin is chiefly
contained in the embryo from which 5 per cent was extracted by sodium-
chloride solution. The preparations of the globulin that were made from
the embryo contained nucleic acid, while those from the whole seed con-
tained none. These nucleic acid compounds from the embryo behaved like
nucleates, for their proportion of nucleic acid was not constant, but varied
with the conditions of preparation. It seemed most probable that the nucleic
acid combines with the basic protein to form salts in the same manner as
other acids are known to do, and that such combinations with the globulin
still retained the solubility characteristic of the globulin. It is quite possi-
ble that other insoluble compounds containing more nucleic acid may have
existed in the embryo or have been formed during extraction, so that the
amount of globulin obtained may not have equaled that actually present in
the embryo. The fact that the globulin from the whole seed was free from
nucleic acid, while that from the embryo was not, was probably due to the
presence of a much larger proportion of protein insoluble in salt solution in
the whole seed compared with that in the embryo, so that the nucleic acid
united with the insoluble protein instead of with the globulin, as happened
when the embryo was extracted.

This globulin is very similar to, if not identical with, that found in the
seeds of rye and barley. It contains over 18 per cent of nitrogen, and re-
sembles, in composition and properties, many of the globulins found in large
proportion in many other seeds.

Owing to the difficulty encountered in preparing large quantities of this
globulin, the products of its hydrolysis have not been studied.

Its ultimate composition is shown by the following figures, which are the
average of several analyses of preparations from the whole seed :

P. ct.

Carbon 5x-03

Hydrogen 6.85

Nitrogen 18.39

Sulphur o. 69

Oxygen 23.04

100.00

THE PROTEINS OF THE WHEAT KERNEL.

This globulin is precipitated by saturating its solutions with magnesium
sulphate, but not with sodium chloride. Dissolved in 10 per cent sodium-
chloride solution, it is partly coagulated by boiling, but is not coagulated at
temperatures below ioo°.

THE PROTEOSE.

The wheat kernel yields a very small amount of proteose when extracted
with water. Whether this is an actual constituent of the seed or is formed
from the other proteins during its extraction and isolation was not definitely
ascertained. This proteose, like leucosin and globulin, is chiefly found in
the embryo. No preparations were made from the entire seed in sufficient
quantity to permit of analysis or examination of its properties.

The proteose obtained from the embryo appeared to be a mixture of two
or more substances. A part was precipitated by saturating its solution with
sodium chloride ; a part was not.

The analyses of these two parts showed the low percentage of carbon
characteristic of proteoses obtained by peptic digestion. Analyses of prepara-
tions thus obtained gave the following results :

Proteose in-
soluble in
saturated

NaCl
solution.

Proteose
soluble in
saturated

NaCl
solution.

Hydrogen

Nitrogen

P.ct.

49-94
6.80

17.08
I.24

24.94

P.ct.
48.99

6. 85
1689

1. 10
26.17

IOO.OO

100.00

THE GLUTEN.

The proteins of the wheat kernel differ from those that have been found
in any other seed by the fact that they may be largely separated as a coherent
elastic mass by washing the dough made from the flour under a gentle stream
of water. This protein mass has long been known as gluten. It consists
chiefly of gliadin and glutenin in combination with about twice their weight
of water, together with more or less starch, which can not be wholly removed
by washing, and also some fat, cholesterin, and lecithin. The non-protein
constituents usually form about 20 to 25 per cent of the crude gluten. The
protein constituents of the gluten, except for the fact that they are combined
with water, are present in the same form in the gluten as in the grain.

SUMMARY. 117

Their proportion is also nearly the same, for practically the same amount of
gliadin can be obtained from the gluten as from the flour, if allowance is
made for the small amount of gluten that is mechanically carried away in
the process of washing out the starch. This loss is largely made up by the
presence of non-protein constituents in the gluten, so that the weight of the
dried gluten usually corresponds closely with the amount calculated for the
sum of gliadin and glutenin, as computed from the nitrogen belonging to
these proteins.

The gluten is not formed from globulins by the action of a ferment in a
manner analogous to the formation of fibrin from fibrinogen, as was asserted
at one time. The observations on which this supposition was founded were
incorrect.

The glutenin probably forms the nucleus to which the gliadin adheres,
and thus binds the gluten proteins into a coherent elastic mass.

Both gliadin and glutenin are necessary for the formation of gluten, for a
dough made with starch and gliadin, or one made with flour from which the
gliadin has been extracted with alcohol, yields no gluten when washed with
water. That the gliadin is capable of taking part in gluten formation under
such conditions is shown by the fact that when dry and finely ground glia-
din was added to wheat flour the amount of gluten obtained on subsequently
washing out the starch was increased by the full amount of the gliadin that
was added.

THE NUTRITIVE VALUE OF THE WHEAT PROTEINS.

It has recently been shown that in the process of digestion the protein
molecule is very largely broken down into amino-acids, and that the animal
forms from these, by the processes of assimilation, the proteins of its blood
and tissues. How this change is effected is not known, nor is it known
whether the food protein is converted into the body protein and then ox-
idized and eliminated, or is partly converted into body protein and partly
burned directly in the form of amino-acids. It is also a question whether
or not the animal has the power to convert one amino-acid into another, and
thus obtain material suitable for the construction of its own body protein.

Directly connected with these important problems are the facts presented
by the determination of the relative amounts of the different amino-acids
yielded by the proteins of wheat flour, for these are used in enormous quan-
tities as food by man, and, as an examination of the analyses of their decom-
position products show, present marked differences from similar analyses of
all the other food proteins thus far examined.

As gliadin and glutenin together form about 85 per cent of the proteins of
wheat flour, they deserve especial consideration in this respect. If it is

THE PROTEINS OF THE WHEAT KERNEL.

assumed that these two proteins are present in equal quantities in wheat
flour, an assumption that in most cases is nearly correct, the relative propor-
tion of the amino-acids yielded by the proteins of wheat flour is closely
shown by the following figures, which are the mean of those found for
gliadin and glutenin. In comparison with these are given the figures, at
present available, for some of the other forms of food protein. Such a com-
parison is at present unsatisfactory, owing to the lack of data now available,
but it will serve a useful purpose in indicating the wide differences between
these food proteins, and will emphasize the importance of obtaining more
information in regard to this question.

Products of hydrolysis of some food proteins.

Glycocoll

Alanine

Amino-valerianic acid.

Leucine

Proline

Phenylalanine

Aspartic acid

Glutaminic acid

Serine

Tyrosine

Cystine

Lysine

Histidine

Arginine

Ammonia

Tryptophane

Wheat
gluten.

Milk
casein.

Egg
albumin.

Zein,
maize.

Excel-

sin,
Brazil

nut.

Phase-
olin,
■white
bean.

Beef
muscle.

Halibut
muscle.

P.ct.
0.44

3-33
0.23

5-78
5-65
2.16

o.75
30.38

0.44

2-73
0.24
0.96
1. 19
3-94
4-56
present

P.ct.
0.00
0.90
I. OO

10.50

3-IO
3.20
I.20
11.00
0.23

4-50
0.06
5-8o

2-59
4.84

i-95
1.50

P.ct.
O.00
2.IO

P.ct.

P.ct.
O.60

2-33

8.70
3-65
3-55
3.85
12.94

P.ct.
0.55
I.80
I.04

956

2.77

3-25
5-24
14-54
0.38
2.17

P.ct.

6.IO
2.25

4.40

I.50
9.10

16.87

II. I

8.9

I. IO
0.20

I.63
present

3.16

O.OO
0.81
I.82
3.61
O.OO

1.64

1-47
16.02
1.80
present

3-59
1.97
4.72
2.06
present

Owing to the lack of data, it is not yet possible to compare the relative
proportion of the amino-acids which the food proteins yield except in respect
to glutaminic acid, ammonia, arginine, histidine, and lysine. The amount
of glutaminic acid which the gluten proteins yield is far greater than that
yielded by any of the other food proteins, with the exception of gliadin from
rye and hordein from barley. The proteins of the legumes and nuts which
are used as food yield from 15 to 20 per cent of glutaminic acid, so that the
mean amount of this amino-acid from the wheat protein is nearly twice as
large. The same also is true of ammonia.

The proportion of arginine from wheat gluten is relatively small compared
with that from most other seed proteins, many of which yield from 10 to 16
per cent of this base.

SUMMARY. 119

The proportion of lysine is likewise small, especially compared with that
obtained from the leguminous seeds. The amount of histidine, however,
does not differ very greatly from that of the other seed proteins. What
significance these differences have in respect to the nutritive value of these
different proteins must be determined by future investigation, for it has only
very recently been discovered that such differences exist.

That a molecule of gliadin can have the same nutritive value as one of
casein would seem impossible if one molecule of food protein is transformed
into one of tissue protein, for in the former lysine is wholly lacking, and
glutaminic acid, ammonia, and proline are in great excess over the amount
required to form any of the tissue proteins of which we know. It would
seem probable that either the animal requires a variety of food, so that the
relative proportion in which the amino-acids are available for its use shall
correspond more nearly to its requirements, or that only a small part of
these amino-acids are converted into its tissue proteins and the rest oxidized
as such. It is possible that feeding experiments with proteins of known
character in respect to the relative proportions of their decomposition prod-
ucts will throw light on these important questions.

MBL WHOI LIBRARY

wh ifin y.

D H- A

lUl " m
■ SEE*

K
££22= KS

Sssssg

■■■.'-•

SBsagaga

pang

Internet Archive Audio

Featured

Top

Images

Featured

Top

Software

Featured

Top

Books

Featured

Top

Video

Featured

Top

Mobile Apps

Browser Extensions

Archive-It Subscription

Save Page Now

Full text of "Carnegie Institution of Washington publication"

See other formats