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Bulletin 339
4S
June, 1932
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CHEMICAL INVESTIGATIONS OF THE
TOBACCO PLANT
III. TOBACCO SEED
Qlnttnprttrut
Agrtrultural iExp^rtm^nt ^tattnn
Nfuj Baupn
CONNECTICUT AGRICULTURAL EXPERIMENT STATION
BOARD OF CONTROL
His Excellency, Governor Wilbur L. Cross, ex-officio. President
Elijah Rogers, Vice-President Southington
George A. Hopson, Secretary Mount Carmel
William L. Slate, Director and Treasurer New Haven
Joseph W. Alsop Avon
Edward C. Schneider Middletown
Francis F. Lincoln Cheshire
S. McLean Buckingham Watertown
STAFF
Administration. William L. Slate, B.Sc, Director and Treasurer.
Miss L. M. Brautlecht, Bookkeeper and Librarian.
Miss Dorothy Amrine, B.Litt., Editor.
G. E. Graham, In Charge of Buildings and Grounds.
Analytical
Chemistry.
E. M. Bailey, Ph.D., Chemist in Charge.
C. E. Shepard 1
Owen L. Nolan I
Harry J. Fisher, Ph.D. yAssistant Chemists.
W. T. Mathis
David C. Walden, B.S. j
Frank C. Sheldon, Laboratory Assistant.
V. L. Churchill, Sampling Agent.
Mrs. a. B. Vosburgh, Secretary.
Biochemistry. H. B. Vickery, Ph.D., Biochemist in Charge.
Lafayette B. Mendel, Ph.D., (Yale University)
Associate.
George W. Pucher, Ph.D., Assistant Biochemist.
Research
Botany.
Entomology.
G. P. Clinton, Sc.D., Botanist in Charge.
E. M. Stoddard, B.S., Pomologist.
Miss Florence A. McCormick, Ph.D., Pathologist.
A. A. DuNLAP, Ph.D., Assistant Mycologist.
A. D. McDonnell, General Assistant.
Mrs. W. W. Kelsey, Secretary.
W. E. Brixton, Ph.D., D.Sc, Entomologist in Charge, State
Entomologist.
B. H. Walden, B.Agr. )
M. P. Zappe, B.S.
Philip Garman, Ph.D.
Roger B. Friend, Ph.D.
Neely Turner, M.A.
John T. Ashworth, Deputy in Charge of Gipsy Moth Control.
R. C. BoTSFORD, Deputy in Charge of Mosquito Elimination.
J. P. Johnson, B.S., Deputy in Charge of Asiatic and Japanese
Beetle Quarantines.
Mrs. Gladys Brooke, B.A., Secretary.
rAssistattt Entomologists.
Forestry. Walter O. Filley, Forester in Charge.
H. W. HicocK, M.F., Assistant Forester.
J. E. Riley, Jr., M.F., In Charge of Blister Rust Control.
Miss Pauline A. Merchant, Secretary.
Plant Breeding. Donald F. Jones, Sc.D., Geneticist in Charge.
W. Ralph Singleton, Sc.D., Assistant Geneticist.
Lawrence C. Curtis, B.S., Assistant.
Mrs. Catherine R. Miller, M.A., Secretary.
Soils. M. F. Morgan, M.S., Agronomist in Charge.
H. G. M. Jacobson, M.S., Assistant Agronomist.
Herbert A. Lunt, Ph.D., Assistant in Forest Soils.
Dwight B. Downs. General Assistant.
Tobacco Substation Paul J. Anderson, Ph.D., Pathologist in Charge.
at Windsor. T. R. Swanback, M.S., Agronomist.
O. E. Street, M.S., Plant Physiologist.
Miss Dorothy Lenard, Secretary.
CONTENTS
Part I The Nutritive Properties of Tobacco Seed . . 609
Lafayette B. Mendel and Hubert Bradford
\^ickery
Part II The Globulin of Tobacco Seed 625
Hubert Bradford Vickery, Alfred J. Wakeman
and Charles S. Leavenworth
Part III Some Nitrogenous Components of the Hot
Water Extract of Fat-Free Tobacco Seed
Meal 637
Hubert Bradford Vickery
Part R'' A Microchemical Study of the Seed of
Nicotiana Tabacum 646
Florence A. McCormick
I
CHEMICAL INVESTIGATIONS OF THE
TOBACCO PLANT
TIL TOBACCO SEED
PART I
THE NUTRITIVE PROPERTIES OF TOBACCO SEED
Lafayette B. Mendel and Hubert Bradford Vickery
Although every part of the tobacco plant has been reported to
contain nicotine, this alkaloid could not be detected by Vickery and
Pucher in the fully ripened seeds of Connecticut shade-grown
tobacco by chemical methods (18), Ilyin (10), who has studied
the distribution of nicotine in the plant, found that immature seed,
and particularly the ovules at an early stage of development, con-
tained appreciable proportions, but that as ripening progressed the
alkaloid content diminished until finally none could be demon-
strated. The physiological and chemical problems presented by this
observation are of great interest, and, as a preliminary to their
further study, it appeared desirable to verify the observations on
the absence of nicotine from ripe tobacco seed by other than chemi-
cal methods. This seemed the more necessary inasmuch as nicotine
can be easily detected in the sprouts and cotyledons of tobacco
seed after only 9 to 11 days of germination.
In view of the well known toxic properties of nicotine the
simplest physiological test for the presence of traces of this alka-
loid in seed appeared to consist in conducting feeding trials on
small animals ; we therefore offered to albino rats a ration that
consisted almost entirely of these seeds. Somewhat to our surprise
the diet was consumed with avidity and without any evident unto-
ward consequences. This observation gave rise to the present
investigation.
Note : The investigations of tobacco described in the present bulletin
were carried out as part of a general project under the title "Cell Chemis-
try", by the Department of Biochemistry of the Connecticut Agricultural
Experiment Station, New Haven, Conn. The Department has enjoyed the
benefit of the close cooperation of the Tobacco Substation. The expenses
were shared by the Connecticut Agricultural Experiment Station and the
Carnegie Institution of Washington, D. C.
610
Connecticut Experiment Station Bulletin 339
FEEDING EXPERIMENTS WITH TOBACCO SEED
METHODS EMPLOYED
The feeding experiments were of the conventional type long
employed in this laboratory. Albino rats were used, the animals
being kept in wire cages equipped with raised, false bottoms so as
to prevent access to the feces. Water was always available. Casual
observations indicated that ingested intact seeds often passed
through the alimentary tract apparently unaltered ; the material
360
Figure 51. The rate of growth of albino rats on rations consisting almost
exclusively of ground tobacco seed. The food mixtures used were as follows :
for Rats C68 and C74, tobacco seed 98, salt mixture 2 per cent ; for Rats
C2286, C2236, C66, C2228 and C73, tobacco seed 98, salt mixture 2 per
cent -j- 10 drops cod liver oil daily.
was therefore customarily ground to an oily paste before being
offered to the animals. In some instances ground seeds that had
been freed from fat by extraction with organic solvents were
employed in the rations.
THE PILOT EXPERIMENTS
Young male rats, 35 days of age and weighing 66 to 71 gm.,
were fed a mixture of ground tobacco seed 98 per cent, and
Osborne-Mendel salt mixture IV (15) 2 per cent. The animals
The Nutritive Properties of Tobacco Seed 611
grew well for several weeks on this unusual food, as indicated by
the graphs of gains in body weight in Figure 51 (Rats C68, C74).
In a number of preliminary experiments cod liver oil — a source
of fat-soluble vitamins — was added as a daily supplement to the
ration. Good growth was secured (Figure 51, Rats C2286, C2236,
C66, C2228, C73). One male animal (Rat C66) on this unique
ration reached a weight of 454 gm. in 374 days (see Figure 52).
The tobacco seed evidently supplies nearly all of the inorganic
elements requisite for growth, as is indicated in Figure 53 by the
records of Rats C81, C75, C94, C76. The sole food of these animals
consisted of ground tobacco seed 99 per cent, CaCOs 0.5 per cent.
Figure 52. A rat that had been fed for 374 days, beginning- at 35 days
of age {6?> gm. body weight) on a ration consisting of tobacco seed 98,
salt mixture 2 per cent + 10 drops cod liver oil daily. At the time the
photograph was taken the animal weighed 454 gm.
and NaCl 0.5 per cent, with addition of 10 drops of cod liver oil
daih^ in the case of the last two animals.
It is clear from these observations that tobacco seed yields all
of the amino acids essential for growth, and that it contains vita-
mins B and G as well as the necessary inorganic elements, with
the possible exception of calcium and chlorine.
DOES TOBACCO SEED CONTAIN VITAMIN A?
The answer to the question whether tobacco seed contains vita-
min A has been sought in several ways. Curative and prophy-
lactic tests were emoloyed with the whole (ground) seed, and cura-
612
Connecticut Experiment Station Bulletin 339
tive tests were conducted with the oil ol)tained therefrom. This
fat, which may be expected to contain any fat-soluble vitamins
present, was extracted from the ground seed by means of cold
ether. The ether was then distilled off in the presence of carbon
dioxide, the final traces being removed by warming the oil in a
vacuum. The residual oil was kept in a refrigerator in dark colored,
tightly stoppered bottles. The "tobacco seed oil," as we shall here-
after designate it, is pale yellow in color. It sometimes deposits a
small amount of deeper colored sediment that has not yet been
130
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Figure 53. The rate of growth of rats on a ration in which tobacco seed
suppHed all of the requisite inorganic elements except those indicated in
the small mineral supplements below. The food mixtures used were as
follows : for Rats C81 and C75, tobacco seed 99, CaCOs 0.5, NaCl 0.5 per
cent; for Rats C94 and C76, tobacco seed 99, CaCOa 0.5, NaCl 0.5 per
cent -f- 10 drops cod liver oil daily.
carefully examined. The clear, filtered oil, which Prof. W. E.
Anderson of the Laboratory of Physiological Chemistry, Yale
University, kindly examined for us, has the following chemical
characteristics :
Specific gravity, 25725° 0.9215
Refractive index, 25° 1.4740
Saponification value 192.0
Iodine number (Hanus) of oil 134.0
Iodine number (Hanus) of unsaturated fatty acids 161.0
The Nutritive Properties of Tobacco Seed 613
Mixed saturated and unsaturated fatty acids 93.2%
Saturated fatty acids (corrected) 9.7%
Unsaturated fatty acids (corrected) 81.5%
Unsaponifiable matter ' 1.1%
A sample of ground seed was also extracted with hot alcohol and
filtered. The extract was concentrated in vacuo to remove the
alcohol, and was then treated with ether to dissolve the "fats." A
sample of this oil, examined by Dr. Lucille L. Reed in our labora-
tory, gave an iodine number of 143.0.
COLOR REACTIONS FOR VITAMIN A
Dr. A. J, Wakeman of our laboratory examined the clear oil by
the antimony trichloride reaction of Carr and Price (5), a pro-
cedure which is assuined by some investigators to indicate the
presence of vitamin A. The deeply colored sediment referred to
above was similarly examined. The results were negative in every
instance. This does not necessarily imply that there is no physio-
logical potency, however, for recent studies have repeatedly shown
that carotinoid pigment, which fails to yield the antimony trichlo-
ride color reaction, may fimction like vitamin A in the animal
organism (14).
ANIMAL TESTS FOR VITAMIN A
A search for vitamin A was made in both the ground tobacco
seed and in the oil extracted therefrom. Young male rats, approxi-
mating 60 gm. in body weight and selected from groups whose
mothers had been kept on a regimen rather poor in vitamin A, were
supplied with a food mixture lacking vitamin A and composed of
casein 18 per cent, salt mixture (15)4 per cent, starch 54 per cent,
and lard 24 per cent, with the addition of 0.2 gm. yeast and 0.001
gm. viosterol daily. On this the animals developed unmistakable
xerophthalmia in 35 to 140 days and exhibited a slowing, if not a
cessation, of growth. When these signs of vitamin A deficiency
were established various curative measures that involved the use
of tobacco seed were instituted. In some cases the carefully pre-
served tobacco seed oil was fed, apart from the rest of the ration
and usually admixed with the dried yeast, in doses not exceeding
25 drops (665 mg.) per day. This corresponds to the oil in 1.56
gm. of the seed. Even with these largest doses curative effects were
not observed ; nor was there any marked improvement in growth.
In other cases, when the deficiency symptoms became marked and
benefit was not secured through administration of tobacco seed oil,
the diet was changed to a mixture of 98 per cent of ground tobacco
seed, plus inorganic salts. This tended to bring about recoveries.
On the assumption of a daily food intake of only 5 gm. of the
614
Connecticut Experiment Station Bulletin 339
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Figure 54. Experiments to test for the presence of vitamin A in tobacco
seed and tobacco seed oil. During the first period of each experiment, indi-
cated by the unbroken Hne, the animals were on an otherwise adequate diet
devoid of vitamin A, and gave evidence of avitarainosis at the end of the
period. Changes in the ration were then instituted as follows : in Period 2
Rats C1559, C329, C1482, C315, C1911 received a supplement of tobacco
seed oil in varying amounts indicated on the graphs (d.= drops). In the
subsequent periods the daily supplement of oil was either increased as indi-
cated, or the diet was replaced by a mixture of tobacco seed 98, salt mixture
2 per cent (T.s. 98 per cent). In the case of Rat C301 during the second
period the supplement consisted of 1 gm. tobacco seed daily. In period 3 the
diet was changed to the tobacco seed mixture indicated above. The effects
are discussed in the text.
The Nutritive Properties of Tobacco Seed 615
tobacco seed food, the oil supplied in the seeds would exceed 2 gm.
per day — a quantity far surpassing that which the rats would con-
sume in extracted form. The efifect on the growth rate was some-
times particularly striking (see Figure 54, Rat C301).
Recovery from deficiency symptoms occurred also on diets in
which the quantity of tobacco seed was limited to one-half the food
mixture.
There are other biological phenomena that serve as indications
of lack of vitamin A in a food. In 1922 Evans and Bishop (7, 8)
noted a new and characteristic test for deficiency in fat-soluble
vitamin A — the constant appearance of cornified cells either
predominantly or exclusively in the vaginal smear. This is in
marked contrast to the orderly succession of different cell types
that are thrown off within the vagina of rats under conditions of
perfect nutrition and a normal oestrous cycle. According to Evans
inadequacy of vitamin A injures the female reproductive system
so that fertilization and implantation take place but resorption
follows.
An investigation of the possible occurrence of vitamin A in
tobacco seed by means of the delicate index of its possible influence
on the vaginal epithelium has been carried out with the helpful
cooperation of Dr. S. D. Aberle of the Yale University School of
Medicine. Preliminary tests were undertaken on the efiiciency of
the procedure in which microscopic examination of the cells secured
through vaginal smears was made by expert observers. In 40
female rats on diets deficient in vitamin A the continual succession
of cornified vaginal cells preceded either loss of weight or signs
of xerophthalmia. In three ovariectomized animals, after an aver-
age of 7Z days on the A-deficient diet, continual cornified cells were
present in the vagina. Yet cornified cells have never been found in
the vagina of ovariectomized rats on diets that contain adequate
supplies of vitamins. The changed picture of the vaginal epithelium
is a result of the dietary deficiency and is not related to ovarian
function.
In the new experiments female rats were kept upon diets that
contained different proportions of tobacco seed, to ascertain
whether the food supplied sufficient vitamin A to prevent continual
cornification of the vagina. Eight rats ranging in age from 28 to
Z7 days were divided into two groups. The first group of four
animals was put on a ration which contained ground tobacco seed
50 per cent, casein 15 per cent, salt mixture (15) 3 per cent, lard
10 per cent, starch 22 per cent ; 0.2 gm. of yeast and 0.001 gm. vio-
, sterol were fed daily (Diet I). Four rats were given food consisting
of tobacco seed 98 per cent and salt mixture 2 per cent (Diet II).
In both groups food and water were kept continually in the cages.
The animals were observed two or three times a week, at first
to determine the date of the opening of the vagina, and subse-
616 Connecticut Experiment Station Bulletin 339
quently to take samples of the vaginal cells. The animals on Diet I,
which contained yeast and viosterol, gained weight more rapidly;
the vaginas opened earlier and continual cornified cells were present
in the vagina sooner than in the animals on Diet II. Although
there was an average difference of 42.5 days in the opening of the
vagina of the two groups, the average weight for both groups at the
time the vagina opened was approximately the same.
No marked soreness of the eyes (xerophthalmia) developed in
the rats on either Diet I or Diet II. Diet II did not produce as
good growth as Diet I. The average ages at which the rats on the
50 per cent and the 98 per cent tobacco seed diets showed continual
cornified vaginal cells were 101 and 125 days respectively. The
delay of 24 days in the group on 98 per cent tobacco seed indicates
a higher content of vitamin A in the latter diet. The vitamin A
content of the 98 per cent tobacco seed, however, was not sufficient
to change the character of the vaginal cells in rats that had been
reared on 50 per cent tobacco seed diets, once the condition had
become established, nor yet to prevent the continual appearance of
cornified cells in the vaginas of rats on that diet. In every instance
ingestion of two drops of cod liver oil daily changed the vaginal
picture within a few days.
The upshot of our own tests seems to us to indicate the occur-
rence of vitamin A potency in low concentration in tobacco seed.
This would account for the occasional slow recoveries from the
effects of vitamin A deficiency that were observed.
ANIMAL TESTS FOR VITAMINS B AND G
The preliminary experiments demonstrated that rats grew satis-
factorily on a diet in which tobacco seed furnished the sole source
of those food factors formerly designated as vitamin B (or the
vitamin B complex). This result implies that the seed contains
what are now distinguished as vitamins B (antineuritic) and G
(antipellagric) and further suggests that the third component of
the complex, claimed to exist by some investigators, is also present
(see Figure 51).
Tobacco seed can be shown in other ways to contain vitamins
B and G. Relatively small daily quantities (0.2 gm.) of the fat-free
seed supplement autoclaved yeast (a source of the more thermo-
stable vitamin G) (Figure 55) ; but when fat-free seed is autoclaved
for 4 hours at 100° temperature and 15 pounds pressure, it will
no longer supplement autoclaved yeast (Figure 55). The presence
of the thermolabile vitamin B in the seed is thus further established.
In order to secure some idea of the relative abundance of vita-
mins B and G in tobacco seed a few quantitative experiments were
carried out. Two types of "basal" rations were used, like those of
Chase (6) and Bourquin (4), described by Quinn, Whalen and
The Nutritive Properties of Tobacco Seed
617
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GROUP B
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Figure 55. Evidence for the presence of vitamin B (Bj) in tobacco seed. In Group
A the four animals received, during the first period, a basal diet supplemented with
autoclaved yeast (a source of the more thermostable vitamin G). When failure of
growth resulted, the animals received additional supplements of fat-free tobacco seed
in Periods 2 and 3. The amount of the daily supplements is indicated in grams. Growth
was promptly restored when the supplement of tobacco seed amounted to 0.2 gm.
In Group B the four animals received during Period 1 a mixture of autoclaved yeast
(a source of vitamin G) and autoclaved fat-free tobacco seed. The cessation of growth
indicates the destruction of vitamin B in the seed by heating. When the autoclaved
fat-free tobacco seed was replaced in Period 2 by a daily supplement of 0.2 gm. unheated
fat-free tobacco seed, growth was promptly restored, indicating anew the potency of the
tobacco seed in vitamin B.
618 Connecticut Experiment Station Bulletin 339
Hartley (16). The B-free ration consists of casein 18 per cent,
autoclaved yeast 15 per cent, cornstarch 53 per cent, butter fat 8
per cent, cod liver oil 2 per cent, salt mixture (15) 4 per cent; the
G-free ration consists of casein 15 per cent, whole wheat 20 per
cent, cornstarch 51 per cent, salt mixture (15)4 per cent, butter fat
8 per cent, and cod liver oil 2 per cent. Young rats were kept on
one or the other of these deficient rations until they began to decline
in body weight ; daily supplements of weighed amounts of ground
tobacco seed — 0.25 gm., 0.5 gm., or 1.0 gm. respectively — were
then supplied for periods of 35 days. Two male rats were used for
each comparison. The outcome was as follows :
Test of Tobacco Seed for Vitamin B
Daily supplement to
B-free ration
Average daily gain
in body weight
gm.
gm.
0.25
2.8
0.5
Z.7
1.0
3.8
Test of Tobacco Seed for Vitamin G
Daily supplement to
G-free ration
Average daily gain
in body weight
gm.
gm.
0.25
1.3
0.5
1.7
1.0
2.6
It is apparent that tobacco seed is relatively richer (in terms of
conventional unitage) in vitamin B than in vitamin G.
VITAMIN D
Preliminary tests for the presence of vitamin D in tobacco seed
were conducted on animals reared on a diet that consisted of corn
685 gm., gluten flour 180 gm., sodium chloride 10 gm., calcium
carbonate 8.69 gm., disodium phosphate (hydrated) 116 gm.,
supplemented by the daily administration of 0.1 gm. butter fat,
0.5 gm. tobacco seed oil, and 0.2 gm. of a mixture of yeast, calcium
carbonate and starch in the ratio of 100 : 0.9 : 13.1. This diet
was made so as to contain calcium and phosphorus in the ratio of
0.3 : 1.0. The control animals received a supplement of corn oil.
The ribs and forelegs of the animals were kindly examined for
us by Dr. E. A. Park of the Johns Hopkins University. Although
this diet was not one upon which rickets is readily produced, the
absence of any notable difference between the bones of the two
lots of animals permitted the tentative conclusion that tobacco seed
does not contain any antirachitic agent.
The Nutritive Properties of Tobacco Seed 619
BREEDING TESTS ^VITAMIN E LACTATION
Various causes of sterility in male and female mammals are
recognized today. A regimen may be adequate in so far as one
can judge from the growth, somatic appearance, and evident well-
being of animals; yet they may fail to produce offspring. Slight
changes in the dietary often result in securing successful pregnancy,
parturition and lactation. Thus the need of a specific accessory
food factor to insure normal reproduction in animals has been
definitely demonstrated for the rat (9, 11, 12, 13). The essential
substance is designated vitamin E. Lack of it leads to degenerative
changes in the testes of the male ; vitamin E is needed "not only for
the multiplication but also for the very existence of the germ cells
themselves." In the female, oestrus, copulation, ovulation, fertili-
zation, tubal journey, and implantation occur, but pregnancy is
interrupted when there is a depletion of vitamin E. Dietary ster-
ility has thus come to have a recognized standing. Infertility does
not give conclusive evidence of the deficiency. Successful mating,
however, offers assurance that the dietary requisites have been
supplied — particularly when the breeding tests are conducted
under circumstances that bring about an exhaustion of previously
acquired "stores" of vitamin E. Dietary sterility once established
in the male is not readily cured ; in the female the capacity to bear
young can frequently be restored.
Our breeding experiments have been conducted with rats for
which tobacco seed supplied the sole source of vitamin E after
the time of weaning. Both males and females were reared on a
diet that consisted of tobacco seed 98 per cent and salt mixture
(15)2 per cent ; 10 drops cod liver oil were also administered daily.
The salt mixture was added to avert the possible inorganic defi-
ciencies of unsupplemented tobacco seed. Cod liver oil, in the
proportions used, has been demonstrated by Evans and Burr, as
well as in this laboratory, to be devoid of effective quantities of
vitamin E ; it was furnished to the animals in order to insure an
adequate intake of vitamins A and D. The pathological changes
induced in the female genital tract, when there is a shortage of
vitamin A, have already been mentioned.
Out of fourteen matings of rats thus reared to maturity, only
a single instance of failure of reproduction was noted ; 104 young
were actually counted. This means an average of 7 young per
litter — a figure identical with the average for our colony.
Much difficulty was experienced in rearing the litters because so
many of the young were eaten by their mothers. This is, however,
by no means a consequence attributable specifically to the tobacco
seed diet, for it is an occasional complaint among breeders, even
in "stock" colonies. Lactation also was far from satisfactory. It
was found that addition of a small amount of yeast to the diet of
620 Connecticut Experiment Station Bulletin 339
the young resulted in better gains in weight. The requirement of
vitamin B is known to be high during lactation, and milk is fre-
quently relatively poor in this factor. We suspect, therefore, that
the mammary secretion of the mother rats was not such as to
permit optimal growth of the suckling young.
Improvement was secured in some instances by attempting a
concentration of the water soluble vitamins in the tobacco seed diet
through elimination of part of the fats therein. It will be recalled
that the fats amount to nearly half the weight of the seed. Ground
tobacco seed was accordingly extracted with gasoline and ether
and much of the "fat" removed. When the "extracted tobacco
seed meal," correspondingly enriched in the residual vitamins B
and G, was fed in place of the usual tobacco seed meal some im-
provement in the growth of the young was secured. The gains of
the young were, however, never up to average, and they could be
increased by the addition of yeast to the ration. However, three
females of the "second generation" of rats that were fed exclusively
on tobacco seed, inorganic salts and cod liver oil, and were mated to
males fed on this diet from the time of weaning, gave birth to litters
of 8 to 10 "third generation" young. The occurrence of vitamin E
in tobacco seed is thus established beyond question.
Incidentally, the rearing of a third generation of animals on a
diet in which so many of the requisites for growth and reproduc-
tion were supplied by a single seed is noteworthy from the stand-
point of animal nutrition and feeds. It is not unlikely that many
other seeds, some of them designated as "weed seeds" and care-
fully excluded from commercial feeds may possess unanticipated
nutrient values.
THE FAT OF RATS FED ON TOBACCO SEED RATIONS
In an elaborate study of the effects of diet upon the body fat
of albino rats, Anderson and Mendel (1) observed that, in some
instances, the resulting fat, judged by the iodine number, resembled
that which was ingested. This was notably true for rations rich in
soy bean, maize, cottonseed, and peanut oils. In the case of other
food fats striking dififerences were found in the deposited body fats.
The present investigation afforded an opportunity to examine the
depot fat of two male rats that had been kept on a ration consisting
of tobacco seed 98 per cent and salt mixture (15) 2 per cent, with
10 drops cod liver oil administered daily. This represented an
intake in which considerably more than half of the energy was de-
rived for a long period from tobacco seed fat (iodine number ca.
143). The fat removed from the subcutaneous, perirenal and genital
regions^ showed small variations in iodine numbers ranging from
133 to 138, according to analyses made for us by Dr. Lucille L.
^The fats were prepared as described by Reed, Yamaguchi, Anderson and Mendel (17).
The Nutritive Properties of Tobacco Seed 621
Reed. Evidently the tobacco seed oil was influential in producing
the deposition of an even more unsaturated fat than that stored by
rats that had ingested large amounts of very unsaturated oils such
as soy bean oil and corn oil (iodine numbers of fat from rats on
these oils were 122 and 112 respectively (1)). Rats forced to
synthesize fat from a diet high in carbohydrate stored a much less
unsaturated fat with an iodine number of 60.
EXPERIMENTS ON MICE
In order to extend these experiments to another species a few
feeding experiments were made for us by Prof. W. E. Anderson
of Yale University on brown mice. The nutritive requirements of
albino mice have been discussed in detail by Beard (2) and by Bing
(3). Our mice were weaned at 23 days of age and thereafter were
kept on a ration consisting of tobacco seed 98 per cent, salt mixture
(15) 2 per cent. Growth continued satisfactorily until the tests
were discontinued at the end of 151 days, long after the animals had
reached adult size, 31 and 32 gm. respectively.
These records confirm the value of tobacco seed as a nutrient
notable in so far as its protein and water soluble vitamins are con-
cerned. There is no reason to assume that other mammalian species
would not derive nutriment equally well from the seeds, provided
that the latter are satisfactorily disintegrated.
EXPERIMENTS ON PIGEONS
A few experiments on pigeons, conducted for us by Prof G. R.
Cowgill of Yale University, have given unequivocal evidence that
tobacco seed is non-toxic for this species also and that, even when
unsupplemented with water soluble vitamins or other proteins, it
can meet the requirements of these birds with respect to the food
essentials indicated. Two pigeons weighing respectively 281 and
312 gm. were given polished rice ad lihitiim and a gelatin capsule
containing the dried residue of hot water extracted muscle (so-
called "meat residue"), a small quantity of the Osborne-Mendel
salt mixture (15), and a small allowance of cod liver oil daily over
a period of 12 days. At the end of this time they had declined in
body weight to 238 and 279 gm. respectively — a result clearly due,
as indicated by curative tests in other experiments on the same
birds, to a shortage of the antineuritic vitamin B. The ration was
then changed to about 15 capsules containing approximately 1 gm.
each of whole tobacco seed daily, the intake varying from 15 to 14
gm. for the smaller pigeon and 17 to 16 gm. for the larger one.
Chicken "grit" and water were always available. On this regimen
the pigeons promptly began to gain in weight, reaching 304 and 346
gm. respectively when the feeding tests with the seed were termi-
622 Connecticut Experiment Station Bulletin 339
nated after 22 days. Some apparently intact seeds were observed
from time to time in the excreta, but the amounts were always
small.
EXPERIMENTS ON CHICKENS
An experiment was conducted for us by Prof. Walter Landauer
of Storrs Agricultural Experiment Station in which a diet made
up of tobacco seed 94 per cent, cod liver oil 2 per cent, calcium
carbonate 2 per cent, sodium chloride 1 per cent, and salt mixture
(15) 1 per cent, was fed to chickens. Two lots of 26 chicks each
were confined in separate parts of an experimental brooder, one lot
being furnished a standard chicken mash to serve as a control. At
the end of 4 weeks, crushed oyster shells were placed before both
lots of birds, and the tobacco seed in the experimental diet was
ground to a paste before being mixed with the cod liver oil.
Table 1. The Average Weight of Chickens Fed on a Tobacco Seed
Diet and on a Standard Chicken Mash
Remarks
26 chicks in each lot
Grit added; seed ground
Experimental chicks
changed to chicken mash
10 " 209.3 diet
11 " 296.3
The data in Table 1 show the relative rates of growth of the
birds on the two diets in terms of the average weight of each lot.
The rate of growth on the experimental diet was very slow but the
prompt resumption of growth when the experimental diet was
replaced by standard chicken mash and the general healthy aspect
of the animals throughout the experiment showed clearly that
tobacco seed contains no factor toxic to chickens. The diet selected,
however, was obviously not satisfactory for the growth of this
species.
Experiments were also conducted by us in which groups of six
chicks were fed on the following experimental diets :
Age
Tobacco seed diet
Controls
gm.
gm.
2 days
35.2
37.2
1 week
45.9
61.1
2 weeks
54.2
99.5
3 "
64.2
151.9
4 "
69.8
205.8
5 "
72.2
290.5
6 "
78.4
381.3
7 "
87.7
447.1
8 "
104.7
558.8
9 "
148.5
The Nutritive Properties of Tobacco Seed 623
1. Extracted tobacco 3. Ground tobacco seed 94 per cent
seed 94 per cent Salt mixture 4 per cent
Salt mixture^ 4 per cent Cod liver oil 2 per cent
Cod liver oil 2 per cent
4. Ground tobacco seed 48 per cent
2. Extracted tobacco Chicken mash 50 per cent
seed 47 per cent Cod liver oil 2 per cent
Cornstarch 47 per cent rj .___._„^
Salt mixture 4 per cent
Cod liver oil 2 per cent
A control group received a standard chick feed fortified with 2
per cent by weight of cod Hver oil. In no case was the growth rate
of the chickens fed upon tobacco seed satisfactory. The diet in
which the oil of the seed had been replaced by cornstarch gave a
slow steady growth at a rate of approximately one-quarter of that
of the control animals. The growth rate on the mixture of tobacco
seed and mash was about the same, but the animals on the other
diets grew very slowly and most of the chicks fed on the extracted
seed diet died. Daily records of the amount of food eaten by the
various groups show very poor consumption of the tobacco seed
diets. This suggests that the factor limiting the growth of the birds
was the small intake of food, possibly owing to a taste that they
found unpleasant. This factor, of course, played no part in the
experiments on pigeons since force feeding was resorted to in that
case.
A tentative conclusion only can be drawn from these admittedly
incomplete experiments ; it seems, however, that diets in which
tobacco seed forms a substantial part of the ingested food are not
satisfactory for young chickens under the conditions of our experi-
ment. No experiments on mature fowl have been attempted.
SUMMARY
Feeding trials on albino rats have shown that this species can be
successfully raised to maturity at a satisfactory rate of growth,
and can reproduce, on a diet that consists almost exclusively of
tobacco seed. Either 1 or 2 per cent of inorganic salts was added,
and a few drops of cod liver oil were administered daily. This was
essential to successful growth because of the deficiency of the seed
in vitamins A and D.
Experiments designed to provide evidence of the presence of
vitamin A in the seed were somewhat inconclusive. It is certain that
the seed does not contain a concentration of this vitamin adequate
for successful growth or for complete protection against xeroph-
thalmia. It is probable, however, that the vitamin is not entirely
absent.
^The salt mixture used in these experiments was composed of 4 parts of the Osborne-
Mendel salt mi;cture (IS), 1 part of sodium chloride, and 1 part of calcium carbonate.
624 Connecticut Experiment Station Bulletin 339
Vitamins B and G were present in tobacco seed in quantities
adequate for growth and well-being. Under circumstances of
unusual demand for vitamins B and G, as, for example, during
lactation, a moderate degree of deficiency was apparent.
Although no final conclusion can yet be drawn it is probable
that tobacco seed is almost, if not entirely, deficient in vitamin D.
Vitamin E is present in tobacco seed in quantities adequate to
provide for reproduction and to protect both sexes from physiologi-
cal changes due to a deficiency of this factor.
Experiments on mice and on pigeons have demonstrated the non-
toxicity of the tobacco seed for other species ; likewise the good
nutritive value of the proteins in the seed. The seed also supplied
the water-soluble vitamins needed by mice, and functioned as an
effective antineuritic agent (proof of vitamin B) for pigeons.
The success of these feeding experiments demonstrates that the
total protein of this seed is of exceptionally good biological quality ;
in this respect it resembles the proteins of other oil seeds for which
experimental data have been obtained.
A few experiments on young chickens showed that tobacco seed
is not a satisfactory source of nutriment for this species.
BIBLIOGRAPHY
1. Anderson, W. E., and Mendel, L. B., Jour. Biol. Chem., 76: 729. 1928.
2. Beard, H. H., Amer. Jour. Physiol., 75 : 645, 658, 668, 682. 1926.
3. BiNG, F. C, and Mendel, L. B., Jour. Nutr., 2 : 49. 1929.
4. BouRQUiN, A., and Sherman, H. C, Jour. Amer. Chem. Soc, 53: 3501.
1931.
5. Carr, F. H., and Price, E. A., Biochem. Jour., 20 : 497. 1926.
6. Chase, E. F., and Sherman, H. C, Jour. Amer. Chem. Soc, 53: 3506.
1931.
7. Evans, H. M., Jour. Biol. Chem., 77 : 651. 1928.
8. Evans, H. M., and Bishop, K. S., Anat. Rec, 23 : 17. 1922.
9. Evans, H. M., and Burr, G. E., The Antisterility Vitamine Fat Soluble
E, Calif. Univ. Mem., 8: 1. 1927.
10. Ilyin, G., U. S. S. R. State Inst. Tobacco Investigations, Bui. 57 (in
Russian). 1929.
11. Mason, K. E., Natl. Acad. Sci. Proc, 11: Z77. 1925.
12. Mason, K. E., Jour. Expt. Zool., 45 : 159. 1926.
13. Mason, K. E., Jour. Nutr., 1 : 311. 1929.
14. Moore, T., Biochem. Jour., 24 : 692. 1930.
15. Osborne, T. B., and Mendel, L. B., Jour. Biol. Chem., 37 : 572. 1919.
16. QuiNN, E. J., Whalen, F. B., and Hartley, J. G., Jour. Nutr., 3: 257.
1930.
17. Reed, L. L., Yamaguchi, F., Anderson, W. E., and Mendel, L. B.,
Jour. Biol. Chem., 87 : 147. 1930.
18. ViCKERY, H. B., and Pucher, G. W., Conn. Agr. Expt. Sta., Bui.
311: 234. 1930.
PART II
THE GLOBULIN OF TOBACCO SEED
Hubert Bradford Vickery, Alfred J. Wakeman
and Charles S. Leavenworth
The few studies of the seed of the tobacco plant that have been
pubHshed refer chiefly to the oil and to the availability of the
oil-free residue for fertilizer or for animal feeding (6, 7). Ilyin
(3) and Vickery and Pucher (10) have given brief reports of
the distribution of nitrogen in this seed, but apparently no particu-
lar attention has been paid to the proteins that it contains.
The seeds employed in the present investigation were those of
Connecticut shade-grown tobacco ; they are brown in color, the
testa being somewhat rough or pebbled. The seeds are ellipsoidal
in shape, frequently flattened from the close packing in the capsule,
and very small, 100 of them weighing only 0.009 gm. A histological
study of the seeds carried out by Dr. McCormick of the Depart-
ment of Botany forms Part IV of this Bulletin.
A proximate chemical analysis of two lots of tobacco seed,
derived from different crops, is given in Table 2.
Table 2. Proximate Analysis of Tobacco Seed on Air-Dry Basis
1929 crop
1930 crop
per cent
per cent
Moisture
3.34
6.87
Ash
3.71
3.31
Total nitrogen
3.89
3.67
Crude protein (Nx5.34)
20.76
19.60
Crude fiber
14.44
14.20
Carbohydrate
Water soluble after acid hydrolysis
(as dextrose)
3.08
2.13
Water insoluble after hydrolysis
(as dextrose)
0.55
1.31
Starch
0.00
0.00
Ether extract
42.23
43.10
Undetermined
11.89
9.48
The fat content of this seed is extraordinarily high and its accu-
rate determination presents considerable difficulty. The figures
given represent the weight of the ether extract after being dried in
a hot water oven. The specimen of the seed taken had been previ-
ously dried in a vacuum oven for several hours, or until no more
water was removed. The extraction was conducted with absolute
ethyl ether in a continuous percolation apparatus for 16 hours;
626 Connecticut Experiment Station Bulletin 339
subsequent extraction for 16 hours gave an insignificant additional
quantity of fat.
An appreciably higher apparent fat content was observed when
samples of ground seed that were dried in a vacuum desiccator over
sulfuric acid were extracted with absolute ethyl ether for 4 days in
a modified Soxhlet apparatus. The fat was then dried in a vacuum
desiccator over sulfuric acid. Under these conditions as much as
48.0 per cent of crude fat could be obtained while, if ordinary ether
were used for extraction, 50.6 per cent was secured.
For the preparation of the protein a convenient quantity of seed
was ground in a poppy seed mill and the paste-like mass was
extracted twice successively with light gasoline. The meal was
filtered ofif and the residual gasoline was removed by washing with
ether ; the meal was then dried in thin layers on pans at room
temperature. The product was of a gray-brown color ; a typical
specimen from the 1929 crop contained 6.35 per cent of moisture
and only a trace of fat. The nitrogen content, calculated moisture-
free, was 6.74 per cent. A specimen from the 1930 crop contained
8.81 per cent moisture and 7.37 per cent nitrogen, calculated mois-
ture-free.
EXTRACTION OF THE SEED MEAL
A preliminary experiment showed that 72.4 per cent of the nitro-
gen of the meal could be extracted by thorough treatment with
molar sodium chloride solution. The residual nitrogen was then
extraordinarily resistant to the action of solvents ; cold 0.05 M
sodium hydroxide removed only 1.3 per cent more of it and cold
0.05 M sodium hydroxide in 70 per cent alcohol only an additional
0.4 per cent. The residue was heated at boiling temperature with
0.05 M sodium hydroxide in 70 per cent alcohol, but even this
ordinarily extremely effective solvent removed only 4.7 per cent
of nitrogen from the residue ; more than 12 per cent of the nitro-
gen of the seed resisted all efforts at extraction. The details of
another extraction experiment are shown in Table 3.
The first extraction with molar sodium chloride was conducted
by repeatedly grinding the fat-free seed through a small plate mill
with the solvent and then shaking by machine for several hours ; a
little toluene was also added. The opalescent extract was centri-
fuged ofif and filtered through a thick pad of paper pulp. On dial-
ysis, 88.5 per cent of the nitrogen of this extract separated in a
form that appeared to be protein. The subsequent extracts were
prepared by shaking the meal with the solvent for several hours.
The saline extracts were opalescent and were filtered clear on pulp
before sampling for analysis ; the first alkaline extract was also
turbid, the others were clear. The extractions with hot alkaline
alcohol were conducted at boiling temperature under a reflux con-
The Globulin of Tobacco Seed
627
denser for 3 and 2 hours respectively. The somewhat large loss
of nitrogen can be accounted for by the filtration of the turbid
saline extracts before sampling and also by loss of ammonia dur-
ing the alkali extractions.
Table 3. Extraction of Nitrogen from Ground Fat-Free Tobacco Seed
(1930 Crop)
Weight of seed taken = 74.4 gm.
Solvent Volume
1.0 M sodium chloride
cc.
2000
800
700
Water 1060
0.05 M sodium hydroxide, cold 1000
750
0.05 M sodium hydroxide in 70%
alcohol, cold 1100
1000
0.05 M sodium hydroxide in 70%
alcohol, hot
Residue
Loss
Nitrogen = 5.00
gm.
Reaction
Nitrogen
Total
extracted
nitrogen
of seed
pH
gm.
per cent
6.1
3.208
64.16
6.4
0.340
6.81
6.5
0.0837
1.67
0.0188
0.38
0.0584
1.17
0.0277
0.55
0.0342
0.68
0.005
0.10
0.329
6.59
0.070
1.41
0.445
8.91
0.379
7.57
This, and several similar experiments, showed that, in general,
about 80 per cent of the nitrogen of tobacco seed could be brought
into solution by suitable solvents. jMost of the extracted nitrogen
is undoubtedly protein ; and that a considerable part of the insol-
uble nitrogen also probably belongs to protein was shown by hydrol-
ysis of the extracted residue with hydrochloric acid. From 85 to
87 per cent of the nitrogen that was thereby obtained in solution
was in the form of amino nitrogen. The failure to obtain in soluble
form a larger proportion of the nitrogen of tobacco seed recalls the
experience of Jones and Csonka (4) who found that not more than
7Z per cent of the nitrogen of feterita and kafir seeds could be
brought into solution with alkaline alcohol.
THE GLOBULIN OF THE TOBACCO SEED
To anticipate slightly it may be said that we have so far demon-
strated the presence of only one definite protein in tobacco seed ;
this is a globulin which, in some respects, resembles edestin from
hempseed. Filtered saline extracts of the seed become turbid on
heating to about 60° and it is therefore probable that a small
amount of coagulable albumin is present. The best yield of purified
globulin obtained as a dry preparation amounted to less than half
the probable protein content of the seed, but whether the balance
represents exclusively proteins of another type, or in part consists
628 Connecticut Experiment Station Bulletin 339
of globulin the solubility of which had become altered during the
operations of extraction, has not yet been determined.
The globulin may be prepared in several different ways. The
following qualitative experiments were designed to show that a
similar product is obtained in each case.
PREPARATION OF SALINE EXTRACT
For the rapid preparation of small quantities of extract 50 gm.
of fat-free meal were stirred into 500 cc. of molar sodium chloride
solution at 50°, two 50 cm. square sheets of filter paper were added
in small pieces, and the whole was reduced to a pulp. The mass was
then squeezed by hand through cheesecloth, the filter paper pulp
serving to retain particles of meal, and 325 cc. of turbid fluid were
secured. The residue, enveloped in the cheesecloth, was pressed
at the hydraulic press, an additional 100 cc. being thus obtained.
The extract was then filtered through a thick pad of filter paper
pulp which had previously been washed with molar sodium chlo-
ride. The first portion of filtrate was collected separately and
returned to the filter, after the extract had run through, to serve
as washing fluid. The filtration proceeded rapidly ; the filtrate was
a perfectly clear, slightly viscous, amber colored fluid that pos-
sessed a strong Tyndall effect. The nitrogen in this extract cor-
responded to 55.2 per cent of the nitrogen of the meal.
A more concentrated extract can be prepared by employing 100
gm. of meal and proceeding in the same way, but the filtration is
then much slower.
PREPARATION OF THE GLOBULIN BY DILUTION
A 500 CC. quantity of clear extract prepared essentially as
described and warmed to 50° was diluted with 10 volumes of water
at 50° ; the solution remained clear for a short time. It was chilled
overnight, the clear top liquid was decanted, and the separated
protein was centrifuged ofif and dissolved in about 200 cc. of molar
sodium chloride. The solution was clear but was, nevertheless,
filtered through pulp previously washed with solvent. The filtrate
was diluted as before and allowed to stand for 24 hours. The pro-
tein was again dissolved and filtered. A third separation by dilu-
tion yielded a product that was instantly soluble in molar sodium
chloride to a clear solution. It was washed with water until nearly
free from chloride, as evidenced by a tendency to pass into colloidal
solution, then with 50 per cent alcohol until free from chloride,
and finally with absolute alcohol and ether. The white powder
was dried by exposure to the air in a thin layer. The preparation
contained 8.00 per cent of moisture and 0.11 per cent of ash. The
nitrogen content, calculated ash- and moisture-free, was 18.73 per
cent and the sulfur content was 0.97 per cent.
The Globulin of Tobacco Seed 629
PREPARATION OF THE GLOBULIN BY DIALYSIS
A similar 500 cc. quantity of clear extract was placed in a cello-
phane dialyzing bag together with a little toluene and dialyzed in
running water for 2 days. The contents of the bag were rinsed
into a centrifuge bottle with water and the separated protein was
centrifuged oiif. It was redissolved in about 200 cc. of molar
sodium chloride and the solution, though clear, was filtered through
pulp. The filtrate was dialyzed a second time, the protein was
collected as before, redissolved, filtered, and the solution was
dialyzed a third time. The final product was completely soluble
in molar sodium chloride and was washed with dilute and with
absolute alcohol and finally with ether; it was then dried in the
air. It contained 6.59 per cent of water and 0.13 per cent of ash;
the nitrogen content was 18.66 per cent and the sulfur content 1.12
per cent, both calculated ash- and moisture-free.
PREPARATION OF THE GLOBULIN BY SALTING OUT
A third 500 cc. quantity of clear extract was treated with a suffi-
cient quantity of dry, powdered ammonium sulfate (132 gm.) to
produce a concentration with respect to this salt of approximately
2 molal. The precipitated protein was centrifuged ofif and was
washed with 2.5 M ammonium sulfate. It was then redissolved in
500 cc. of molar sodium chloride, in which it was wholly soluble,
was filtered through pulp and the protein was precipitated as before.
The solution of the protein in molar sodium chloride was this time
slightly turbid, due to the presence of a little denatured protein. It
was filtered clear and the protein was precipitated a third time. The
product was dissolved in 700 cc. of molar sodium chloride, was
filtered from a trace of denatured protein and was dialyzed into
running water until sulfate ion could no longer be detected. The
separated protein was centrifuged off, was washed successively
with water, 50 per cent alcohol, absolute alcohol, and ether, and
dried in the air. It contained 6.46 per cent of moisture and 0.15 per
cent of ash. The nitrogen content was 18.54 per cent and the
sulfur content 1.00 per cent calculated ash- and moisture-free.
These three methods of preparation yielded products of essen-
tially the same physical and chemical properties. The average nitro-
gen content was 18.64 per cent and the average sulfur content 1.03
per cent. The preparations were all soluble in molar sodium chlo-
ride, but were practically insoluble in water or alcohol ; the custom-
ary color tests were all positive and the protein responds in every
way to the definition of a globulin. Although none of the above
described preparations was well crystallized all contained a few
630
Connecticut Expcrhnent Station Bulletin 339
crystals ; the greater part of each preparation consisted of tiny
spherical particles. The preparation of wholly crystalline material
is described below and an illustration of the crystals is shown in
Figure 56.
Figure 56. The crystalline globulin of the tobacco seed (x 500).
PREPARATION OF CRYSTALLINE GLOBULIN
Although it is relatively easy to prepare specimens of tobacco
seed globulin that are largely crystalline it was found unusually
difficult to secure material that consisted wholly of large, perfectly
formed crystals of uniform size. A number of factors appear to
influence the outcome of experiments designed to provide such a
product but, unfortunately, the exact conditions under which vege-
table globulins may best be crystallized have not yet been defined.
In the case of the tobacco seed globulin the method of dilution of a
warm molar sodium chloride extract of the seed is superior to any
The Globulin of Tobacco Seed 631
other for this purpose, and we have found that a number of circum-
stances have an effect upon the appearance of the final preparation.
The saHne solution of the protein must be relatively dilute and
perfectly clear. It is best to work with ground seed from which the
fat has not been removed by ether extraction, and better results
appear to be obtained with material that has stood a few days after
grinding. The temperature conditions, and rate of cooling are very
important, but the range of reaction within which good crystals
can be secured is quite broad and, apparently, little attention need
be paid to this factor when working with a fresh extract from the
seed.
The finest preparations of crystals were secured by stirring 50
gm. of ground seed with 350 cc. of molar sodium chloride at 56°
in a warm centrifuge bottle. The pasty mass was then centrifuged ;
the oil at the surface was sucked off through a capillary tube and
the clear extract was decanted from the firmly packed residue
through a pad of paper pulp on a Buchner funnel ; 275 cc. of per-
fectly clear extract of a reaction at pH 5.9 were secured. This was
warmed to 55°, was mixed with 5 volumes of water at 55°, and
rapidly filtered through pulp, whereby a sparkling clear filtrate was
obtained. A small quantity of toluene was stirred in and the solu-
tion, the reaction of which was now at pH 6.3, was allowed to stand
at room temperature overnight. The protein then formed a dense
deposit at the bottom of the beaker ; the supernatant fluid was
clear and yielded little or no turbidity on further dilution. Under the
microscope the regular octahedral crystals were uniform in size and
no amorphous material nor spheroidal particles were visible. The
crystals were collected and washed once with 50 per cent alcohol by
centrifugation, were then transferred to a filter, and thoroughly
washed with absolute alcohol and ether. After being air-dried in a
thin layer they were found to have suffered little or no damage.
PREPARATION OF THE GLOBULIN BY EXTRACTION WITH ALKALI
A 50 gm. portion of the fat-free meal was stirred into 500 cc.
of 0.05 M sodium hydroxide and a sufficient quantity of filter paper
clippings was added so as to make a soft mass that could be envel-
oped in cheesecloth. The extract was expressed by hand and the
residue was re-extracted with 500 cc. more of alkali; the final
residue was pressed at the hydraulic press. The combined extracts,
after centrifugation to remove gross particles, were filtered per-
fectly clear on pulp. Very dilute hydrochloric acid was then slowly
added, during rapid stirring, until the protein had flocculated. The
protein was removed and washed and was then dissolved in dilute
alkali and reprecipitated twice successively. It was finally de-
hydrated with alcohol and ether.
Although great care had been taken during the acid precipita-
632 ' Connecticut Experiment Station Bulletin 339
tions, the product was extensively denatured, very little of it being
soluble in molar sodium chloride. The preparation contained 9.17
per cent of moisture and 0.31 per cent of ash. The nitrogen and
sulfur content were 18.28 per cent and 0.83 per cent respectively.
This method of preparation leads to a largely denatured product
of lower nitrogen and sulfur, and higher ash content than those
obtained by the other methods described.
BASIC AMINO ACIDS OF TOBACCO SEED GLOBULIN
A quantity of 70.82 gm, (corrected) of the globuHn prepared
by the salting-out method was hydrolyzed by boiling 8 N sulfuric
acid in the customary way and the greater part of the acid was
removed as barium sulfate; this was extensively washed. The
solution and washings were brought to 2 liters and the basic amino
acids were isolated according to the technic described in previous
papers from this laboratory (8). The results are shown in Table 4.
Table 4. The Basic Amino Acids of Tobacco Seed Globulin
From nitrogen
From weight
in fraction
of salt
per cent
per cent
Histidine
1.28
1.09^
Arginine
16.04
13.62'
Lysine
2.39
0.92'
The proportion of arginine given contains the small correction
customarily added to allow for the solubihty of arginine silver in
the alkaline fluid from which it is precipitated. The recrystallized
lysine picrate decomposed at 265° and the proportion given cor-
responds to the sum of the weight of the recrystallized material
and the small quantity in the mother Hquor calculated from the solu-
bility of lysine picrate.
Although this globulin resembles those from a number of other
oil seeds in its high content of arginine, it yields unusually low pro-
portions of histidine and of lysine. Amandin from the almond
approaches it most closely in this respect, but even this protein
yields 1.87 per cent of histidine (5). It is, of course, possible that
the histidine figures in the old analyses are somewhat high since
they are all based upon nitrogen determinations on fractions that
almost certainly contained some cystine (9) ; nevertheless there
is little doubt that the tobacco seed globulin yields distinctly less
histidine than the other oil seed globulins of which analyses have
been reported. There is no apparent possibility of classifying these
proteins on the basis of the ratio of the molecular proportions of
the basic amino acids, as has been attempted for a series of keratins
by Block and Vickery ( 1 ) ,
^The histidine diflavianate contained 8.18 per cent sulfur, theory 8.17 per cent.
^The arginine flavianate contained 6.72 per cent sulfur, theory 6.56 per cent.
'The recrystallized lysine picrate decomposed at 265°.
The Globulin of Tobacco Seed 633
THE ISOELECTRIC POINT OF THE GLOBULIN
Attempts to locate the exact point at which tobacco seed globulin
is isoelectric did not lead to conclusive results. When phosphate
buffers were employed according to the method of Csonka, Murphy
and Jones (2), the point of minimum solubility of a previously
dried specimen of the protein was at pH 5.6. A freshly prepared
moist specimen, tested with phthalate buffers, appeared to be least
soluble at pH 5.4. In another series of experiments determinations
were made of the nitrogen that passed into solution when specimens
of dried or freshly prepared globulin were shaken at room temper-
ature with M/20 phosphate or with M/10 citrate buffers for a
definite period of time, usually 3 hours. The solubility did not vary
significantly over the range pH 5.2 to 5.5; beyond these limits it
increased. A few experiments in which approximately 1 gm. of
fresh moist protein was shaken with 100 cc, of citrate buffer solu-
tion for 3 hours showed that the effect upon the reaction of the
buffer was minimal in the range pH 5.3 to 5.5. Outside of this
range appreciable changes in the reaction occurred ; for example,
the reaction of the buffer at pH 5.26 changed to 5.30 and that of
the buffer at pH 5.65 changed to 5.60.
The available evidence suggests, therefore, that tobacco seed
globulin is isoelectric at a point in the vicinity of pH 5.4, the
accuracy of the determination probably being not greater than
±0.1 units. The isoelectric point of this protein therefore falls
within the range observed for many of the seed globulins by
Csonka, Murphy and Jones,
THE BEHAVIOR OF THE GLOBULIN TOWARD SALT SOLUTIONS
A filtered extract prepared from 500 gm. of fat-free meal by
means of 4500 cc. of molar sodium chloride solution was treated
with dry ammonium sulfate, with careful stirring, until the con-
centration with respect to this salt was 1.5 molal. The flocculent
precipitate, which had first appeared at approximately 1 M con-
centration, settled fairly well, but when a little more of the salt
was added to the nearly clear solution a small turbidity developed.
The concentration was therefore raised to 2 M and the precipitate
(A), after centrifuging, was washed with 2 M ammonium sulfate
solution. The addition of more ammonium sulfate to the main
solution produced a small turbidity; the concentration was there-
fore raised to 4.5 M when a small precipitate (B) that settled
fairly readily had formed ; this was removed. The filtrate, on stand-
ing overnight, deposited a little more precipitate (C). Fraction A
was dissolved in warm molar sodium chloride, filtered at 60°
through paper pulp from a trace of denatured protein, and the pro-
tein was then precipitated by dilution with 5 volumes of water at
634 Connecticut Experiment Station Bulletin 339
60°. The protein was reprecipitated twice more by dilution and
was finally obtained, after washing in the customary way with
alcohol and ether, for the most part in crystalline form, although
some spheroidal particles were present. The preparation contained
18.61 per cent of nitrogen, moisture-free, and weighed 55.3 gm.,
or 11 per cent of the fat-free meal taken.
Fraction B was dissolved in 300 cc. of water and was again
treated with ammonium sulfate. At 2 M concentration a bulky
flocculent precipitate had separated which was centrifuged off. On
adding more ammonium sulfate the solution became turbid but no
flocculation occurred even at 5 M. The slimy precipitate was
centrifuged as well as possible from the turbid fluid and was placed
in a cellophane dialyzer. After complete dialysis of the salts the
fluid was still turbid and a very small slimy precipitate had separat-
ed. This was instantly soluble in molar sodium chloride but, even
on dialysis of this solution, no satisfactory separation could be
obtained. The behavior described suggests that the greater part of
the protein extracted from tobacco seed meal is a globulin that is
thrown out of solution nearly completely at 2 M concentration of
ammonium sulfate. In addition there is a small amount of protein-
like material of much less clearly defined solubility.
In another experiment 4600 cc. of an extract of 500 gm. of
meal was diluted to 20 liters with water at 60° and the moist
protein, that separated on standing, was collected and dissolved
in 500 cc. of molar sodium chloride. Analysis of the solution
showed that 530 cc. of water were present. Dry ammonium
sulfate was added as before ; a permanent precipitate first formed
when the concentration reached 0.7 M and nearly all the protein
had been precipitated at 1.5 M; only a little more precipitate
separated at 2 M and subsequently only traces were obtained even
at 4.3 M. The protein, which separates on dilution of a saline
extract of tobacco seed, consists almost entirely therefore of globu-
lin that is salted out between the limits of ammonium sulfate con-
centration 0.7 to 1.5 M.
This moist specimen of protein was treated with 300 cc. of water,
in which it dissolved, and dry sodium chloride was added with
stirring until the solution was saturated ; a small slimy precipitate
separated from the highly colloidal but clear solution. This pre-
cipitate, together with the excess sodium chloride, was triturated
with 100 cc. of saturated sodium chloride solution, whereupon
much of it dissolved. The insoluble residue was then separately
treated with 100 cc. of water; nearly all passed instantly into
solution and the small part that remained, apparently denatured
protein, was filtered off. This solution was then diluted to 600 cc.
with water at 60°. On standing, small well-formed crystals of
the globulin separated.
The colloidal sodium chloride solution, from which the small
The Globulin of Tobacco Seed 635
precipitate had been removed, was treated with magnesium sulfate
in an amount necessary to produce a 0.5 molal concentration
calculated on the water (approximately 300 cc.) originally present.
A fine-grained white precipitate separated ; this was centrifuged
off and a second quantity of magnesium sulfate equal to the first
was added. A very small precipitate was thereby produced and
the filtrate was free from protein as shown by a negative biuret
test.
The precipitate produced by the first portion of magnesium
sulfate was dissolved in 400 cc. of water at 60° and the clear
solution was diluted to 2 liters. After being chilled the protein
separated in small, rather poorly formed octahedral crystals, and
in spherules. This preparation, after washing with alcohol, weighed
35.6 gm. (8.0 per cent of the meal) and contained 18.41 per cent
nitrogen moisture-free.
From these observations it appears that the globulin of the
tobacco seed is almost entirely soluble in saturated sodium chloride,
very little of it being precipitated under the conditions of the
experiment. The whole of the protein is, however, thrown out of
such a solution by the addition of a relatively small concentration
of magnesium sulfate.
BEHAVIOR OF THE GLOBULIN ON HEATING
A Specimen of a molar sodium chloride extract of the seed
at pH 5.5, and of 2 per cent concentration with respect to the
protein, was carefully heated with continuous stirring in a water
bath. At 60° the solution was faintly turbid and at 77° definite
flocks separated. After centrifugation the clear fluid was
heated to 80° when flocks again separated. The operations
of heating very slowly a few degrees and then centrifuging
were repeated, flocculations being removed at 84°, 85°, 89°,
90°, and 94°, and a small residual coagulum after boiling. The
reaction had now become more acid (pH 5.3). With the possible
exception of the initial turbidity that forms at 60° there was no
definite evidence of the presence of proteins that coagulate at
different temperatures ; the behavior suggested rather that the
globulin is slowly and progressively denatured at temperatures
above 77° in a manner analogous in many ways to the behavior
of other seed globulins.
SUMMARY
The seed of the tobacco plant contains a protein of the globulin
type that can readily be prepared in crystalline form by the
classical methods. The crystals are regular octahedra occasionally
modified into flattened tablets of hexagonal outline. After com-
636 Connecticut Experiment Station Bulletin 339
plete acid hydrolysis the protein yields 1.09 per cent of histidine,
13.62 per cent of arginine, and 0.92 per cent of lysine; color tests
indicate that tryptophane, cystine, and tyrosine are likewise present.
The isoelectric point of the globulin lies near pH 5.4. It is almost
completely salted out of a relatively concentrated solution by
ammonium sulfate between the limits 0.7 to 1.5 M, but is soluble
in saturated sodium chloride solution. On heating its solution in
molar sodium chloride at pH 5.5 it is slowly and progressively
denatured at temperatures higher than 77° .
BIBLIOGRAPHY
1. Block, R. J., and Vickery, H. B., Jour. Biol. Chem., 93: 113. 1931.
2. CsoNKA, F. A., Murphy, J. C, and Jones, D. B., Jour. Amer. Chem.
Soc, 48: 763. 1926.
3. Ilyin, G., U. S. S. R. State Inst. Tobacco Investigations, Bui. 57 (in
Russian). 1929.
4. Jones, D. B., and Csonka, F. A., Jour. Biol. Chem., 88 : 305. 1930.
5. Osborne, T. B., Leavenworth, C. S., and Brautlecht, C. A., Amer.
Jour. Physiol., 23 : 180. 1908.
6. Paris, G., Bol. tec. (R. ist. sper. coltiv. tabacchi, Scafati), 17: No. 1.
101. 1920.
7. Preissecker, K., and Brezina, H., Fachl. Mitt, osterr. Tabakregie, No.
4 1917.
8. Vickery, H. B., and Block, R. J., Jour. Biol. Chem., 86 : 107. 1930.
9. Vickery, H. B., and Leavenworth, C. S., Jour. Biol. Chem., 83 : 523.
1929.
10. Vickery, H. B., and Pucher, G. W., Conn. Agr. Expt. Sta., Bui. 311 :
234. 1930.
PART III
SOME NITROGENOUS COMPONENTS OF
THE HOT WATER EXTRACT
OF FAT-FREE TOBACCO SEED MEAL
Hubert Bradford Vickery
The literature on the composition of water extracts made from
plant seeds is surprisingly meager. Most investigations of seeds
have dealt exclusively with the detection or determination of a
single type of substance, such as allantoin or an alkaloid, but no
comprehensive chemical investigation of the water extract of
seeds has come to our attention. The desirability of such in-
vestigations is manifest; seeds form a large part of the nutriment
of animals and, although the greater part of their food value arises
from the carbohydrate, protein, fat, and inorganic salts they con-
tain, there is little doubt that substances present in minute amounts,
such as the vitamins, are also of great importance in animal
nutrition. The significance of chemical studies of seeds far tran-
scends this, however. The seed provides the store of food upon
which the growth of the embryo of the plant depends until its
organs of assimilation are developed — every necessary constituent
of the food, save water and oxygen, is therefore actually or poten-
tially present, and the study of the chemical composition of seeds
is really, therefore, the study of the composition of the nutriment
furnished to the developing embryo.
The present investigation had its origin in the observation (4)
that tobacco seeds contain no nicotine, but, after being allowed to
germinate for 9 to 11 days, quite appreciable quantities of this
alkaloid develop in the seedlings. The normal metabolism of the
tobacco plant therefore produces nicotine even at the earliest stages
and, furthermore, a chemical mechanism is present whereby nico-
tine can be synthesized from non-alkaloidal precursors stored
within the seed. It seemed of importance to attempt to identify
these precursors. Although the preliminary search was unsuccess-
ful the experience gained has indicated how the problem can be
again attacked with hope for a more successful outcome.
PREPARATION OF HOT WATER EXTRACT
Seed obtained from shade-grown tobacco of the 1929 crop was
ground in a poppy seed mill. The pasty mass was weighed and
extracted with light gasoline twice successively; the material was
then washed with a liberal quantity of ether and spread out in a
thin layer to dry. After all the ether had evaporated the meal
638 Connecticut Experiment Station Bulletin 339
was again passed through the mill. Treated in this way 2955 gm.
of freshly ground tobacco seed yielded 1738 gm. of air-dry meal.
The loss of 1217 gm. or 41.2 per cent represents the oil removed
by the solvents. The air-dry meal contained 7.01 per cent of
moisture; after being completely dried it contained 7.11 per cent
of nitrogen.
The meal was slowly introduced into about 13 liters of boiHng
distilled water and was boiled for IS minutes. Enough dilute
sulfuric acid to bring the reaction to pH 4 was then added and,
after being allowed to mascerate for several hours, the insoluble
residue was filtered off. This was boiled as before with 15 liters
of water to which enough sulfuric acid had been added to bring
the reaction to pH 4, and the operation was repeated a third time.
The final residue was enveloped in filter cloth and pressed dry at
the hydraulic press, the cakes were then stirred up with water and
were pressed a second time.
The acidity of the extracts was reduced to pH 5 to 6 by the
addition of barium hydroxide and the solutions were evaporated,
at first in open dishes and then in vacuum stills, to a volume of
about 10 liters ; the solution was then filtered and further con-
centrated to 2 liters. Alcohol to make a total volume of 5 liters
was added and the precipitate, which settled after the solution had
been chilled overnight, was removed. This was thoroughly washed
with alcohol and dried. It weighed 232 gm., or 13.6 per cent of
the fat-free meal, and contained 39.75 per cent of ash and 4.11
Table 5. Composition of the Hot Water Extract Prepared
FROM Tobacco Seed Meal
Extract 1
Extract 2
Total
gm.
gm.
gm.
Whole seed
2955
2140
5095
Fat-extracted meal
1710
1284
2994
Nitrogen in fat-extracted meal
121.6
84.74
206.3
Ash
101.7
76.4
178.1
Alcohol precipitate
232.1
158.2
380.3
Nitrogen in alcohol precipitate
9.56
6.38
15.94
Ash _ "
92.26
63.72
155.9
Nitrogen in water extract
6.77
6.50
13.27
Ammonia nitrogen in water extract
0.321
0.294
0.615
Amide " " " "
0.743
0.576
1.319
Amino " " " "
1.38
0.86
2.24
Organic solids " " "
115.9
111.9
227.8
Ash " "
12.6
13.1
25.7
per cent of nitrogen. If the nitrogen is assumed to be in the form
of coagulated protein the product must have contained at least
80 gm., or 33 per cent, of non-protein organic substances, some
of which probably consisted of substances allied to the pectins.
The alcoholic solution was concentrated to a sirup, was diluted
and filtered from a trace of insoluble material, and was then made
Nitrogenous Components of Hot Water Extract 639
to a definite volume for analysis. A second extract from 1284 gm.
of fat-free seed was similarly prepared and analyzed and the two
were united after the removal of the barium hydroxide-alcohol
precipitates described in the following section.
In Table 5 are given the analytical data on these two extracts
together with sums of the separate items. The combined solutions
were then examined in detail according to the methods of fractiona-
tion in current use in this laboratory.
THE BARIUM HYDROXIDE-ALCOHOL PRECIPITATION
The procedure followed in removing the substances that are
precipitated by barium hydroxide from an alcoholic (50 per cent)
solution was substantially that already described in detail on
page 173 of Bulletin 323 (5) of this Station; this Bulletin like-
wise contains the data obtained from the detailed analysis of the
precipitate.
THE NEUBERG REAGENT PRECIPITATION
The combined filtrates from the barium hydroxide-alcohol pre-
cipitations were treated with mercuric acetate, sodium carbonate
and alcohol according to the technique described by Neuberg and
Kerb (1). This procedure has been found to effect an important
concentration of the nitrogenous substances and a purification of
these from the non-nitrogenous substances present in the extract.
The precipitate produced was decomposed by hydrogen sulfide
and was found to contain 8.27 gm. of nitrogen, or 62.3 per cent
of the water soluble nitrogen of the seed, distributed as is shown
in Table 6.
Table 6. The Composition of the Neuberg Precipitate
gtn.
Total nitrogen
8.27
Ammonia "
0.76
Amide "
0.578
Amino "
1.86
Peptide ' "
1.81
Organic solids
76.88
Ash
6.01
This fraction contains those substances that possess an amino
group in the a position to a carboxyl group, that is, the amino
acids ; peptides of these together with basic substances, such as the
purines and pyrimidines ; and a variety of substances the nature
of which is still unknown are also present. The separation of the
individual components of such a mixture has provided an analytical
problem that is still far from solution. At the present time it is
640 Connecticut Experiment Station Bulletin 339
possible only to divide the mixture into subfractions that contain
substances of essentially similar types, and to isolate a few of the
components the properties of which are sufficiently well known
to permit of this. For the rest it is necessary to depend upon
indirect methods of analysis where these can be supplied.
SUBFRACTIONATION BY MEANS OF SILVER COMPOUNDS
Previous experience in this laboratory has shown that fairly
definite subfractions of the mixture usually present in mercuric
acetate precipitates can be obtained by precipitation of silver com-
pounds between selected pH limits. This procedure has found
wide application in dealing with the relatively much simpler
mixtures of substances in protein hydrolysates, and the particular
pH limits chosen in the present work are those that have been
found most useful in the analysis of proteins ; such a selection is,
of course, purely arbitrary in the present case.
The actual technique employed was substantially that described
by Vickery and Leavenworth (3). A preliminary precipitation of
the silver compounds was made at strongly alkaline reaction ; the
material so obtained was then fractionated into silver compounds
insoluble at pH 4, pH 7 , and pH 12. These three fractions are
designated the Purine, Histidine, and Arginine fractions respective-
ly, and the combined filtrates form the Lysine fraction.
THE PURINE FRACTION
The purine fraction prepared by precipitation of the silver
compounds at pH 4 contains, in addition to true purines, other
substances that form acid insoluble silver salts. To separate the
true purines from these the solution was made alkaline with
ammonia and an excess of silver oxide was added ; the precipitate
produced was then examined for purines. Adenine and guanine
were found in the amounts shown in Table 7 .
Table 7. The Purine Fraction
gin.
Total nitrogen of the fraction 2.08
Nitrogen of adenine 0.254
" " guanine 0.024
" " filtrate from ammoniacal silver precipitate 1.452
It is clear that the true purines form only a small part of the
nitrogenous substances of this important fraction. Attempts to
isolate definite substances from the non-purine part were un-
successful ; a small amount of organic acid was present, and
positive color tests were obtained with diazobenzenesulfonic acid
and with Folin's phosphotungstic acid reagent; substances that
Nitrogenous Components of Hot Water Extract 641
contain nitrogenous ring structures, perhaps of the imidazole or
pyrimidine type, therefore probably occurred in it. Attempts at
isolation of definite compounds with such reagents as flavianic
acid or mercuric sulfate showed that basic substances were present,
but no crystalline products could be brought to separate.
THE HISTIDINE FRACTION
This fraction contained only 0.593 gm. of nitrogen. It gave
positive color tests for imidazole compounds, but treatment with
mercuric sulfate in the manner customarily employed for the
isolation of histidine precipitated less than one-tenth of the nitrogen
of the fraction and no histidine diflavianate could be obtained from
the precipitate. The filtrate from the mercuric sulfate precipitate
yielded allantoin, by direct crystallization, in an amount equivalent
to 0.102 gm. of nitrogen, or 17.3 per cent of the nitrogen of the
fraction. The product was identified by its decomposition point
of 232°, which was unchanged by admixture with authentic
allantoin. This substance has previously been reported as a con-
stituent of tobacco seed by Scurti and Perciabosco (2).
The filtrate from the allantoin contained an appreciable amount
of peptides of amino acids, inasmuch as its amino nitrogen was
increased from 19.1 to 44.7 per cent of the total nitrogen by severe
hydrolysis.
THE ARGININE FRACTION
This fraction contained 1.12 gm. of nitrogen or 13.5 per cent
of the nitrogen of the Neuberg reagent precipitate. A small amount
of arginine (flavianate, S 6.60, theory 6.56 per cent) was isolated
by treatment of this solution with flavianic acid. An oily flavianate
which appeared to contain a peptide of arginine likewise separated.
After complete hydrolysis the amino nitrogen in this material was
increased from 27.3 to 56.7 per cent, and subsequent treatment
with flavianic acid afiforded pure arginine flavianate (S 6.56 per
cent) in an amount equivalent to the non-amino nitrogen of the
solution after hydrolysis.
After removal of the substances precipitated by flavianic acid,
and also of the reagents, the solution was subjected to direct
crystallization whereby allantoin of decomposition point 231°
separated (C 30.30, H 3.93, N 35.14; theory C 30.33, H 3.83,
N 35.49 per cent) in an amount equivalent to 0.137 gm. of nitrogen.
The mother liquor was hydrolyzed with acid whereby the amino
nitrogen was increased from 17.1 to 43.1 per cent of the total
nitrogen; peptides of amino acids were therefore present.
The arginine fraction contained 0.219 gm. of arginine nitrogen,
0.137 gm. of allantoin nitrogen, and 0.194 gm. of peptide nitrogen,
making in all 49 per cent of the total nitrogen in assigned forms.
642
Connecticut Experiment Station Bulletin 339
THE LYSINE FRACTION
The filtrates from the silver precipitates at alkaline reaction
were combined and freed from barium and silver. The basic
substances were then precipitated by phosphotungstic acid and the
precipitate was treated, in the usual way, with acetone. A part of
it failed to dissolve; this was therefore removed and decomposed
by treatment in aqueous suspension with barium hydroxide. The
two fractions were subsequently dealt with separately.
The results of indirect analysis of these fractions can be most
concisely presented in tabular form (Table 8). The losses of
nitrogen in the operations of regenerating the basic substances
from the phosphotungstic acid precipitates were severe; 1.62 gm.,
or Z6 per cent of the total nitrogen disappeared, probably for the
most part adsorbed on the precipitates, although a part of this
may have been present as ammonia which volatilized from the
alkaline solutions. Similar severe losses have been encountered
in dealing with phosphotungstic acid precipitates obtained from
other plant extracts.
The solution derived from the acetone soluble phosphotungstates
was treated in the usual way with picric acid but no crystalline
material could be obtained from it. It was therefore subjected to
severe hydrolysis, the basic substances were precipitated by phos-
photungstic acid, and an attempt was again made to isolate lysine
as the picrate. Even after seeding with pure lysine picrate nothing
could be brought to crystalHzation and it was evident that this
fraction contained little, if any, lysine.
Table 8. The Lysine Fraction
Nitrogen 4.48 gm.
Acetone
soluble
phospho-
tungstates
Acetone
insoluble
phospho-
tungstates
Filtrate from
phosphotungstic
acid
(Mono amino
acid fraction)
gm.
gm.
gm.
Total nitrogen
Amino "
0.548
0.122
0.543
0.055
1.771
0.649
Peptide "
0.196
0.094
0.253
THE MONO AMINO ACID FRACTION
The filtrate from the phosphotungstic acid precipitate was freed
from reagents and evaporated to a sirup. In order to see if any
asparagine had survived the previous operations this sirup was
seeded with a small fragment of asparagine. No crystallization
occurred even after treatment with alcohol, and it was therefore
inferred that asparagine could have been present at most in only
very small amounts.
Nitrogenous Components of Hot Water Extract 643
The fraction was next subjected to severe hydrolysis and an
attempt was made to separate the dicarboxylic acids by precipitation
with barium hydroxide and alcohol. The precipitate contained
15 per cent of the total nitrogen of the fraction, but yielded no
aspartic acid when treated in the customary way with copper
hydroxide. It seems evident, therefore, that asparagine made up
no significant part of the original amide nitrogen of the tobacco
seed extract.
The filtrate from the dicarboxylates, after being freed from
reagents, was evaporated to a sirup. A mixture of amino acids
separated but the total quantity was too small to permit isolation
of individual substances.
THE FILTRATE FROM THE NEUBERG REAGENT PRECIPITATE
After removal of reagents the basic substances in this filtrate
were precipitated by phosphotungstic acid and the solution obtained
by decomposition of the phosphotungstates was investigated for
quaternary bases.
Nicotinic acid could not be detected in this solution. The chief
component, choline, was isolated for the most part by direct
crystallization of the picrate. Recourse was then had to Stanek's
periodide procedure to separate the rest of the choline from
the other basic substance present; this turned out to be betaine.
The choline picrate was identified by its melting point (248°) and
by analysis for nitrogen (16.8 per cent; theory 16.87 per cent).
The betaine picrate melted at 182 to 183° and contained C 38.38,
H 4.11, N 16.19 per cent; theory C 38.15, H 4.04, N 16.18 per cent.
The quantities of these two bases that were isolated from this
fraction are shown in Table 9; together they account for 86.5
per cent of the basic nitrogen of the filtrate from the Neuberg
reagent precipitate.
Table 9. Choune and Betaine Isolated as Picrates from the
Betaine Fraction
gm.
Total nitrogen of the fraction 0.528
Nitrogen of choline from main crystallization of picrate 0.331
" " " " periodide precipitate 0.059
" betaine " " " 0.067
" " mother liquor of periodide 0.016
Total nitrogen isolated 0.457
DISCUSSION
The chief results of the fractionation of this extract of tobacco
seed are shown in Table 10. It is clear that a large part of the
nitrogen belongs to substances that are essentially basic in their
behavior towards the customary precipitants. A disappointingly
644 Connecticut Experiment Station Bulletin 339
small part of the total nitrogen was isolated as definite crystalline
substances, and the evidence points unmistakably to the presence
of large proportions of nitrogenous compounds of wholly unknown
nature. It is unlikely that these belong to types strange to organic
chemistry ; obviously new methods of attack must be devised in
order to secure some idea of what this unknown material may be.
Table 10. Distribution of Nitrogen in Fractions
gm.
per cent
gm.
per cent
Total nitrogen of extract
13.27
100.
Barium hydroxide-alcohol precipitate
1.625
12.2
Neuberg reagent precipitate
8.27
100.
8.27
62.3
Purine fraction
2.08
25.2
Histidine "
0.593
7.2
Arginine "
1.12
13.5
Lysine "
1.19
14.4
Mono-amino fraction
1.77
21.4
Filtrate from Neuberg reagent
precipitate
1.28
100.
1.28
9.67
Choline fraction
0.528
41.2
0.528
3.98
The present methods gave satisfactory results only in the
detailed analysis of the choline fraction. This fraction is much
simpler in composition than the others ; by far the greater part
of the nitrogen belonged to the two substances choline and betaine.
Although traces of other quaternary bases may have been present,
at the outside these could account for considerably less than 1 per
cent of the nitrogen of the original tobacco seed extract.
The substances isolated are shown in Table 11. In all they
account for 2.55 per cent of the organic solids and 8.98 per cent
of the nitrogen of the seed extract.
Table 11. Substances Isolated from Tobacco Seed
Weight
Nitrogen
gm.
gm.
Choline
3.367
0.390
Betaine
0.559
0.067
Adenine
0.491
0.254
Guanine
0.051
0.024
Allantoin
0.676
0.239
Arginine
(free)
0.323
0.104
"
(combined)
0.358
0.115
Total 5.825 1.193
A somewhat more encouraging result is shown in Table 12 which
gives the distribution of the nitrogen in known forms ; about
60 per cent of the nitrogen is accoimted for. The estimation of
the peptide nitrogen is derived from independent data secured
from an extract of the same tobacco seed. The quantity of amino
nitrogen reported has been corrected for the small quantity of
amino nitrogen contributed by the isolated substances.
Nitrogenous Cotnponents of Hot Water Extract 645
Table 12. Distribution of Nitrogen in Known Forms
gm.
Total nitrogen
13.27
Ammonia "
0.62
Amide "
1.32
Amino "
(corrected)
2.20
Peptide "
(estimated)
2.38
Isolated substances
1.19
Other nitrogen
5.29
SUMAIARY
per cent
100.
4.67
9.93
16.6
17.9
8.98
39.8
A hot water extract prepared from fat-free tobacco seed has
been examined with respect to the forms of nitrogen it contains.
Approximately 60 per cent of this nitrogen was accounted for in
terms of well-recog'nized nitrogenous groupings and nearly 9 per
cent was isolated in the form of pure crystalline substances. It
seems certain that a large part of the unknown forms of nitrogen
is contained in basic substances many of which contain nitrogen in
cyclic structures. No evidence was secured of the origin of the
nicotine that is produced very soon after sprouting of this seed
occurs.
BIBLIOGRAPHY
1. Neuberg, C, and Kerb, J., Biochem. Ztschr., 40 : 498. 1912.
2. Scurti, F., and Perciabosco, F., Gaz. chim. ital., 36 : II, 626. 1906.
3. Vickery, H. B., and Leavenworth, C. S., Jour. Biol. Chem., 76 : 707.
1928.
4. Vickery, H. B., and Pucher, G. W., Conn. Agr. Expt. Sta., Bui. 311 :
234. 1930.
5. Vickery, H. B., and Pucher, G. W., Conn. Agr. Expt. Sta., Bui. 323.
1931.
PART IV
A MICROCHEMICAL STUDY OF THE SEED
OF NICOTIANA TABACUM
Florence A. McCoraiick''
The interest of the Department of Biochemistry in the chemistry
of tobacco seed has given rise to a number of questions concerning
the structure of the seed and the identification and location in it
of various substances, especially protein and fat. It was the desire
to have at hand a concrete picture of such facts that led to the
request for the present brief study.
Splendore (5) in his paper on the genus Nicotiana has photo-
graphs of seeds of many varieties of N. Tabacum and he gives
comparative weights and measurements of them. He does not
consider the interior of the seed. Behrens (1) gives a short
description of the markings of the testa and of the embryo and
endosperm. Jensen (2) adds more details concerning the size
and shape of the seed and the markings of the testa. .Behrens and
Jensen give the weight of the individual seed as about 0.08 mg.
and Splendore's weights are slightly less.
In the present investigation Connecticut Havana tobacco seed
was studied. The mature seeds are a cinnamon brown. There is
considerable variation in shape, but they are commonly elliptical-
ovate (Plate 18, Fig. 6) with the longer diameter in 10 seeds
averaging 714/a and the shorter 571/^. The seeds are anatropous
with the short, often curved, funiculus remaining attached at the
narrow end. Radiating from the base of the funiculus and extend-
ing over the entire seed is a network of thickened wavy cell walls
of the testa which, under low magnification, give a roughened
appearance (Plate 18, Fig. 1, 2). The raphe, usually represented
by a ridge in anatropous seeds, is not externally apparent in this
seed.
Seeds were killed for sectioning in a saturated alcoholic solu-
tion of picric acid, in Benda's solution, in Fleming's strong solu-
tion, and in acetic acid-alcohol. For the microchemical tests,
thoroughly dried seeds, not previously treated with any killing
agent, were kept in melted paraffin in an electric oven at a tem-
perature of 52 °C for 24 hours before embedding, and these were
then sectioned with a rotary microtome, a section of a seed so
treated being represented in Plate 18, Figure 6. Such seeds, left
in the continuously melted paraffin from October 28, 1931, to
-■Of the Department of Botany.
Microchemical Study of the Seed 647
June 18, 1932, germinated when put into a moist chamber. Sec-
tions of the untreated seeds were cut S/x, to 25/*, 20/^ being the
most favorable for general study. Thinner sections of seeds killed
in the reagents were used for comparison..
Testa. The epidermis of the ovule undergoes a marked change
during the development of the embryo. The outer walls remain
thin and, in the mature seed, usually only fragments of them are
left. The inner and lateral walls, however, become greatly thick-
ened and to the latter ones are due the conspicuous markings of
the testa mentioned above. This increase in thickness of the inner
and lateral walls can be seen by comparing Figures 3 and 4 in
Plate 18 with any sections of the mature seed, particularly Figure
6. In Figure 3 there is represented an ovule containing a small
embryo. The epidermal cells are large with the inner and lateral
walls beginning to thicken. Figure 4 shows a decided thickening
of the same walls, while sections of the mature seeds show rem-
nants of the outer walls and the greatly thickened inner and lateral
walls. The crushed empty cells beneath the epidermis appear
almost homogenous in the photographs of the mature seed
(Figs. 7 and 8).
Embryo and endosperm. The embryo (Fig. 6, em) is usually
somewhat kidney-shaped with the concave side lying toward the
raphe. It is a thickened and elongated body with the large coty-
ledons (Fig. 6, c) extending about one-third its length and
with no apparent plumule. It is about 613ix long and 250ix broad
and comprises about one-fourth the volume of the seed. Endo-
sperm (Fig. 6, end) completely surrounds the embryo, but there
is relatively little of it over the narrowed tip of the radicle (Fig.
6, rad) and the rounded ends of the cotyledons. The cells of the
embryo are small and are filled with granules varying in size, but
on the whole considerab^ smaller than those in the endosperm.
The region of the raphe (Fig. 7, r) is marked by a single layer
of outermost cells. These cells are somewhat smaller than the
contiguous cells of the endosperm, are more nearly rectangular in
shape, and contain granules which are more uniform in shape and
smaller even than those in the embryo. Figures 7 and 8 are
enlarged views of parts of the endosperm, Figure 7 showing a
part of the region of the raphe (r). of the endosperm (end) and
of the embryo (em), and Figure 8 showing a part of the side
opposite the raphe.
MICROCHEMICAL TESTS
Microchemical tests were made for starch, protein, and fat.
648 Connecticut Experiment Station Bulletin 339
STARCH
The mature seed contains no starch. In the ovule, while the
embryo is still immature, there is starch in the nucellar tissue
(Figures 3 and 4, s), but this disappears before the embryo is
fully developed. Both figures are photographs of a section of an
ovule stained v^^ith iodine alone, thus dififerentiating the starch
grains (s) w^hich are limited, at this stage to the periphery. Seeds,
germinated on moist filter paper show, when the seedlings are
about l}'i mm. to 2 mm. long and before the cotyledons have
emerged, a trace of starch about in the middle of the hypocotyl
(Figure 5, s). The section from which this photograph was made,
was also stained in iodine alone and was of a seedling the cotyledons
of which were similar to those shown in Plate 19, Figure 16.
Thus we see that there is starch in the ovule, none in the mature
seed and that it early reappears in the hypocotyl of the young
seedling.
PROTEIN
For the most part Klein's Prakticum der Histochemie (3) was
followed in the study of the distribution of protein. The Enzyklo-
padie der inikroskopischen Technik (4) and Pflansenmicrochemie
by Tunmann (6) were also consulted.
As stated above, sections of seeds embedded in parafifin but not
previously treated with any killing agent, were used in the micro-
chemical tests. Sections were tested both with and without the
removal of paraffin, these showing, however, no appreciable dif-
ference in the results. The tests used indicate that the granules
which fill the cells of the mature seed are protein, but the granules
in the cells of the embryo, the endosperm and the region of the
raphe differ in their reactions to the microchemical tests. The
relatively large granules of the endosperm stain more deeply and,
on prolonged treatment with the reagent, show even a different
color from that of the smaller granules of the embryo and the still
smaller granules in the cells of the raphe. The granules in the
embryo and raphe stain more nearly alike, but those of the raphe,
while staining the same color, retain a somewhat Hghter shade.
Plate 19, Figure 14 represents a transverse section of a seed show-
ing the difference in staining of these three regions. The embryo
(Fig. 14, em) is surrounded by the darker endosperm (Fig. 14,
end) and a part of the region of the raphe is shown at r. The
section from which the photograph was made was stained in
gentian violet alone, but shows typical differences in the staining
of the three regions. A similar differentiation is exhibited by sec-
tions stained with the biuret reagents. The difference in size of
the granules can scarcely account for this distinction in staining.
Microchemical Study of the Seed 649
The difference in size of the granules and the difference in the
reaction to microchemical tests suggest that there may also be a
chemical difference.
Biuret test. The protein of the endosperm, when treated with
the biuret reagents, becomes a deep lavender and the embryo a
gray with a tinge of lavender. With prolonged staining the endo-
sperm becomes deeper in color and the lavender tone in the embryo
disappears, leaving it a light grayish brown. The raphe is lighter
than the embryo and has no lavender tone. During germination
the disintegration can be seen progressing from the micropylar
end of the seed toward the chalazal end (Figs. 12, 13, and 14)
and, at this stage, there is an increased difference in staining
between the embryo and endosperm, indicating that in the growing
seedling the protein in the embryo is used sooner than that in the
endosperm. The embryo gives a very slight protein reaction while
the endosperm still retains granules that stain a deep lavender.
Millon's reagent. With this reagent the endosperm and embryo
become an orange yellow, the endosperm containing more red
and the raphe staining lighter than the embryo, so that three
intensities of color may be seen in the section.
Xantho-protein test. When nitric acid alone is put on sections
of tobacco seed the testa immediately becomes a deep yellow
developing into an orange. The embryo and endosperm more
slowly become a light yellow. Wlien ammonia is added the color is
intensified, the endosperm being slightly darker than the embryo.
When 50 per cent caustic soda, or 50 per cent caustic potash is
used instead of ammonia both regions become orange, the endo-
sperm staining more deeply than the embryo.
Picric acid. An aqueous solution of picric acid stains the entire
section yellow. However, the endosperm is a clear lemon yellow
while the embryo and raphe are a grayish yellow deeper in tone
than the endosperm.
Vanillin test. Both embryo and endosperm stain a distinct
violet. The endosperm stains a much deeper shade than the embryo.
FAT
The seeds killed in Benda's solution as well as in Fleming's
strong solution show in sections a surprisingly small area blackened
with the osmic acid. The largest amount of fat reaction is at the
micropylar end and in the outermost layer of endosperm, which
becomes an intense black. The rest of the endosperm and the
embryo stained either a light brown or not at all. The result was
the same even though the vacuum pump was used and the seeds
were left in the killing agent 48 hours with a change to fresh
killing solution after 24 hours. This is evidentlv due to the lack
650 Coiniccficuf Experiment Station Bulletin 339
of penetration of the osmic acid in these solutions and not to a
lack of fat in the slightly or uncolored areas, for sections made
from the material killed in these osmic acid solutions, when put
into water containing a few drops of 2 per cent osmic acid alone,
stained a deep black over the entire section. Sections of seeds,
embedded in paraffin, but not killed, were treated with osmic acid
alone; these also became uniformly black in endosperm and embryo.
Sudan III and Oil Red O were likewise used and indicated fat
both in endosperm and embryo.
Figure 17 is a photograph of a cross section of a seed that had
been embedded in paraffin. The tissues had not been killed before
embedding and the paraffin had not been removed when the photo-
graph was taken nor had any staining reagent been added. To
prevent loss of fat the slide was left on the stage of the microscope
and a drop of osmic acid was then put on this section. To avoid
smearing no cover glass was added. Figure 18 is a photograph of
this section after being treated with osmic acid. The intense
blackening demonstrates the presence of fat both in the endospc-rm
and the embryo.
RESULTS
The seeds of Nicotiana Tabacinn contain no starch. There is
starch in the ovule and it develops very early in the hypocotyl of
the young seedling.
Both endosperm and embryo contain an abundance of protein.
The difference in size of the protein granules in these two parts
and the difference in reaction to the tests suggest that there may
be a chemical difference. The amount of protein in the raphe is
obviously small, but there is an indication that it is also unlike
that of the endosperm and more nearly resembles that of the
embryo. The granules are smaller and they stain more lightly
than those of the embryo, which may imply that they are also
chemically distinct from those in the embryo and endosperm.
There is present both in endosperm and embryo a large amount
of fat, which only appears in globules after the microchemical
tests have been applied. No attempt has been made to estimate
the relative amounts in the two regions.
BIBLIOGRAPHY
L Behrens, J., Landw. Vers. Sta., 41 : 191. 1892.
2. Jensen, H., Proefsta. Vorstenland. Tabak (Dutch East Indies), Meded.,
28. 1917.
3. Klein, G., Prakticum der Histochemie. Wien and Berlin 1929.
4. Krause, R., Enzjfklopadie der mikroskopischen Technik. (Berlin) 1926.
5. Splendore, a., Bol. tec. (R. ist. sper. coltiv. tabacchi. Scafati), 5: No.
4-5. 1906.
6. TuNMANN, O., Pflanzenmikrochemie. (Berlin) 1913.
PLATE 18
■•in
^'*t. ,..■^'^5
PLATE 19
Explanation of Plates 651
EXPLANATION OF PLATES
PLATE 18
Figure 1. Mature seeds of Nicotiana Tabacum; x 30.
Figure 2. Surface view of testa of seed showing the wavv lateral walls ;
x62.
Figure 3. Section of ovule of tobacco showing s, starch ; x 62.
Figure 4. Part of section of ovule showing s, starch ; x 165.
Figure 5. Part of hypocotyl showing s, starch ; x 165.
Figure 6. Section of seed embedded in paraffin but not killed: t, testa; r,
raphe ; end, endosperm ; em, embryo ; rad, radicle ; c, cotyledons ; x 62.
Figure 7. Part of section of seed : t. testa ; r, raphe ; end, endosperm ; em,
embryo ; x 165.
Figure 8. Part of seed opposite that of Fig. 7: t, testa; end, endosperm;
em, embryo ; x 165.
Figure 9. Part of section of seed : rad, radicle ; end, endosperm ; x 165.
PLATE 19
Figure 10. Protein granules from endosperm ; x 500.
Figure 11. Protein granules from embryo; x 500.
Figure 12. Section of seedling showing the partial disintegration of pro-
tein in the endosperm at the radicle end ; x 165.
Figure 13. Section of slightly older seedling showing increased disintegra-
tion of protein ; x 165.
Figure 14. Cross section of seed embedded in paraffin but not killed show-
ing the difference in staining reaction of end, endosperm ; r, raphe : em, em-
bryo ; X 165. This section was stained with gentian violet alone and shows a
typical difference resulting from microchemical tests.
Figure 15. Cross section of seedling slightly older than that of Fig. 13 ;
t, testa ; end, endosperm ; r, raphe ; c, cotyledons ; x 165.
Figure 16. Section of still older seedling ; x 165. Most of the protein of
the endosperm is gone but that in the raphe, r, is still apparent. At this stage
starch is apparent in the hypocotyl as shown in Fig. 5.
Figure 17. Cross section of seed embedded in paraffin but not killed. This
photograph was made of an unstained section and without the removal of
paraffin ; x 165.
Figure 18. Same section as that of Fig. 17 after the addition of osmic
acid, thus showing fat present both in endosperm and embryo ; x 165.
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