J. Biol. Chem.-1937-Loring-733-54

ISOLATION OF CRYSTALLINE TOBACCO MOSAIC VIRUS
PROTEIN FROM TOMATO PLANTS*

BY HUBERT S. LORING AND W. M. STANLEY
(From the Department of Animal and Plant Pathology of The Rockefeller
Institute for Medical Research, Princeton)
(Received for publication, December 15, 1936)
The isolation of a crystalline protein possessing the properties

of tobacco mosaic virus has been described (1, 2). This crystalline
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protein was obtained from the globulin fraction of extracts of
diseased Turkish tobacco plants, and was found to be over 100
times as active as the crude juice from the diseased plants used as
starting material. The chemical composition, optical rotation,
and infectivity of the crystalline protein remained unchanged after
ten successive recrystallizations. These facts have suggested
that the protein is essentially pure and is the agent responsible for
the tobacco mosaic disease.
The tobacco mosaic disease, as is well known, occurs in plants
belonging to many different genera of the solanaceous family.
The disease was first described in tomato plants by Clinton (3),
who showed that the infectious material obtained from tomato
plants infected with tobacco mosaic virus produced the same
symptom complex on healthy tobacco plants as that caused by
infectious material from diseased tobacco plants. As the juice
obtained from diseased tomato plants is highly infectious, such
plants offered the possibility of providing a new source of material
for the isolation of the tobacco mosaic virus protein. If the pro-
tein is the infectious agent, then it would necessarily be present
in mosaic-diseased tomato plants. The isolation of a similar
highly infectious, crystalline protein from diseased tomato plants
would prove that this protein is associated with the tobacco mosaic
disease in tomato plants and would provide additional evidence
* A preliminary announcement of this work was published in Science, 83,
85 (1936).
733
734 Mosaic Virus Protein from Tomato Plants
for the identity of the protein with the infectious agent. The
present paper describes the isolation of a crystalline protein from
tobacco mosaic-diseased tomato plants and presents the results
of comparative studies on the properties of this protein and that
isolated from mosaic-diseased tobacco plants.
EXPERIMENTAL
Method of Isolation-The same experimental procedure used in

the improved method for the isolation of the crystalline protein
from tobacco plants (4) was applied to diseased tomato plants,

Lycopersicon esculentum, Miller, var. Bonny Best, which had been
grown in the field. 2 months after inoculation with the ordinary
strain of tobacco mosaic virus (Johnson’s tobacco Virus l), the
plants were harvested. They were frozen, ground, and extracted.
The first extract from 200 tomato plants gave 105 liters of juice,
containing 2.6 mg. of total nitrogen per cc. (by Kjeldahl) and 0.65
mg. of protein nitrogen per cc., while a second extract gave 105
liters of juice containing 1.0 mg. of total nitrogen per cc. and 0.2
mg. of protein nitrogen per cc. The globulin fraction in each
extract was precipitated with (NH&S04 and filtered. The com-
bined yield of crude globulin amounted to 350 gm. or approxi-
mately 0.2 per cent of the weight of the freshly cut plants.
Protein nitrogen was determined by precipitation of the protein
with hot 5 per cent trichloroacetic acid. The suspension was
immediately cooled and the denatured protein was centrifuged
and dissolved in about 1 cc. of 0.2 N NaOH. It was then reprecipi-
tated with about 5 cc. of 10 per cent trichloroacetic acid and again
centrifuged. The protein residue was mixed with about 5 cc. of
5 per cent trichloroacetic acid and again centrifuged. It was then
transferred to a micro-Kjeldahl flask with 0.2 N NaOH and di-
gested, etc., as for a Kjeldahl analysis.
According to the improved method, the crude globulin was
purified by two precipitations with ammonium sulfate, by adsorp-
tion on celite at pH 4.5, and by treatment with 0.1 per cent CaO.
The application of this procedure to the crude globulin first
obtained from mosaic-diseased tomato plants resulted in a yellow
pigmented preparation which failed to crystallize. However, it
was found that if the procedure described above was repeated
several times, the yellow color was gradually lost and the protein
H. S. Loring and W. M. Stanley 735
could then be crystallized. The first crystalline material obtained
from tomato plants still contained an appreciable amount of yellow
color. This could be removed by further fractionation with
ammonium sulfate, celite, and CaO.
The application of the improved method to diseased tobacco
plants grown under the same conditions in the field as were the
tomato plants mentioned above also led finally to white crystalline
preparations, but, as in the case of the tomato plants, the crude
material had to be fractionated with ammonium sulfate, celite,
and CaO more often than when greenhouse plants were used.

The amount of treatment necessary to remove the colored impuri-
ties from the tobacco protein was, however, considerably less than
that required for the tomato protein. Whereas, in the case of the
crude tobacco globulin, four to six precipitations with (NH&SO4
and two or three precipitations on celite were sufficient to give a
white opalescent preparation, the crude tomato globulin required
approximately twice as much treatment with (NH&S04 and celite.
Measurement of Infectivity-The relative infectivity of the crys-
talline virus protein obtained from tomato and tobacco plants, as
described above, was determined by the local lesion method of
Holmes (5) as modified by Samuel and Bald (6). If infectivity
is a characteristic of the crystalline protein, then one would
expect it to possess the same infectivity regardless of the plant
source. The experiments were, therefore, planned to detect
whatever difference, if any, there might be in the infectivity of the
different preparations. Samples of virus protein from tobacco
plants were compared with samples of virus protein from tomato
plants. Solutions to be tested were prepared in dilution series
representing concentrations of 10k4, 10w5, lo-“, and 1O-7 gm. of
protein per cc. In each test one solution was rubbed on the right
halves of all the leaves of half the plants and on the left halves of
all the leaves of the remaining half of the plants, while the solution
with which it was compared was rubbed on the remaining un-
inoculated right and left halves of the leaves. Before infectivity
measurements were made, the protein solutions were dialyzed
until free of salt. Dilutions were then made with 0.1 M phosphate
at pH 7. At least twenty, and in some cases from twenty-eight
to forty-one, half leaves of Nicotiana glutinosa, L., or Phaseolus
vulgaris, L., var. Early Golden Cluster were used for each concen-
tration in each test. The relative infectivity of two solutions was

then determined by the average number of necrotic lesions pro-
duced on the half leaves on which each solution had been rubbed.
In order to evaluate the data obtained, the results of each test at
each dilution were treated statistically by ‘Student’s” method (7)
on the basis of half leaf units. In this way the mean difference
in the number of lesions between the half leaves at one dilution
and the standard deviation of the mean difference at that same
dilution were obtained. The ratio of the mean difference to the
standard deviation was calculated and the odds for significance

were determined from Fisher’s table of t (7). The infectivities of
two samples were considered significantly different if ratios greater
than 2.1, corresponding to odds of about 2O:l or more, were
obtained consistently for two or more dilutions in each of two or
more tests.
Relative Infectivity of Virus Protein from Tomato and Tobacco
Plants Grown in Field-The results of a few typical tests on the
infectivity of different samples of virus protein from the tomato
and tobacco plants described above are shown in Table I. The
samples of virus protein from tomato plants, Tomato 1 and 2, as
well as those from tobacco plants, Tobacco 1 and 2, were prepared
from different batches of crude globulin obtained from plants
harvested on different dates. All four samples were purified by
the same general procedure. The tomato samples, as mentioned
above, required more extensive fractionation than the tobacco
samples before they could be crystallized. The preparation,
Tomato 3, was obtained from that labeled Tomato 2 by further
fractionation with celite and (NH&,S04.
The results show that the crystalline virus protein obtained
from the tomato plants was deiinitely less infectious than that
isolated from the tobacco plants. The data presented also show
that repeated and prolonged fractionation of the virus protein
resulted in a gradual loss of activity. The crude globulin obtained
from old tomato plants grown in the field contained much dark
colored pigment and the virus protein could not be crystallized
without the prolonged fractionation mentioned above. Several
other attempts to obtain more active preparations from this
crude globulin likewise led to crystalline but comparatively in-
active products.
Isolation of Virus Protein from Young Greenhouse Plants-The
results obtained from old tomato plants grown in the field showed
that an active crystalline protein similar in properties to that
present in mosaic-diseased tobacco plants, but of lower infectivity,
TABLE I
Relative Infectivity of Crystalline Virus Protein from Tomato and Tobacco

Plants Grown in Field
Concentration
km. protein per cc.)
Preparation Test plant

lo-4 10-s 10-6
--__
Tomato 1 Nicotiana 68.2* 6.7 2.6 Tomato 1, slightly yellow

Tobacco 1 glutinosa 92.7 14.3 8.45 crystalline preparation
No. of half 24 24 23 first obtained from plants
leaves grown in field. Tobacco
M.DJS.D.1. 2.92 6.19 4.13 1, sample comparable to
Tomato 1 but prepared by
less drastic procedure
Tomato 2 Phaseolus 69.9 23.7 7 Tomato 2, second prepara-
Tobacco 2 vulgaris .06.3 37.3 12 tion from tomato plants.
No. of half 22 22 20 Tobacco 2, second prepa-
leaves ration from tobacco
M.D./S.D. 4.80 3.02 3.48 plants
“
Tomato 2 83.3 38.8 7.5 Tomato 3, sample obtained
I( 67.3 24.3 3.7 from Tomato 2 after pro-
31
No. of half 24 22 24 longed fractionation, as
leaves described in text
M.D./S.D. 2.04 4.42 4.15
* Numbers opposite a particular preparation represent the average

number of necrotic lesions per half leaf obtained on inoculation with the
designated preparation and concentration.
t To show a significant difference between the mean number of lesions in
any one experiment, the ratio of the difference of the mean (M.D.) to the
standard deviation of the difference (S.D.) should not be less than 2.1.
1 The concentration of Tomato 3 in these tests was 4 times as much as
indicated.
could be obtained from mosaic-diseased tomato plants. Because

of the difficulties encountered in purifying the protein, however,
it could not be determined from the above samples whether the
preparations from the two sources represented the same protein,
one with a lowered activity due to the more drastic treatment, or
whether they represented different proteins with different activi-
ties. It was also possible that the tomato virus protein differed
from the tobacco virus protein, because the tomato plants had
been infected for a longer time previous to cutting than the
tobacco plants. As shown by Wyckoff, Biscoe, and Stanley (S),
there is some variation in the sedimentation constants of the virus
proteins obtained from tobacco plants of different ages at different
intervals after inoculation. Virus protein obtained from plants 2
weeks after inoculation gave a higher sedimentation constant
than that obtained at 4 or 5 weeks after inoculation.

It was desirable, therefore, in comparing the virus protein from
diseased tomato and tobacco plants to use plants of approximately
the same size, to inoculate them at the same time, and later to
harvest the plants after the same period of infection. When
young plants grown under greenhouse conditions were used as the
source of virus protein, it was found that the purification procedure
used for old plants could be simplified and the crystalline protein
could be readily obtained from either tobacco or tomato plants.
The procedure used in this case was similar to the improved
method (4) but differed in several respects. The detailed pro-
cedure is described below.
Two flats each of young tobacco and tomato plants, planted
twenty-five to a flat, were inoculated when about 3 inches high
with 5 cc. of diluted, filtered juice from a mosaic-diseased tobacco
plant. The juice contained 0.22 mg. of protein nitrogen per cc.
Both the tobacco and tomato plants were cut 4 weeks later, after
they had shown symptoms of tobacco mosaic for about 3 weeks.
The plants were placed in burlap bags and frozen in a refrigerator
at -8”. Each group of plants was ground and extracted as
previously described (2). The extracts from the tomato plants
after filtration through Hyflo Super-Cel contained a total of 0.82
gm. of protein nitrogen, while those from the tobacco plants con-
tained 1.05 gm. of protein nitrogen. The globulin present in each
extract was then precipitated by the addition of 40 gm. of
(NH&S04 per 100 cc. of extract. The globulin was removed by
filtration with the aid of suction on a layer of Hyflo Super-Cel, and
the precipitate was washed with about 200 cc. of 40 per cent
(NH&Sod. The celite was extracted with 1 liter of 1 per cent
NatHPOd, u.s.P., and the extract was filtered through a second
layer of celite. The globulin in the filtrate was next precipitated
with 20 gm. of (NH&S04 per 100 cc. of extract and removed by
filtrationonStandard Super-Cel (celite). The filter cakewaswashed
with about 100 cc. of 20 per cent (NH&S04 and extracted twice
with 300 cc. and 200 cc., respectively, of 0.1 M phosphate buffer
at pH 7.0. The suspension was filtered each time through a thin
layer of celite. The protein in the combined extracts was next
precipitated with 15 gm. of (NH&S04 per 100 cc. of extract
and filtered as before on celite. The filter cake was washed with
15 per cent (NH&S04 solution and extracted again with 0.1 M

phosphate buffer. The protein was next precipitated with 13 gm.
of (NH&S04 per 100 cc. and filtered as before on celite, and the
celite was washed with a small amount of 13 per cent ammonium
sulfate solution. The phosphate extract from the 13 per cent
(NH&S04 precipitation gave a white opalescent solution of
protein with only a trace of yellow color. As the protein was
freed from brown pigment, it became less soluble and could be
precipitated with a lower concentration of (NH&S04. The use
of lower concentrations of ammonium sulfate also aided in the
removal of the remaining brown pigment, for the latter was more
soluble in the dilute salt solution.
The protein was next precipitated with 20 per cent (NH&S04
and was removed by gravity filtration on filter paper. It was
scraped from the paper, dissolved in water at pH 7 to 8, and
precipitated on an equal weight of celite by adjustment of
the hydrogen ion concentration of the solution to pH 4.5.
The celite and protein were filtered out on a layer of celite on a
Buchner funnel and suspended in 400 cc. of water at pH 7 to 8.
The suspension after being thoroughly mixed was filtered, and the
virus protein in the filtrate was crystallized by the addition of
enough saturated (NH&S04 dropwise to cause a slight silkiness
in the appearance of the solution, and then by the addition of
enough 10 per cent acetic acid in one-half saturated (NH&S04 to
lower the hydrogen ion concentration to about pH 5.5, Crystal-
lization was completed by the addition of sufficient saturated
(NH&SO4 to bring the salt concentration to approximately 20 per
cent by weight. The virus protein from either tomato or tobacco
plants may be recrystallized repeatedly without change of activity
if the solution is kept cold and the hydrogen ion concentration is
maintained between pH 5 and 7.5. In fractionation experiments

in which virus protein from tobacco plants was recrystallized
fifteen times, the final sample proved to have the same infectivity
as the original material. The results of the infectivity compari-
TABLE II _
Relative Infectivity of Virus Protein from Tobacco Plants after One
Crystallization and Fifteen Recrystallizations
-
Concentration
Experi- (pm. protein per cc.)
Test No. Preparation
ment No.

10-s 5 x 10-s
-
1* Crystallized once 66.6t 43.3
I‘ 15 times 68.4 49.0
No. of half leaves 42 42
M.D./S.D.l 0.44 1.4
Crystallized once 38.6 34.5
‘I 15 times 35.5 32.9
M.D./S.D. 1.05 0.47
2* Crystallized once 79.8 38.4
“ 15 times 72.5 47.0
M.D./S.D. 1.59 2.57
Crystallized once 51.1 42.4
‘I 15 times 55.3 41.9
M.D./S.D. 0.98 0.12
* In Experiment 1, 30 per cent and in Experiment 2, 81 per cent of the

original amount of virus protein was lost in the mother liquor during
recrystallization.
t Numbers opposite a particular preparation represent the average num-
ber of necrotic lesions per half leaf obtained on Phaseolus vulgaris on in-
oculation with the designated preparation and concentration.
$ To show a significant difference between the mean number of lesions
in any one experiment, the ratio of the difference of the mean (M.D.) to the
standard deviation of the difference (s.D.) should be not less than 2.1.
sons for two such samples are shown in Table II. In one experi-
ment 30 per cent and in the other 81 per cent of the original weight
of protein was lost during recrystallization. The recrystalliza-
tions were carried out by the same procedure used for the original
crystallization, with the exception that the solutions were kept at
about 10’. The crystallized protein was removed by centrifuga-

tion and the fifteen recrystallizations in each case were completed
in 2 days. It may be seen that in both experiments the fifteen
times recrystallized sample had the same infectivity as the
original, once crystallized material.
The yield of crystalline virus.protein obtained from 1.8 kilos of
tomato plants by the procedure described above was 1.7 gm., or
about 35 per cent of the protein nitrogen present in the extracts.
In the case of the tobacco plants, a yield of 3.4 gm.of crystalline
virus protein, or approximately 54 per cent of the protein nitrogen
FIQ. 1, a FIG. 1, b
FIG. 1. (a) Tobacco mosaic virus protein from tobacco plants; (b)
tobacco mosaic virus protein from tomato plants. X 520. (Photographed
by J. A. Carlile.)
present in the extracts, was obtained from 1.6 kilos of plant mate-
rial. Photomicrographs of several times recrystallized samples
from these young tomato and tobacco plants are shown in Fig. 1.
Relative Inject&dies of Mosaic Virus Protein from Young Tomato
and Tobacco Plants-The relative infectivities of the crystalline
protein obtained from the young tomato and tobacco plants were
determined by the method outlined above. The results of four
separate tests on Phaseolus vulgaris and Nicotiana glutinosa are
shown in Table III. In one experiment, in which from thirty-
seven to forty-one half leaves of Phaseolusvulgaris were used for
TABLE III
Relative Infectivity oJ Cr?ystalline Virus Protein Jrom Greenhouse Tomato and
Tobacco Plants
concentration
(gm. protein per cc.)
Preparation Test plant Remarks
10-4 10-s 10-s 10-7
Tomato 5 Phnseolus 96 1*161.7 46.1 17.6 Tomato 5 and To-

Tobacco 3 vulgaris 74.8 146 9 36.7 14.9 bacco 3, samples
No. of half 37 40 38 41 from young green-
leaves house plants

M.D./S.D.t 1.73 1.96 2.11 1.28
“
Tomato 5 133.0 36.3 5.4
Tobacco 3 123 8 38.4 3.5
No. of half 28 36 41
leaves
M.D./S.D. 1.01 0.48 2.62
Tomato 5 Nicotiana 60.0 95.4 43.1
Tobacco 3 glutinosc 50.6 103.6 41.7
leaves
M.D./S.D. 1.95 1.40 0.55
“
Tomato 5 60 79.8 18.7
Tobacco 3
No. of half
leaves
M.D./S.D. 1.08 0.27 2.86
Tomato 5 Phaseolus Q4.6 150.5 97 5 39.4 Tobacco 2, same sam-
Tobacco 2 vulgaris 18.0 81 6 35.1 10.2 ple from old tobacco
No. of half 19 24 22 20 plants described in
leaves Table I
M.D./S.D. 7 87 7.55 9.55 6.0;
‘i
Tomato 6 51.7 167.7 93.9 Tomato 6, once crys-
‘< 7 61.7 150 1 53.2 tallized sample.
No. of half 35 37 40 Tomato 7, same
leaves sample after re-
M.D./S.D. 0.85 2 81 7.58 peated treatment
with celite
* Numbers opposite a particular preparation represent average number

of necrotic lesions per half leaf obtained on inoculation with the designated
preparation and concentration.
t To show a significant difference between the mean number of lesions
in any one experiment, the ratio of the difference of the mean (M.D.) to the
each of the four dilutions tested, no significant odds for differences

in their infectivities were found in three of the four dilutions
compared. In another test, in which from twenty-eight to forty-
one half leaves of Phaseolus vulgaris were used for each of three
dilutions tested, only one of the three dilutions gave significant
odds for difference. Similar results were found when Nicotiana
glutinosa was used as the test plant. In two separate tests, in
which from twenty-one to thirty-three half leaves of Nicotiana
glutinosa were used for each of three dilutions tested, only one of
the six dilutions gave significant odds for difference. In this and

in the other two instances in which differences were found, the
odds, however, were not highly significant. As shown in Table
III, Tomato 5 and Tobacco 2, the infectivity of the virus prepara-
tion from young tomato plants grown in the greenhouse was also
compared with that of the protein obtained from old tobacco
plants grown in the field. The data show that the virus protein
obtained from young plants was significantly more active than
that obtained after more prolonged treatment from old plants.
In experiments in which the virus protein from young plants was
repeatedly treated with celite, it was found that this protein, like
that from old plants, became partially inactivated. The infectiv-
ity of one such preparation, Tomato 7, which had been recrystal-
lized twelve times and filtered on celite each time, compared to
the original sample, Tomato 6, is shown in Table III.
Relative Infectivity of Juice from Greenhouse Tomato and Tobacco
Plants-The percentage yield of crystalline protein from the young
tomato plants was appreciably smaller than that from the young
tobacco plants. The yields as mentioned previously were 35 and
54 per cent, respectively, of the total protein nitrogen in the tomato
and tobacco extracts. The lower yield in the case of the tomato
plants may have been due to a greater loss of the tomato virus
protein during fractionation or to its lower relative concentration
in the extract. The relative infectivities of extracts from tomato
and tobacco plants were, therefore, determined to compare by
infectivity measurements the relative concentration of virus
present. Both crude freshly expressed juice, obtained by pressing
the juice from the ground plants through bandage gauze, and the
same after filtration through Hyflo Super-Cel were compared.
The extracts from tomato and tobacco plants were analyzed for
protein and were diluted so that all contained the same protein
concentration before they were rubbed on the test plants. The
concentrations tested and the method of testing were the same as
for the crystalline preparation described above. The results of
several tests are shown in Table IV. It may be seen that the
crude tobacco extract was significantly more infectious than the
TABLE IV
Relative Infectivity of Juice from Greenhouse Tomato and Tobacco Plants
Concentration

km. protein per cc.)
Preparation Remarks
10-4 10-s 10-s

-___~
Tomato S.J. 1 lOl.l* 60.2 19.3 Freshly expressed samples of juice

Tobacco S.J. 1 140.2 80.3 37.3 strained through bandage gauze
leaves
M.D./SD.7 4.29 2.15 3.94
Tomato S.J. 2 191.7 50.6 30.6 Different from Tomato S.J. 1 and
Tobacco S.J. 2 233.0 85.3 34.3 Tobacco S.J. 1, but prepared in
No. of half 22 22 24 same way
leaves
M.D./&D. 2.65 3.85 0.97
Tomato F.J. 148.8 53.4 19.6 Same as Tomato S.J. 2 and To-
Tobacco F.J. 244.6 88.0 37.5 bacco S.J.2 after filtration
No. of half 22 22 20 through Hyflo Super-ccl
leaves
M.D./SD. 5.68 4.27 4.26
* Numbers opposite a particular preparation represent the average

number of necrotic lesions per half leaf obtained on Phaseolus vulgaris on
inoculation with the designated preparation and concentration.
i To show a significant difference between the mean number of lesions in
any one experiment, the ratio of the difference of the mean (M.D.) to the
crude tomato extract on a total protein basis. Since crystalline

protein of the same infectivity was obtained from both these
extracts, it appears that the difference in infectivity was due to a
difference in concentration of the infectious protein. The total
protein in the extract of 1.8 kilos of tomato plants was not only
lessthan that in the extract of 1.6 kilos of tobacco plants, but the
relative amount of infectious protein was also less. Thus it
appears that the tobacco mosaic virus reaches a higher concen-
tration in tobacco than in tomato plants. The fact that as much
as 54 per cent of the total protein in the extract of the tobacco
plants was isolated in crystalline form indicates that the major
portion of the protein present consists of virus protein.
General Properties of Crystalline Virus Protein from Tomato
Plants-The crystalline material isolated from mosaic-diseased
tomato plants is similar to that obtained from mosaic-diseased
tobacco plants, in that it has the general properties of a globulin.
It gives a positive test with the usual protein color reactions, such

as Millon’s, xanthoproteic, glyoxylic acid, and biuret. The
Elementary Analysis of Crystalline Tobacco Mosaic Virus Protein from

Tomato and Tobacco Plan.ts
Preparation* C H N (Dumas) P Ash
per cent per cent per cent per cent per cent
Tomato 5 50.93 7.58 16.70 0.21 1.29
Tobacco 3 50.74 7.56 16.56 0.01 0.53
Tomato 2 51.57 6.91 16.20 1.64
51.37 6.95 16.35 1.43
* The tomato and tobacco samples are the same as those described in
Tables I and III. Tomato 5 and Tobacco 3 were dialyzed against distilled
water at pH 7 for 7 days and precipitated and washed with acetone. To-
mato 2 was prepared similarly, but was also dialyzed for about 2 days at
pH4.
purified protein is soluble in distilled water at pH 7 and in dilute

ammonium sulfate solutions. It is precipitated at pH 7 by con-
centration of (NH&S04 greater than 13 per cent, or by saturation
with MgSOd. As the hydrogen ion concentration is increased
below pH 7, the protein is precipitated by increasingly smaller
amounts of (NH&S04. At pH 3.3 it is practically insoluble even
in the absence of salt.
The results obtained from chemical analyses for C, H, N, P, and
ash of the highly active crystalline proteins from young tomato and
tobacco plants, together with the analysis of one of the lessactive
samples from old tomato plants grown in the field, are shown in
Table V. It may be seen that all samples have about the same
chemical composition. The values for C, H, and N of the highly
active tomato sample are in good agreement with those obtained
for the tobacco sample of comparable activity.
Opticab Activity-The results of several comparative determina-
tions of optical activity for preparations of quite different infectivi-
ties from both old and young plants are shown in Table VI. They
were obtained by determining the rotation of a solution of the
purified protein prepared by the addition of 2 drops of 2 N NaOH
to 5 cc. of approximately a 0.5 per cent dialyzed suspension of the
protein. These likewise show all the samples to have about the

same specific rotation, regardless of their infectivity or whether
they were prepared from tomato or tobacco plants. It is interest-
ing to note that completely inactive tobacco mosaic virus protein
TABLE VI
Optical Activity of Different Samples of Crystalline Tobacco Mosaic
Virus Protein
Preparation* [a]: (per mg. N) Conoentration pH of solution
degrees gm. per 100 cc.

Tomato 1 -0.43 0.57 11.2
“ 2 -0.42 0.88 11.45
I‘
3 -0.44 0.46 11.65
L‘
5 -0.42 0.49
Tobacco 3 -0.45 0.40
- -!-
* The preparations are the same as those described in Tables I and III.
prepared by treatment with ultraviolet light, formaldehyde, or

nitrous acid (9) likewise has a specific rotation of -0.42” per mg.
of nitrogen.
Isoelectric Point-Cataphoresis experiments were carried out by
means of the Northrop-Kunite apparatus on dilute suspensions
of crystals of virus protein. The suspensionswere prepared by
the addition of 2 drops of saturated (NH&S04 solution and 3 to 4
drops of 0.2 N HCl to 10 cc. of a 0.4 per cent solution of the puri-
fied protein dialyzed at pH 7 to 8. The suspensionswere then
diluted with 90 cc. of distilled water adjusted to pH 3.3 with 6
drops of 0.2 N HCl. Portions of this suspensionwere adjusted to
various hydrogen ion concentrations with 0.02 N NaOH and 0.02
N HCl, and were examined in the cataphoresis cell. Under these
conditions, dilute suspensions of tiny crystals easily visible under

the microscope were obtained. The hydrogen ion concentration
was measured with a MacInnes type glass electrode.
The values found for the isoelectric points of different prepara-
tions from both tomato and tobacco plants have varied from about
pH 3.2 to about pH 3.35. All the samples examined from either
tomato or tobacco plants showed consistent migration towards the
negative pole below pH 3.2 and towards the positive pole above pH
3.35. The less active samples obtained from old plants gave isoe-
lectric points in the lower range, while the more active samples ob-

tained from young plants gave values towards the higher range.
The most active samples isolated from young tomato and tobacco
plants grown in the greenhouse gave the same value of about 3.3.
The isoelectric point of an individual preparation was found to vary
depending on the salt concentration of the buffer mixture and the
nature of the salt present. In experiments in which acetic acid,
as well as (NH&S04 and NaCl, was present, the isoelectric point
was raised from 0.1 to 0.3 of a pH unit depending on the concen-
tration of (NH&S04 and acetic acid. Eriksson-Quensel and
Svedberg (10) have found the isoelectric point of a many times
recrystallized sample in (NH&S04-acetate buffer to be pH 3.49.
Best (ll), from precipitation studies, has reported the isoelectric
point of a purified but not crystalline tobacco mosaic preparation
to be pH 3.4. Takahashi and Rawlins (12), in experiments in
which the migration of virus particles at various hydrogen ion
concentrations was determined by infectivity measurements,
found the virus to be isoelectric below pH 4.
Sedimentation Constants-The sedimentation constants and
molecular weights of samples of crystalline tobacco mosaic virus
protein from both the old and young tomato plants described
above have been determined by Dr. Wyckoff and Mr. Biscoe (8).
The sedimentation constant for the crystalline protein isolated
from old tomato plants is slightly larger than that found for the
protein from tobacco plants. However, the sedimentation con-
stants of the virus proteins prepared under comparable conditions
from young tobacco and tomato plants were found to be exactly
the same.
Samples of the virus proteins from young tomato and tobacco
plants were submitted to Dr. Svedberg for an ultracentrifugal
analysis. During their preparation these samples were main-

tained between pH 6 and 8 in an effort to retain their molecular
homogeneity. The samples, however, arrived too late to be
included in the report by Eriksson-Quensel and Svedberg (10).
Dr. Svedberg has kindly consented to have the results of the
ultracentrifugal study, which was carried out by Mrs. Eriksson-
Quensel, presented here. Although the new samples of virus
protein were found to be somewhat inhomogeneous with respect
to molecular weight, they were more homogeneous than the virus
protein previously studied. The sedimentation constant,,

Xzo’ X 1013,of virus protein from tobacco plants in solution at pH
6.8 was found to be 197 for the sample submitted in solution and
190 for the sample submitted in the form of a paste. That of
virus protein from tomato plants submitted and tested in solution
at pH 6.8 was found to be 202. These constants correspond to a
molecular weight of about 17,000,OOO. At pH 9.8 the sample
from tomato @ants was found to have formed two components
having constants of 185 and 125, respectively, and at pH 11.7 it
was found to have been disintegrated and split into low molecular
weight components having constants of 8.1 and 3.8, respectively.
The ultracentrifugal analyses of Dr. Svedberg and Dr. Wyckoff
indicate that the sedimentation constant of virus protein obtained
from young tomato plants is probably the same as that of virus
protein obtained under comparable conditions from young tobacco
plants.
Serological Experiments-Both precipitation and absorption
experiments were performed to compare the serological properties
of the crystalline virus protein preparations obtained from tobacco
and tomato plants. Antisera were obtained from rabbits which
had been injected with 200 mg. of each preparation, in weekly
doses of 50 mg. each, dissolved in physiological saline solution
at pH 7 to 8. The antiserum from the tobacco virus protein was
then tested for precipitating antibodies both with tobacco virus
protein and with that obtained from tomato plants, according to
the precipitation technique used by Chester (13). The converse
experiment was also performed for the antiserum to the tomato
virus protein. The results of a typical experiment are shown in
Table VII. As previously found (2), the antiserum to tobacco
virus protein gave a precipitate with 2 X 10e6 gm. of the homol-
ogous antigen. Likewise, the antiserum to tomato virus protein

gave a precipitate with 2 X 1OW gm. of its homologous antigen.
When antisera to either the tobacco virus protein or the tomato
virus protein were mixed with the other antigen, a precipitate was
likewise formed in each case with but 2 X lO-‘j gm. of the antigen.
In the absorption experiments, both antisera were completely
absorbed with their heterologous antigens. The absorbed anti-
serum was then tested with a small amount of its homologous
antigen. In neither case was there an additional precipitate.
One of two such experiments performed is outlined in Table VIII.

TABLE VII
Results of Precipitin Tests with Crystalline Tomato and Tobacco
Virus Proteins
Coneentrat&;of protein Antiserum to Antiserum to

tomato virus tobacco virus
protein protein
gm.
Tomato virus protein
2 x 10-d ++++ ++++
2 x 10-G + +
2 x 10-e f f
Tobacco virus protein
2 x 10-b ++++ ++++
2 x 10-s + +
2 x 10-6 f f
. .. .
i-i-+-/- indicate a very heavy precipitate; + a moderate precipitate;
A a very slight precipitate; - absence of a precipitate.
The results demonstrate that both the virus protein from tobacco
and from tomato plants served equally well in absorbing the anti-
bodies formed after the injection of either protein, and that the
injection of virus protein from either plant host caused the forma-
tion of no antibodies which were not precipitated by the virus
protein from the other.
Solubility Experiments-The solubilities of the crystalline virus
proteins obtained from tomato and tobacco plants were deter-
mined to find whether the two samples would give the same solu-
hility. Preliminary experiments, in which the virus proteins
obtained from the old tomato and tobacco plants grown in the
TABLE VIII
Absorption Experiment on Crystalline Tomato and Tobacco Virus Protein
T
Procedure Result Procedure Result
(I) I cc. antiserum to tomato virus protein + 2.33 20+t (1) 1 cc. antiserum to tobacco virus protein + 2.33 2o+t
cc. (9.3 mg.) tobacco virus protein + 0.3 cc. cc. (9.3 mg.) tomato virus protein + 0.3 cc.
merthiolate (preservative), * mixed, incubated merthiolate (preservative), mixed, incubated
at 37”for 1 hr., stored in refrigerator overnight at 37” for 1 hr., stored in refrigerator over-
and centrifuged night, and centrifuged
(2) Supernatant from (1) mixed with 1.0 cc. (3.99 +t (2) Supernatant from (1) mixed with 1.0 cc. (3.99 +t
mg.) tobacco virus protein, incubated, etc., as mg.) tomato virus protein, incubated, etc., as
in (1) in (1)
(3) Supernatant from (2) treated as in (2) a (3) Supernatant from (2) treated as in (2) G
(4) “ ‘( (3) “ (‘ (‘ (2) t (4) “ “ (3) “ (( “ (2) t
(5) “ “ (4) mixed with 1.0 cc. (3.99 t (5) ‘I “ (4) mixed with 1.0 cc. (3.99 t
mg.) tomato virus protein, incubated, etc., as mg.) tobacco virus protein, incubated, etc., as
in (1) in (I)
* A control experiment, in which normal serum was mixed with the same concentration of merthiolate, failed to give a
precipitate.
t The precipitates from (1) and (2) in each experiment were combined, washed twice with physiological salt solution,
and analyzed for nitrogen. That from the antiserum to tomato virus protein contained 2.14 mg. of N, while the other con-
tained 2.06 mg. of N. 20+ indicates a large precipitate, + a small precipitate, and t a trace of precipitate.
$ The trace of precipitate which continued to be formed after the absorption of the antibodies probably consisted of
denatured protein.

field were compared, had shown the former to have a solubility
about four times that of the latter. However, the tomato virus
protein, as was mentioned before, carried more yellow pigment
than did the tobacco sample. It was felt, therefore, that the
difference might be due to the presence of the yellow impurity in
the tomato sample or possibly to its lower infectivity. Subse-
quent efforts to purify this tomato sample had resulted in prepara-
tions that gave white solutions, but, as mentioned before, possessed
still less virus activity than the original crystalline material.
Additional solubility determinations were, therefore, carried out

on the virus proteins obtained from young tobacco and tomato
plants. To 9.0 cc. of dialyzed solutions of the tomato and tobacco
virus proteins, each containing 63.8 mg. of protein, was added 1 cc.
of 1 M phosphate buffer at pH 5.6 and 625.0 mg. of (NH&S04.
Kach solution was stirred during the addition of the (NH&S04,
and in each case a part of the protein crystallized. Both suspen-
sions were then stirred for 4 hour and centrifuged for a total of 4
hours. At the end of 2, 3, and 4 hours, respectively, samples were
removed from each and analyzed for protein nitrogen.
The supernatant liquid from the tobacco protein contained 2.37
mg. of protein per cc. after centrifugation for 2 hours, while that
from the tomato protein contained 1.84 mg. per cc. Additional
centrifugation decreased the former value to 2.24 and finally to
1.47 mg. per cc., whereas the tomato decreased to 1.63 and 1.19
mg. per cc. Several experiments of this type in which the amount
of ammonium sulfate was varied from 901 mg. to 590 mg. per 10
cc. were performed. In each case it was found that the protein
concentration of the supernatant liquid decreased continually
during 4 hours of centrifugation, and in all cases the values found
for the tobacco protein were slightly higher than those found for
the tomato protein. In some cases attempts were made to ap-
proach equilibrium from the undersaturated side by extraction of
the residue obtained after centrifugation with solvent of the same
composition as the solution from which it had separated. In
these experiments the protein concentration after 3 hours of
centrifugation was always less than the lowest value found when
equilibrium was approached from the supersaturated side.
The above experiments provide a rough comparison of the solu-
bilities of the virus protein from tomato and tobacco plants and
show that they are of the same order. Additional data must be
obtained, however, before it can be said with certainty whether
the two proteins have identical or slightly different solubilities.
DISCUSSION
The highly active crystalline protein obtained from young

tomato plants by careful fractionation agrees very closely in
properties with that isolated from young tobacco plants grown
under the same conditions. The general properties of both pro-
teins are the same. They have the same elementary composition,

the same optical activity, and the same isoelectric point. The
properties of the virus proteins which are most likely to show
differences between two preparations are their infectivities, sero-
logical reactions, and solubilities. These have been compared
for both the tomato and tobacco proteins. A number of infectiv-
ity measurements at various concentrations have shown each
preparation to have very much the same infectivity. The mean
difference between the number of lesions obtained with the same
concentration of tomato and tobacco virus protein was always,
with but three exceptions, too small to indicate any significant
difference in the infectivity of the two samples.
The serological experiments have, likewise, shown the two
preparations to have identical serological properties. It is well
known that antiserum to one strain of tobacco mosaic virus will
give a precipitate when tested either with the same or with a
different strain of mosaic virus. However, it has recently been
shown by Chester (14) that some strains of tobacco mosaic virus
may be differentiated by absorption experiments. It was found
that antiserum to ordinary tobacco mosaic virus gave a precipi-
tate with this virus after the serum had been completely absorbed
with aucuba mosaic virus. Aucuba mosaic virus, therefore, does
not completely absorb the antibodies present after the injection of
ordinary tobacco mosaic virus. Absorption experiments on
tomato and tobacco virus protein, however, have failed to show
differences between these proteins. The antibodies formed
after the injection of one protein were completely removed by
absorption with the other.
The results of the solubility experiments suggest that the virus
protein from tomato plants is slightly less soluble under the same
conditions than virus protein from tobacco plants, However,

the differences found were small and could be explained in a
number of ways. More experimental data must be obtained
before a definite conclusion can be drawn regarding the solubilities
of the two proteins. It is entirely possible that the virus protein
from tomato plants may differ slightly from that present in tobacco
plants but still possess the same physiological properties. As
shown by Northrop (15), crystalline pepsins prepared from differ-
ent animal species possess the same physiological properties but
have different solubilities and are therefore different proteins.

It seems possible that slightly different proteins might also carry
the same virus activity.
The isolation of crystalline protein preparations with varying
degrees of infectivity from old tomato plants has shown that the
loss of activity does not affect the ability of the protein to crystal-
lize. This result is comparable to what has been found for aucuba
mosaic virus (16) and even more strikingly for tobacco mosaic
virus treat,ed with hydrogen peroxide or nitrous acid (9). Aucuba
mosaic virus protein, like tobacco mosaic virus protein, becomes
less active on repeated fractionation, while tobacco mosaic virus
protein becomes entirely inactive when treated with hydrogen
peroxide or nitrous acid. Both the less active aucuba and the
entirely inactive hydrogen peroxide-treated proteins are well
defined crystalline preparations. The fact that the changed
protein obtained after hydrogen peroxide treatment resembles
very closely the partially inactive samples suggests that the
partial inactivation may be due to a similar type of reaction.
Repeated treatment with celite as used in the course of fractiona-
tion may well catalyze the oxidation by air of unstable groups in
the molecule.
SUMMARY
The isolation from tobacco mosaic-diseased tomato plants of a

crystalline protein possessing the properties of tobacco mosaic
virus is described. It was found that the most active crystalline
material could be best obtained from young rapidly growing green-
house plants by a procedure involving a minimum amount of
treatment with celite.
The properties of the proteins obtained from tomato and tobacco
plants grown under the same conditions and treated by the same
procedure have been compared. The proteins have been shown
to possess the same infectivities and to have identical serological
properties and very nearly the same solubilities. They likewise
have the same chemical composition, optical activity, and iso-
electric point, and give the same sedimentation constant.
Repeated fractionation of the virus protein with celite at pH 4.5
and 8.0 results in a gradual inactivation of the protein, which
remains soluble and may still be crystallized. It has been shown
in other fractionation experiments, however, in which as much as

81 per cent of the original sample has been lost during the course of
fifteen recrystallizations, that the crystals which remained pos-
sessed the same infectivity as the original sample.
The comparison of the relative infectivities of the juices of
diseased tomato and tobacco plants on a total protein basis indi-
cates, in agreement with the percentage yields of crystalline virus
protein isolated, that the tobacco mosaic virus reaches a higher
concentration in tobacco than in tomato plants.
BIBLIOGRAPHY
1. Stanley, W. M., Science, 81, 644 (1935).

2. Stanley, W. M., Phytopathology, 26, 305 (1936).
3. Clinton, G. P., Connecticut Agric. Exp. Stat. Biennial Rep., pt. 12, 857
(190748).
4. Stanley, W. M., J. Biol. Chem., 116, 673 (1936).
5. Holmes, F. O., Bot. Gaz., 87, 39 (1929).
6. Samuel, G., and Bald, J. G., Ann. Appl. Biol., 20, 70 (1933).
7. Fisher, R. A., Statistical methods for research workers, Edinburgh
and London, 6th edition (1936).
8. Wyckoff, R. W. G., Biscoe, J., and Stanley, W. M., J. Bid. Chem., 117,
57 (1937).
9. Stanley, W. M., Science, 83, 626 (1936).
10. Eriksson-Quensel, I., and Svedberg, T., J. Am. Chem. Sot., 68, 1863
(1936).
11. Best, R. J., AustralianJ. Exp. Biol. andMe& SC., 14,l (1936).
12. Takahashi, W.‘N., and Rawlins, T. E., Hilgardia, 4, 441 (1930).
13. Chester, K. S., Phytopathology, 26, 686 (1935).
14. Chester, K. S., Phytopathology, 26, 778 (1936).
15. Northrop, J. H., J. Gen. Physiol., 16, 615 (1933).
16. Stanley, W. M., J. Biol. Chem., 117, 325 (1937).

J. Biol. Chem.-1937-Loring-733-54

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ISOLATION OF CRYSTALLINE TOBACCO MOSAIC VIRUS

PROTEIN FROM TOMATO PLANTS*

(Received for publication, December 15, 1936)

The isolation of a crystalline protein possessing the properties

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Method of Isolation-The same experimental procedure used in

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tration in each test. The relative infectivity of two solutions was

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Relative Infectivity of Crystalline Virus Protein from Tomato and Tobacco

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Tomato 1 Nicotiana 68.2* 6.7 2.6 Tomato 1, slightly yellow

* Numbers opposite a particular preparation represent the average

could be obtained from mosaic-diseased tomato plants. Because

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maintained between pH 5 and 7.5. In fractionation experiments

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* In Experiment 1, 30 per cent and in Experiment 2, 81 per cent of the

about 10’. The crystallized protein was removed by centrifuga-

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Tomato 5 Phnseolus 96 1*161.7 46.1 17.6 Tomato 5 and To-

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* Numbers opposite a particular preparation represent average number

each of the four dilutions tested, no significant odds for differences

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10-4 10-s 10-s

Tomato S.J. 1 lOl.l* 60.2 19.3 Freshly expressed samples of juice

* Numbers opposite a particular preparation represent the average

crude tomato extract on a total protein basis. Since crystalline

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Elementary Analysis of Crystalline Tobacco Mosaic Virus Protein from

Preparation* C H N (Dumas) P Ash

purified protein is soluble in distilled water at pH 7 and in dilute

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Preparation* [a]: (per mg. N) Conoentration pH of solution

degrees gm. per 100 cc.

prepared by treatment with ultraviolet light, formaldehyde, or

conditions, dilute suspensions of tiny crystals easily visible under

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analysis. During their preparation these samples were main-

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ogous antigen. Likewise, the antiserum to tomato virus protein

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Coneentrat&;of protein Antiserum to Antiserum to

Procedure Result Procedure Result

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The highly active crystalline protein obtained from young

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conditions than virus protein from tobacco plants, However,

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The isolation from tobacco mosaic-diseased tomato plants of a

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1. Stanley, W. M., Science, 81, 644 (1935).

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